thesis karthika revised - Murdoch University...Karthika Krishnasamy , Richard Bell and Qifu Ma...
Transcript of thesis karthika revised - Murdoch University...Karthika Krishnasamy , Richard Bell and Qifu Ma...
SODIUM AND CULTIVAR EFFECTS ON POTASSIUM NUTRITION OF WHEAT
THIS THESIS IS PRESENTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF MURDOCH UNIVERSITY
By
KARTHIKA KRISHNASAMY
Bachelor of Technology in Horticulture;
Honours in Environmental Science
School of Veterinary and Life Sciences
Murdoch University
2015
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DECLARATION
I declare that this thesis is my own account of my research and contains as its
main content work which has not previously been submitted for a degree at any
tertiary education institution.
........................
Karthika Krishnasamy
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Papers and Presentations from this research
Journal Paper
1. Krishnasamy, K., Bell, R and Ma, Q (2014). Wheat responses to sodium vary with
potassium use efficiency of cultivars. Frontiers in Plant Science, 5: 631.
doi: 10.3389/fpls.2014.00631.
http://journal.frontiersin.org/Journal/10.3389/fpls .2014.00631/abstract#
International Conference Presentations
1. Richard Bell, Qifu Ma and Karthika Krishnasamy (2013) Wheat and barley genotypes
differ in growth response to soil potassium supply under low to moderate sodium supply, in:
XVII. International Plant Nutrition Colloquium (IPNC) held on 19- 22 August, 2013 at the
Istanbul Convention and Exhibition Centre (ICEC), Istanbul, Turkey.
2. Karthika Krishnasamy , Richard Bell and Qifu Ma (2013), Moderate sodium has positive
effects on wheat grown in a potassium deficient split-root system, in: XVII. International
Plant Nutrition Colloquium (IPNC) held on 19- 22 August, 2013 at the Istanbul Convention
and Exhibition Centre (ICEC), Istanbul, Turkey.
3. Karthika Krishnasamy , Richard Bell and Qifu Ma (2013), Low to moderate sodium is
beneficial to wheat genotypes grown under potassium deficient conditions, in: Combio, 2013
held on 29 September to 3rd October at Perth Convention and Exhibition Centre, Perth,
Western Australia.
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ABSTRACT
In arid and semiarid regions, soil salinity is largely due to excessive sodium chloride (NaCl)
which, apart from osmotic and specific Na+ and Cl- ion effects, has a detrimental effect on
potassium (K) uptake and nutrition of most crops. However, in K deficient soils, Na+ can
substitute for some functions of K+, provided that plants have the ability to take up,
translocate, and compartmentalise Na+ into the vacuoles where it mainly replaces the
biophysical functions of K+ in maintaining cell turgor, ionic balance, regulating osmotic
potential and improving water balance via stomatal conductance. Potassium deficiency and
soil salinity stress have become increasingly common in agricultural lands of Western
Australia (WA) and many parts of the world, but the role of Na in K nutrition of wheat
(Triticum aestivum L.) is not well understood. The interaction between K and Na in wheat
genotypes differing in K-use efficiency has not been researched previously. This research
focussed mainly on low to moderate concentrations of Na in wheat K nutrition and less
emphasis is placed on Na toxicity effects as there is a large body of research available on Na
toxicity effects. A series of glasshouse experiments were designed for both soil and solution
culture where Na was supplied at a range of concentrations at low and adequate K levels. The
responses of K-efficient and K-inefficient Australian wheat cultivars were examined. Plant
responses were assessed by measuring plant growth, leaf gas exchange, shoot and root K and
Na concentrations and their contents. High soil Na levels (100 and 200 mg Na/kg) greatly
reduced the plant growth in wheat cultivars predominantly at low soil K (40 mg K/kg). By
contrast, low to moderate Na levels (25 and 50 mg Na/kg in soil culture and 2 mM Na in
solution culture) stimulated wheat growth at low K supply, particularly in K-efficient
cultivars compared with K-inefficient cultivars. Roots were more responsive to low
concentrations of Na than shoots in experiments where growth stimulation was observed.
Low to moderate Na supply also increased leaf net photosynthesis and stomatal conductance
at low K supply, with the measured values similar to those observed under adequate K
condition both in soil and solution culture. In the split-root experiment, the positive effects of
moderate soil Na on growth and K uptake of low K plants were evident when K and Na were
supplied in the same or different parts of the root system. In low K soil, low to moderate Na
levels increased plant K content, particularly shoot K content, which may account for the
increased leaf net photosynthesis rate, stomatal conductance, and plant dry weight. In contrast
to previous reports, which attributed Na stimulation of plant growth at low K to increased
Na+ uptake and utilisation in place of K+, in wheat, Na+ increased K+ uptake in soil culture,
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and it increased Rb+ uptake (as a tracer of K+) in solution culture experiments. Hence we
attribute most of the benefits of low to moderate Na application in wheat to improved K
uptake and K nutrition. The main mechanism for Na+-stimulated K+ uptake under limited K
availability with low external Na supply in wheat is likely the effect of Na+ on K+
transporters, both on high-affinity and low-affinity K+ uptake transport systems. In this study,
K-use efficiency among wheat cultivars showed varied responses to Na supply at low K, with
increased stimulation in root growth, shoot K concentrations, K uptake and leaf
photosynthesis in K-efficient cultivar relative to K-inefficient cultivar. Genotypic differences
in K-use efficiency also influenced Na uptake and salt tolerance with K-efficient cultivars
being more salt tolerant than K-inefficient cultivars. The current research on K+ substitution
by Na+ in wheat physiological processes is of great importance in fertiliser management
strategies. The application of expensive K fertilisers is limited by poor farmers especially in
developing countries, and partial substitution of K by Na in plant nutrition can decrease the
cost of production. Based on this study, when K-efficient wheat cultivars are grown under
low to moderately saline conditions, the substitution of K by Na was not strong enough to
recommend Na-based fertilisers in place of K in wheat. Nevertheless, the alleviation of K
deficiency symptoms in wheat by addition of moderate Na provides a trigger for conducting
further studies. The present research based on glasshouse experiments needs to be evaluated
under field conditions with further studies under varying soil and agro-climatic conditions to
define critical soil levels of Na that stimulate wheat growth.
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TABLE OF CONTENTS
Page numbers
DECLARATION……………………………………………………………………. ii
ABSTRACT…………………………………………………………………………. iv
TABLE OF CONTENTS……………………………………………………………. vi
LIST OF FIGURES………………………………………………………………….. xii
LIST OF TABLES…………………………………………………………………… xv
LIST OF ABBREVIATIONS AND SYMBOLS…………………………………… xx
ACKNOWLEDGEMENTS………………………………………………………….. xxiii
Chapter 1. Introduction………………………………………………………………. 1
1.1 Potassium nutrition in plants…………………………………………….... 1
1.2 Sodium nutrition in plants……………………………………………….... 1
1.3 Interaction between potassium and sodium………………………………. 2
1.4 K deficiency in soils………………………………………………………. 3
1.5 Salinity issues of WA……………………………………………………... 3
1.6 Research aim and scope of the study……………………………………… 4
1.7 Layout of the thesis………………………………………………………... 4
Chapter 2. Literature review…………………………………………………………... 6
2.1 Introduction………………………………………………………………... 6
2.2 K functions in plants………………………………………………………. 6
2.2.1 K and enzyme activation………………………………………… 7
2.2.2 K and protein synthesis………………………………………….. 7
2.2.3 K and stomatal activity………………………………………….. 7
2.2.4 K and photosynthesis…………………………………………..... 8
2.2.5 K and stress tolerance in plants………………………………….. 8
2.2.5.1 Anti-oxidant activity…………………………………... 9
2.2.5.2 Drought and heat stress……………………………….. 10
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2.2.5.3 Low temperature and frost stress…………………….. 11
2.2.5.4 Disease and pest resistance……………………………. 12
2.3 K deficiency in plants……………………………………………………... 12
2.4 K uptake and accumulation by plants…………………………………….. 13
2.5 Forms of K in soil………………………………………………………..... 14
2.5.1 Unavailable/mineral K…………………………………………... 15
2.5.2 Slowly available/non-exchangeable/fixed-K………………….... 15
2.5.3 Readily available/ exchangeable K……………………………… 15
2.5.4 Soil solution K…………………………………………………... 15
2.6 Removal of K from soil…………………………………………………… 16
2.7 Na functions in plants…………………………………………………….. 17
2.8 Interaction between K and Na……………………………………………. 18
2.8.1 High Na………………………………………………………….. 19
2.8.1.1 Na toxicity effects…………………………………….. 19
2.8.1.2 Imbalance in K/Na ratios…………………………….... 19
2.8.2 Low to moderate Na…………………………………………….. 20
2.9 Functions of K replaced by Na…………………………………………… 20
2.10 Plant responses to Na at low K………………………………………….. 22
2.11 K and Na transporters…………………………………………………… 30
2.12 Genotypic variation in Na substitution of K……………………………. 30
2.13 Salinity and duplex soils…………………………………………………. 31
2.14 Research scope, aim and research questions……………………………. 32
2.15 Conclusion………………………………………………………............. 33
Chapter 3. Wheat responses to sodium vary with potassium use efficiency of cultivars………………………………………………………………………………..
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3.1 Introduction……………………………………………………………..... 34
3.2 Materials and methods……………………………………………………. 35
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3.2.1 Potassium and sodium treatments………………………………. 36
3.2.2 Measurements…………………………………………………… 36
3.2.3 Statistical analysis………………………………………………. 37
3.3 Results…………………………………………………………………….. 37
3.3.1 Plant growth…………………………………………………….. 37
3.3.2 Leaf gas exchange……………………………………………….. 43
3.3.3 K and Na concentrations in shoots and roots……………………. 43
3.3.4 Soil exchangeable cations after K and Na addition……………... 49
3.4 Discussion…………………………………………………………………. 50
3.5 Conclusion………………………………………………………………… 55
Chapter 4. Split-root experiment
Moderate sodium increased K uptake, leaf gas exchange and plant growth of wheat cv. Wyalkatchem grown in a K-deficient split-root system……………………………
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4.1 Introduction……………………………………………………………...... 56
4.2 Materials and methods……………………………………………………. 57
4.2.1 Potassium and sodium treatments………………………………. 58
4.2.2 Measurements…………………………………………………… 58
4.2.3 Statistical analysis……………………………………………….. 59
4.3 Results……………………………………………………………………... 59
4.3.1 Plant growth……………………………………………………... 59
4.3.2 Leaf gas exchange……………………………………………….. 62
4.3.3 K and Na concentrations and accumulation…………………….. 63
4.4 Discussion…………………………………………………………………. 68
4.5 Conclusion………………………………………………………………… 74
Chapter 5. Column experiment
Potassium response of wheat grown in columns with drying topsoil and varying subsoil K and Na levels………………………………………………………………..
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5.1 Introduction………………………………………………………………. 75
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5.2 Materials and methods…………………………………………………… 76
5.2.1 Treatments………………………………………………………. 77
5.2.2 Measurements…………………………………………………… 79
5.2.3 Statistical analysis………………………………………………. 79
5.3 Results…………………………………………………………………….. 79
5.3.1 Plant growth…………………………………………………….. 79
5.3.2 Leaf gas exchange………………………………………………. 86
5.3.3 K and Na concentrations………………………………………… 91
5.4 Discussion…………………………………………………………………. 99
5.5 Conclusion………………………………………………………………… 102
Chapter 6. Solution culture short-term response
Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium (Na+) supply using rubidium (Rb+) tracer in solution culture experiments: short-term responses……………………………………………………………………………..
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6.1 Introduction………………………………………………………………. 103
6.2 Materials and methods…………………………………………………… 104
6.2.1 Plant culture……………………………………………………. 104
6.2.2 Basal nutrient solution…………………………………………. 105
6.2.3 Potassium and sodium treatments……………………………… 105
6.2.4 Measurements………………………………………………….. 105
6.2.5 Statistical analysis……………………………………………… 106
6.3 Results……………………………………………………………………. 106
6.3.1 Experiment 1…………………………………………………… 106
6.3.1.1 Plant growth………………………………………….. 106
6.3.1.2 Leaf gas exchange……………………………………. 106
6.3.1.3 K, Na and Rb concentrations in shoot and root………. 108
6.3.1.4 K, Na and Rb contents in shoot and root…………….. 110
6.3.2 Experiment 2…………………………………………………… 112
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6.3.2.1 Plant growth …………………………………………. 112
6.3.2.2 Leaf gas exchange…………………………………… 112
6.3.2.3 K, Na and Rb concentrations in shoot and root……… 114
6.3.2.4 K, Na and Rb contents in shoot and root……………. 117
6.4 Discussion……………………………………………………………….. 119
6.5 Conclusion……………………………………………………………….. 122
Chapter 7. Solution culture experiment long-term response
Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium (Na+) supply using rubidium (Rb+) tracer in a solution culture experiment: long-term responses………………………………………………………………………………
123
7.1 Introduction………………………………………………………………. 123
7.2 Materials and methods……………………………………………………. 124
7.2.1 Plant culture…………………………………………………….. 124
7.2.2 Basal nutrient solution………………………………………….. 124
7.2.3 Potassium and sodium treatments………………………………. 124
7.2.4 Measurements…………………………………………………… 125
7.2.5 Statistical analysis………………………………………………. 125
7.3 Results……………………………………………………………………. 126
7.3.1 Plant growth…………………………………………………….. 126
7.3.2 Leaf gas exchange………………………………………………. 130
7.3.3 K, Na and Rb concentrations……………………………………. 132
7.3.4 K, Na and Rb contents………………………………………….. 137
7.4 Discussion…………………………………………………………………. 141
7.5 Conclusion………………………………………………………………… 144
Chapter 8. General discussion and conclusions………………………………………. 145
8.1 Introduction………………………………………………………………. 145
8.2 Growth stimulation by Na………………………………………………… 145
8.3 Na effects on K deficient wheat………………………………………….. 147
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8.4 Stimulation of K uptake by Na……………………………………………. 148
8.5 Possible mechanisms of Na+-induced K+ uptake………………………… 150
8.6 Toxicity effects of Na…………………………………………………… 152
8.7 Na effects on cultivars differing in K-use efficiency…………………… 153
8.8 Implications of low to moderate Na for plant K nutrition……………… 153
8.9 Conclusions and recommendations……………………………………… 154
8.9.1 Conclusions……………………………………………………. 154
8.9.2 Further research recommendations…………………………….. 156
References…………………………………………………………………………….. 157
Appendices……………………………………………………………………………. 166
Appendix 1
1.1 K and Na concentrations in leaves, spikes and stem……………………… 166
1.2 Ca and Mg concentrations in young and old leaves………………………. 170
Appendix 2
Root: Shoot ratios of wheat grown in columns harvested at 5 WAS………… 173
Appendix 3
Experimental setup used in solution culture experiments…………………….. 174
Appendix 4
4.1 Plant growth (Experiment- 1) …………………………………………….. 175
4.2 Pre-treatment leaf gas exchange measurements (Experiment- 1)…………. 178
4.3 Plant growth (Experiment- 2)……………………………………………... 179
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LIST OF FIGURES
Figure 2.1 Role of potassium in resisting plant stresses (Wang et al., 2013)………….. 9
Figure 2.2 Role of K in drought stress (Wang et al., 2013)…………………………… 11
Figure 2.3 Schematic representations of different forms of soil K [modified figure from Department of Environment and Primary Industries report, 2014]………………
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Figure 3.1 Wyalkatchem (K-efficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg) under soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks…………………………………………………………………………………...
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Figure 3.2 Gutha (K-inefficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg) under soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks…………………………………………………………………………………...
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Figure 3.3 Shoot dry weight (g/plant) (upper sub-figures), and tillers/plant (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1 for analysis of variance results……………………….
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Figure 3.4 Root dry weight (g/plant) (upper sub-figures) and root: shoot ratio (n=3) (lower sub-figures) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1 for analysis of variance results…………………….
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Figure 3.5 Leaf photosynthesis (upper sub-figures) and stomatal conductance (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1 for analysis of variance results…………………….
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Figure 3.6 K concentration (mg/g, dry weight) in shoot (upper sub-figures) and root (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.2 for analysis of variance results………………..
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Figure 3.7 K content (mg/plant) in shoot (upper sub-figures) and root (lower sub-figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.2 for analysis of variance results……………………………
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Figure 4.1 Potassium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems (±SE, n=4)……………………….
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Figure 4.2 Sodium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems (±SE, n=4)……………………….
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Figure 4.3 Wheat (cv. Wyalkatchem) at six weeks after transplanting grown in a split-root system treated with 40 mg K/kg and nil, 50 mg Na/kg. The picture shows the growth difference with and without Na addition (50 mg Na/kg)……………………..
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Figure 4.4 Correlation between shoot dry weight/plant (g) harvested at 6 weeks after transplanting and the shoot K concentration (mg K/g, dry weight) measured in low soil K (40 mg K/kg) split-root treatments……………………………………………...
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Figure 5.1 Column experiment of wheat cv. Wyalkatchem at 3 weeks after sowing (left). Column set-up with plastic tubes used for subsoil watering, commencing at 5 weeks after sowing (right)……………………………………………………………...
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Figure 5.2 Shoot dry weight (g) and tiller number per plant at 5 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1. See Table 5.2 for statistical analysis………………………………………………………………………………….
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Figure 5.3 Columns were supplied with low K (40 mg K/kg) in the whole profile with varying subsoil Na levels: a) nil Na, b) 50 mg Na/kg, and c) 200 mg Na/kg. Shoot growth and tillering was depressed by 200 mg Na/kg at 5 weeks after sowing………..
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Figure 5.4 Root dry weight (g/plant) of wheat cv. Wyalkatchem in different sections of column (0- 20, 20- 40 and 40- 60 cm) at 5 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1. See Table 5.2 for statistical analysis………..
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Figure 5.5 Shoot dry weight (g) and tillers per plant at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1 and see Table 5.2 for statistical analysis……………………………………………………………………………….....
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Figure 5.6 Root dry weight (g/plant) in different sections of column (0- 20, 20- 40 and 40- 60 cm) at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1……………………………………………………………………………..
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Figure 5.7 Root: shoot ratios of wheat at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer to Table 5.1……………………………………………….
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Figure 5.8 Leaf photosynthesis, stomatal conductance, and transpiration at 5 weeks after sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and for statistical analysis refer Table 5.3…………………………………………………………………
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Figure 5.9 Leaf photosynthesis, stomatal conductance, and transpiration at 7 weeks after sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and for statistical analysis refer Table 5.3…………………………………………………………………
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Figure 5.10 Leaf photosynthesis, stomatal conductance, and transpiration at 11 weeks after sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and Table 5.3 for statistical analysis……………………………………………………………………..
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Figure 5.11 Correlation between leaf net photosynthesis rates and shoot Na concentrations (mg Na/g, dry weight) at final harvest at 11 weeks after sowing…………………………………………………………………………………
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Figure 6.1 Leaf net photosynthesis, stomatal conductance, and transpiration of wheat cultivars Wyalkatchem and Gutha treated with low K (0.2 mM K) for two weeks, followed by two K levels (0.2 and 2 mM) and three Na levels (0, 10 and 20 mM) (±SE, n=4) and measured 42 hours after the treatments (Experiment 1)………………
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Figure 6.2 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for two weeks, followed by two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) (±SE, n=4) and measured 42 hours after the treatments (Experiment 2)……………………………………………………………………….....
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Figure 7.1 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (harvested 32 days after transplanting, pre-rubidium addition) (±SE, n=4)…………………………………………………………………………………….
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Figure 7.2 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium harvest), and after Rb treatment for 48 hours (post-rubidium harvest, 35 days after transplanting) (±SE, n=4)…………………………….
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Figure 7.3 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (±SE, n=4)……………………………….......
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LIST OF TABLES
Table 2.1 Removal of K through grain and hay harvest of different crops……………. 17
Table 2.2 Functions of K replaced and not replaced by Na……………………………. 21
Table 2.3 Sodium response at low K in various crop species………………………….. 24
Table 3.1 Statistical summary of plant growth and leaf gas exchange in four wheat cultivars (Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks……………………………………………………………………………………
40
Table 3.2 Statistical summary of K and Na concentrations and contents in four wheat cultivars (Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two levels of soil K (40, 100 mg K/kg) and five levels of Na (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks……………………………………………………………………………………
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Table 3.3 Shoot and root Na concentrations (mg/g, dry weight) and contents (mg/plant) of four wheat cultivars treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks (n=3). See Table 3.2 for statistical summary of main effects and interactions of the treatments………………..
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Table 3.4 Shoot and root K/Na ratios of four wheat cultivars treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks (n=3). See Table 3.2 for statistical summary of main effects and interactions of the treatments………………………………………………………………………………...
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Table 3.5 Concentrations of exchangeable cations in non-planted soils (n=3) with or without 50 mg Na/kg at two K levels (40, 100 mg K/kg) after one week of incubation. Means with different letters differ at P≤0.05…………………………………………..
50
Table 4.1 Split- root treatments experimental design………………………………….. 58
Table 4.2 Shoot dry weight (g) and number of tillers per plant of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems. Means (n=4) in a column with different letters differ at P≤0.05………………………………………
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Table 4.3 Total root dry weight (g) per plant and their root: shoot ratios of wheat cv. Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). One-way analysis of variance was conducted to assess the effects of split-root treatments. Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in total root dry weight and root: shoot ratios between the 11 split-root treatments and the specific root responses between the two compartments were compared within each split-root treatment. Means (n=4) with different letters differ at P≤0.05…………………………………………….
61
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Table 4.4 Leaf net photosynthesis rate (Pn), stomatal conductance (Gs) and transpiration (E) of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems. Means (n=4) with different letters differ at P≤0.05………………..
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Table 4.5 Shoot and root K concentrations (mg K/g) of wheat cv. Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in shoot K concentrations between the 11 split-root treatments and the specific root K concentrations between the two compartments were compared within each split-root treatment. Means (n=4) with different letters in a column differ at P≤0.05..………………………………………………………………
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Table 4.6 Shoot and root Na concentrations (mg Na/g) of wheat cv. Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in shoot Na concentrations between the 11 split-root treatments and the specific root Na concentrations between the two compartments were compared within each split-root treatment. Means (n=4) with different letters in a column differ at P≤0.05………………………………………………………………
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Table 4.7 K/Na ratios of wheat (whole plant) cv. Wyalkatchem were compared between the split-root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and 200 mg Na/kg). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………………………
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Table 5.1 Experiment design showing topsoil watering, topsoil K (mg K/kg) and subsoil K (mg K/kg) and Na (mg Na/kg) treatments harvested at 5 and 11 weeks after sowing ………………………………………………………………………………….
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Table 5.2 Statistical summary of plant growth at 5 and 11 weeks after sowing treated with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table 5.1…………………………...
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Table 5.3 Statistical summary of leaf gas exchange at 5, 7 and 10 weeks after sowing treated with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table 5.1……………………
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Table 5.4 Shoot K and Na concentrations and accumulation in wheat cv. Wyalkatchem harvested at 5 weeks after sowing. Means (n=3) with different letters differ at P≤0.05…………………………………………………………………………
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Table 5.5 Statistical summary of shoot K and Na concentrations and content at 5 and 11 weeks after sowing in wheat plants treated with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table 5.1. Note only whole shoots and roots were analysed at 5 weeks……………………………………………………………………………………
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Table 5.6 K concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Means (n=3) with different letters differ at P≤0.05……………
95
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Table 5.7 Shoot K and Na accumulation in wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Values are means of 3 replicates. Means (n=3) with different letters differ at P≤0.05. …………………………………………………………………
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Table 5.8 Na concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Means (n=3) with different letters differ at P≤0.05……………
97
Table 5.9 Shoot K/Na ratios in wheat cv. Wyalkatchem harvested at 11 weeks after sowing. Means (n=3) with different letters differ at P≤0.05…………………………..
98
Table 6.1 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for a further 48 hours (Experiment 1)…………….………………………….
107
Table 6.2 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5mM) for a further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters differ at P≤0.05…………………………………..
109
Table 6.3 Statistical summary of shoot and root K, Na, Rb concentrations and contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for 48 hours (Experiment 1; n=4)…………………………………………………………………………………….
110
Table 6.4 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters differ at P≤0.05…………………………………..
111
Table 6.5 The whole plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters differ at P≤0.05…………………………………..
112
Table 6.6 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM K), two Na levels (0, 2 and 10 mM Na) and Rb (0.5mM) (n=4) added for 48 hours (Experiment 2)…………………………………….
113
xviii
Table 6.7 Statistical summary of shoot and root K, Na, Rb concentrations and contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2; n=4).…………………………………………………….
115
Table 6.8 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05…………………………………..
116
Table 6.9 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05…………………………………..
118
Table 6.10 Plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………
119
Table 7.1 Statistical summary of plant growth in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium harvest), and after Rb treatment for 48 hours (post-rubidium or final harvest).…………………………………………………………………………………
128
Table 7.2 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3), number of tips and forks in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested after Rb treatment for 48 hours (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05…………………………………………………
130
Table 7.3 Shoot and root K and Na concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………
133
Table 7.4 Statistical summary of shoot and root K, Na and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks and then treated with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-rubidium harvest), and harvested after Rb treatment for 48 hours (Post-rubidium harvest)……………………………………….
134
xix
Table 7.5 Young leaf, old leaf, and the rest of shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested 48 hours after Rb addition (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05…………………………………………………………………………
136
Table 7.6 Shoot and root K and Na contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, and harvested after treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05………………………………………………………………….
138
Table 7.7 Statistical summary of shoot and root K, Na and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb harvest) and harvested 48 hours after Rb treatment (post-rubidium or final harvest)……………………………………………..
139
Table 7.8 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested 48 hours after Rb addition (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05……………………………………
140
xx
LIST OF ABBREVIATIONS AND SYMBOLS
µ Micro
µM Micromolar
˚C Degree Celsius
AAS Atomic absorption spectrophotometer
Al Aluminium
ANOVA Analysis of Variance
B Boron
Ca Calcium
CAM Crassulacean acid metabolism
CAT Catalase
CEC Cation exchange capacity
CO2 Carbon dioxide
DAT Days after transplanting
DI De-ionized water
E Transpiration
FC Field capacity
GRDC Grains Research and Development Corporation
GRS Grains Research Scholarship
Gs Stomatal conductance
HAK/HKT High affinity potassium transporter
H2O2 Hydrogen peroxide
hsd honest significant difference
ICP Inductively coupled plasma
K Potassium
KCl Potassium chloride
M Molar
xxi
Mg Magnesium
min Minute
mM Millimolar
Na Sodium
NaCl Sodium chloride
NAD(P)H Nicotinamide adenine dinucleotide phosphate
nmol Nanomoles
NO3 Nitrate
n.s Not significant
O2 Oxygen
O2- Superoxide radical
OH- Hydroxyl radical
Pn Photosynthesis
PVC Poly vinyl chloride
ROS Reactive oxygen species
SE Standard error
SOD Superoxide dismutase
SOPIB Sulphate of Potash Information Board
SWA South Western Australia
vs Versus
WA Western Australia
WAS Weeks after sowing
WAT Weeks after transplanting
WUE Water use efficiency
xxii
ACKNOWLEDGEMENTS
This study was supported by the Grains Research and Development Corporation (GRDC
Project UMU00035), and a Grain Research Scholarship (GRS- 10268), and the Sulphate of
Potash Information Board (SOPIB).
I am deeply grateful to my supervisor, Professor Richard Bell for his constant guidance,
encouragement and valuable suggestions in the course of experiment and writing of thesis.
His excellent supervision and support motivated to publish journal and present conference
papers. I am truly thankful to have conducted this research under such an expert and
knowledgeable person.
I extend my greatest thanks and appreciation to Dr. Qifu Ma, my co-supervisor for his ideas,
motivation and valuable advice. He was easy to approach and his suggestions were helpful in
setting experiments and thesis writing, and his guidance during field visit helped to gain
practical exposure and made this whole research journey an enjoyable one.
I would like to extend my sincere thanks to Professor Giles Hardy, Sonia Aghighi for helping
with WinRhizo root scanning, Andrew Foreman for providing training on flame photometer
and also, to Wendy Vance and other fellow post-graduate students and friends of ‘Land
management group’ for their generous help and support during the research. Special thanks to
people from DAFWA, in particular Craig Scanlan for helping in soil collection, and farmers
of Dowerin for letting to collect soil for glasshouse experiments.
I am grateful to my family and friends for their support and motivation throughout this
journey. In particular to my husband, Pradeep for his unending support and understanding
which made the completion of this study and to my little person Eeshva for his valuable
distractions and being good to let me do final stages of writing and revisions.
1
CHAPTER 1
INTRODUCTION
1.1 Potassium nutrition in plants
Potassium (K) is an essential element for higher plants since plants are unable to complete
their life cycle in its absence, and its function cannot be fully replaced by any other element.
Potassium, unlike other nutrients (apart from chloride) does not become a part of the
chemical structure of compounds, but, plays regulatory roles within the plant as a cation.
Potassium is taken up in large quantities in plant tissues (Wakeel et al., 2011). It is a
dominant cation in the cytoplasm of plant cells constituting up to 100 g/kg of plant dry
weight (Very & Sentenac, 2003). Plant cytoplasm concentrations are tightly regulated and
maintained at ~100 mM, however, vacuolar K concentrations are highly variable reflecting
plant K status (Marschner, 1995).
Potassium plays an important role in photosynthesis, protein synthesis, enzyme activation,
osmoregulation, stomatal movement, phloem loading and transport and stress tolerance in
plants (Mengel & Kirkby, 2001; Römheld & Kirkby, 2010). It is highly mobile in plants and
is readily re-translocated from source to sink organs. When plant K concentration is lower
than 10 g/kg dry weight, most species will show deficiency symptoms with interveinal
chlorosis in older leaves. With the progression of deficiency, necrosis and death of tips and
margins of leaves may occur (Gierth & Mäser, 2007). Plant K status is dependent upon soil K
availability and K uptake by roots.
1.2 Sodium nutrition in plants
The role of sodium (Na) in plant nutrition and its status as an essential element is still being
debated. Plant species are characterized as natrophilic or natrophobic depending on their
growth response to Na and their capacity for uptake and transport. Natrophilic plants absorb
Na+ but translocation to shoots is slow and Na+ is compartmentalised in root vacuoles
(Wakeel et al., 2011). In contrast to K, Na is only beneficial for certain plant species
characterized by C4 and CAM photosynthetic pathways (Marschner, 1995), but Na is
beneficial in relatively low concentrations for many plants and it is toxic to the majority of
plants at high concentrations (Mäser et al., 2002). In some plant species where Na is
beneficial, the main functions are in growth stimulation, osmotic regulation, better water
balance and some other non-specific functions (Kronzucker et al., 2013; Wakeel et al., 2011).
2
Under abundant Na availability, growth of many plants is limited due to water stress and
specific Na+ ion toxicity when plant cytoplasmic Na+ concentrations are above 20 mM
(Benito et al., 2014). However, other studies reported no negative or even positive effects of
Na typically at low Na concentrations with partial substitution of K by Na when K supply
was low and plants suffered at least partial K deprivation (Kronzucker et al., 2013).
1.3 Interaction between potassium and sodium
Potassium and Na ions are similar in ionic radius and ionic hydration energies (Marschner,
1995), and because of this chemical similarity, it is assumed that the both ions compete with
each other (Subbarao et al., 2003). Since K+ and Na+ exhibit homologous behaviour they
share some physiological functions (Almeida et al., 2010). Potassium is required in high
concentrations for plant growth and development, whereas Na is beneficial to certain
halophytes at relatively low concentrations (Greenway & Munns, 1980; Mäser et al., 2002).
In halophytes, presence of Na in the environment and its uptake can reduce the plant K
requirement to meet the plants metabolic requirements (Benito et al., 2014).
Sodium can have either negative effects on plant growth at high levels of supply in soil or
positive effects at low to moderate supply in low K soil, but both these effects vary with plant
tolerance to salinity and with soil K and Na levels (Kronzucker et al., 2013). In arid and
semiarid regions, soil salinity is largely due to excessive NaCl which, apart from osmotic and
specific Na+ and Cl- ion effects, has a detrimental effect on potassium (K+) uptake and
nutrition of most crops (Römheld & Kirkby, 2010). Soil salinity, mainly associated with
sodium chloride (NaCl) is a major environmental stress that affects K+ uptake and transport
by plants (Szczerba et al., 2009). In saline soils, plant physiological functions such as enzyme
activation and protein synthesis are inhibited due to depression in K+ uptake by competing
Na+ ions (Al-Rawahy et al., 1992; Blumwald et al., 2000).
It has been widely reported that K+ counteracts Na+ stress in plants while there are few
reports that Na+ can in turn, alleviate K+ deficiency symptoms (Ali et al., 2009). Although the
complete role of Na+ in plant metabolism still awaits resolution, it is commonly assumed that
Na+ can substitute biophysical functions of K+ in non-halophytic plants, given that the plants
have the ability to take up Na+, translocate it to the shoot, and compartmentalise it in the
vacuoles (Subbarao et al., 2003). Sodium can replace K+ in the vacuole as an alterative
inorganic osmoticum under K-limited conditions, and the released K+ is then available for
more K-specific processes (Benito et al., 2014). Sodium can alleviate K deficiencies in some
3
species such as sugar beet, lettuce, cotton, ryegrass, spinach, marigold, tomato, celery, carrot
(Benito et al., 2014; Idowu & Aduayi, 2007; Marschner, 1995; Mundy, 1983; Tahal et al.,
2000) and barley (Ma et al., 2011). Sodium is beneficial to plant growth when available K is
deficient, but the degree of this beneficial effect varies between crop species, and even
between genotypes of the same plant species (Marschner, 1995).
1.4 K deficiency in soils
Large areas of agricultural soil in the world are reported to be K-deficient and unbalanced K
fertilization may result in significant K depletion from soil reserves and decreased soil
fertility (Zörb et al., 2014). Potassium concentration of top soils is usually > 1 % (10 g K/kg)
in most soils of the world, whereas the top soils of WA contain < 0.1 % (1 g K/kg) and
concentrations > 1 % are relatively rare reflecting the highly weathered state of these soils
(Pal, 1999). Moreover K deficiency is further worsened due to continued removal of grain,
hay/straw, without adequate K replacement and therefore K fertilisation management is
required for profitable cropping (Ma et al., 2011).
1.5 Salinity issues of WA
Soil salinity in arid and semi arid areas is a major constraint of crop productivity in many
parts of the world. The global estimate for agricultural land threatened by or already lost to
salinity exceeds 900 million ha (Kaya, 2002). The Land Monitor method has estimated that
the current area affected by salinity in Western Australia is about 1 million ha and the annual
rate of increase is about 14,000 ha (McFarlane et al., 2004). Sodic soils and duplex soils are
also major soil constraints for crop production in Australia. Sodic soils are common in
Western Australia, particularly in south-west agricultural area (Cochrane et al., 1994).
Duplex/ texture-contrast soils account for about 12 % of the land area of Australia (Dracup et
al., 1992) and half to 2/3rd of the cultivated area in south-Western Australia (Belford, 2005).
The duplex soils have varying concentrations of nutrients due to differences in clay content
and mineralogy. However, considering the interactive effects of K and Na, there is a
possibility that K requirements in moderately saline and in sodic soils may be decreased due
to the presence of Na. This thesis had an emphasis on moderately saline-sodic soils and
interactions between K and Na under such conditions are investigated.
4
1.6 Research aim and scope of study
In saline/sodic soils and low fertility soils, the partial substitution of K+ by Na+ in
physiological processes of wheat would have substantial practical implications for K fertiliser
management.
This study is a part of two research projects funded by the Grains Research and Development
Corporation (GRDC) and the Sulphate of Potash Information Board (SOPIB) to examine K
nutrient management in low K and saline/sodic soils in the drought-prone environments of
south-west Australia. As this research looks into important soil constraints prevailing, the
research findings of this study with further field studies would help in decision making for
nutrient management to improve crop productivity in saline/sodic soils and drought-prone
environments.
In K deficient soils, Na can to a certain degree substitute the role of K in some plants
(Marschner, 1995; Subbarao et al., 2003). In the Chenopodiaceae family, crops like spinach,
beet and sugar beet have received detailed attention in terms of K and Na interactions
(Kronzucker et al., 2013). Wheat (Triticum aestivum L.) is a major cereal grown worldwide
and it is cultivated in semi-arid regions of Western Australia. However, little is known about
K requirements of wheat cultivars grown under saline and sodic soils (Ma et al., 2011).
Understanding the Na and cultivar effects on K nutrition in wheat is the main aim of this
thesis. The potential of K substitution by Na in wheat nutrition may offset the requirement of
expensive K fertiliser and therefore may help in profitable as well as sustainable cropping in
agriculture.
1.7 Layout of the thesis
A review of K and Na nutrition and functions in plants, and of K deficiency effects is
presented in Chapter 2 along with a review on Na effects on K nutrition and their
interactions. Previous research on K substitution by Na in wheat and other plant species will
also be reviewed. The main objectives and research questions of the present thesis are
identified.
Chapter 3 reports on an experiment with wheat cultivars treated with soil K and Na levels. In
this chapter, Na effects on four wheat cultivars differing in K-use efficiency were examined
under low and adequate K supply in terms of growth behaviour, leaf gas exchange
measurements, ion concentrations and content. Sodium levels that are beneficial to wheat
cultivars along with the cultivar effect will be identified. The effects of external Na supply on
5
soil exchangeable K availability was also investigated in a short-term soil incubation
experiment.
In Chapter 4, the split-root experiment consisted of 2 K and 3 Na levels to examine whether
K replacement by Na would depend on both these cations being present in the same or
different parts of the root system.
A column experiment with varying subsoil K and Na levels under topsoil water deficit is
reported in Chapter 5. This experiment aimed to understand the relationship between K
responses and subsoil Na because low K soils commonly contain significant exchangeable Na
in the subsoil.
Following the detailed experiments in soil-based systems, a series of solution culture
experiments were conducted (Chapters 6 and 7). These experiments aimed to investigate
whether supply of low external Na conditions would alter wheat K uptake using Rb as a
tracer. Wheat cultivars differing in K-use efficiency and short-term versus long-term
responses were compared. The experiments gathered evidence on whether Na induced
increased K uptake by wheat cultivars and on cultivar effects on K uptake.
A general discussion is undertaken in Chapter 8 where the main issues are discussed with
other published findings. Also the main findings and conclusions are brought together in
Chapter 8 with further research recommendations outlined.
6
CHAPTER 2
LITERATURE REVIEW
2.1 Introduction
To provide food for an expanding global population, a massive increase in crop production is
required in a more resource-efficient way. In this context, K is an important macronutrient
that plays a critical role in a number of physiological and biochemical processes required for
growth and yield of plants. Potassium constitutes about 2.1 to 2.3 % of the earth’s crust, and
is the seventh or eighth most abundant element (Zörb et al., 2014). Plants can only acquire K+
from solution and its availability in soils is dependent on nutrient dynamics and total K
content. Although soil K reserves may be large, extensive areas of the world are reported to
be deficient in K availability for plants. A proper understanding and management of K
nutrition and its interactions is needed to counter the declining soil fertility and improve food
security.
This review focuses on the role of K in plants, including physiological functions and
deficiency effects in plants. Potassium availability for plant growth, forms of K in soil, and K
uptake by plants are also discussed. This review also discusses Na nutrition in plants,
interaction between K and Na in various plants, and the potential for partial substitution of K
by Na. The main aim and research questions are identified at the end of the literature review.
2.2 K functions in plants
Potassium is the most abundant inorganic cation in plant cells and is vital for plant growth. It
is a highly mobile element in plants, and highest concentrations are found in young and
developing tissues indicative of its role in cell metabolism and growth. Potassium plays a
major role in physiology and biochemistry of plants. Regulation of stomatal opening and
closing, leaf movements, and also other plant tropisms are driven by K+-generated turgor
pressure in cells (Maathuis & Sanders, 1996; Zhao et al., 2001). It acts as an osmoticum in
maintaining turgor pressure, and influencing solute transport and water balance in plants. The
maintenance of turgor pressure is essential for continued cell expansion, and growth of plant
cells (Römheld & Kirkby, 2010). Potassium is essential in activating numerous enzymes,
including those involved in photosynthesis, energy metabolism, protein synthesis and starch
synthesis (Mengel & Kirkby, 2001). It is also essential in maintenance of transmembrane
voltage gradients for cytoplasmic pH homeostasis (Römheld & Kirkby, 2010). The important
K functions in plants are reviewed briefly.
7
2.2.1 K and enzyme activation
The activation of enzymes is a major, critically important, irreplaceable role of K in plant
growth and development. Concentrations of K+ in the cytosol are maintained relatively
constant at around 100 mM which is optimal for the function of cytosolic enzymes (Ashley et
al., 2005). Potassium is important in activation of a large number of enzymes involved in
energy metabolism, protein synthesis and solute transport (Römheld & Kirkby, 2010) by
inducing conformational changes in the enzymic proteins (Marschner, 1995). It is known that
more than 70 important enzymes involved in plant growth are activated by K+ (Anschütz et
al., 2014). Some of the enzymes activated by K+ include pyruvate kinase and
phosphofructokinase involved in carbohydrate metabolism, asparaginase involved in N
metabolism, starch synthase, and membrane-bound proton- pumping ATPases (Blevins,
1985; Marschner, 1995).
2.2.2 K and protein synthesis
Potassium is believed to regulate every major step of protein synthesis, including the
synthesis of ribosomes, and aminoacyl-tRNA binding to ribosomes, peptidyl transfer,
guanosine-5’- triphosphate (GTP) utilization, protein synthesis from charged tRNA transfer,
and messenger RNA turnover (Blevins, 1985). It is claimed that protein synthesis requires
higher concentrations of K+ than for enzyme activation and the “reading” of genetic code in
plant cells to produce proteins is not possible without adequate K concentrations (Marschner,
1995). A probable function of K is in polypeptide synthesis in the ribosomes, since that
process requires a high K+ concentration (Wyn Jones & Pollard, 1983). In K-sufficient plants
high-molecular weight compounds like proteins are increased, whereas, in K-deficient plants
low-molecular weight compounds like amino acids, amides and nitrate accumulate instead of
proteins (Wang et al., 2013).
2.2.3 K and stomatal activity
Potassium plays an important role in opening and closing of stomates, the pores through
which the leaves exchange CO2, O2 and water vapour with the atmosphere. When K+ moves
into the guard cells around the stomata, the cells accumulate water and swell, causing the
pores to open, allowing gases to move freely in and out. When water supply is short, K+ is
pumped out of the guard cells. The pores close tightly to prevent loss of water and minimize
drought stress to the plant. This stomatal movement is essential for transpiration, and CO2
uptake for photosynthesis (Humble & Raschke, 1971). Under K deficiency, stomatal closure
8
may take longer and be incomplete, and plants are more susceptible to water stress (Mäser et
al., 2002). Potassium deficient plants show decrease in turgor pressure and become flaccid
due to impaired stomatal functioning (Römheld & Kirkby, 2010).
2.2.4 K and photosynthesis
Potassium plays an essential but complex role in regulating the rate of photosynthesis of
higher plants. It is the dominant counter-ion to the light-induced H+ flux across the thylakoid
membranes and is also required for the establishment of the transmembrane pH gradient
necessary for the synthesis of adenosine triphosphate (ATP) and activation of enzymes
involved in photosynthesis (Marschner, 1995). The primary effect of K in photosynthesis is
in maintaining the stomal K concentration of the chloroplast to allow CO2 fixation. Potassium
deficiency reduces ATP production, photosynthetic activity, chlorophyll content and
translocation of fixed carbon in plants (Zhao et al., 2001). This depression in photosynthesis
causes an excessive accumulation of light energy and photo reductants in the chloroplasts
which results in the formation of reactive oxygen species (ROS), and chloroplast damage
(Cakmak, 2005). This is discussed in more detail below.
2.2.5 K and stress tolerance in plants
Potassium plays a major role in protecting plants against environmental stresses such as
drought, frost, heat, salinity, high light intensity, and nutrient limitations. It is also claimed
that high K status in crops decreases the incidence of diseases and pests (Römheld & Kirkby,
2010). There is evidence that K also plays a regulatory role in plant stress responses as
discussed briefly below (Fig. 2.1).
Plants exposed to environmental stresses suffer from oxidative damage catalyzed by ROS
which impairs cellular function and causes plant growth depression (Cakmak, 2005).
Reactive oxygen species are extremely cytotoxic and can seriously disrupt normal
metabolism through oxidative damage to lipids, nucleic acids and proteins (Heidari &
Jamshidi, 2011). Examples of ROS are hydroxyl radical (OH-), singlet oxygen/superoxide
radical (O2-), and hydrogen peroxide (H2O2). In plants, ROS are predominantly produced
during photosynthetic electron transport and activation of membrane bound NAD(P)H
oxidases (Römheld & Kirkby, 2010). When K is deficient there is a severe reduction in
photosynthetic CO2 fixation, impairment in partitioning of fixed carbon to sink organs, and
decreased utilisation of photosynthates. According to Cakmak (2005), there is a decrease in
net photosynthesis under K deficiency due to reduced stomatal conductance, increased
9
mesophyll resistance, and lowered ribulose bisphosphate carboxylase activity. Such
disturbances result in excessive photosynthetically-produced electrons and thus increased
ROS production by intensified transfer of electrons to O2. Also there is an increase in
NADPH oxidation under K deficiency, up to 8-fold when compared with K-sufficient plants
(Cakmak, 2005).
Fig. 2.1 Role of potassium in resisting plant stresses (Wang et al., 2013)
2.2.5.1 Anti-oxidant activity
Under stress conditions, plants have evolved molecular defence systems that limit the
formation of ROS and promote their removal. Chloroplasts are the major organelles
producing ROS when plants are exposed to environmental stress conditions, and the
consequences include membrane damage, chlorophyll degradation, and development of leaf
chlorosis and necrosis (Cakmak, 2005). The plant enzymatic defences include production of
antioxidant enzymes such as phenol peroxidases (POX), superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPX), ascorbate peroxidase (APX), which together
with other enzymes of the ascorbate-glutathione cycle promote the scavenging of ROS
(Heidari & Jamshidi, 2011). Catalase (CAT) activity decreased when K levels increased
under salt stress, in millet (Heidari & Jamshidi, 2011).
10
2.2.5.2 Drought and heat stress
Potassium is an important mineral nutrient contributing to osmotic adjustment under drought
stress in many plant species (Damon et al., 2011). Drought stress causes stomatal closure and
therefore decreases CO2 fixation in plants. The formation of ROS is intensified due to
inhibited CO2 reduction by drought stress with consequent oxidative damage to chloroplasts
(Cakmak, 2005). Plants suffering from drought have a larger internal requirement for K, and
adequate K supply will enhance plant adaptation to drought by ensuring improved control of
stomatal opening and closing. For example, the increase in K supply in external solution from
0.2 to 6 mM K alleviated the drought stress in wheat (Gupta et al., 1989). Under drought
stress, root growth and rate of K+ diffusion are restricted, limiting K+ acquisition (Wang et
al., 2013) and increasing K application reduces the damage by significantly increasing the
depth of root penetration, root surface exposed to soil and K absorption (Valadabadi &
Farahani, 2010). However, increasing root penetration when there is limited sub-soil water
available is of questionable value.
Plants exhibit several resistance mechanisms for survival during mild to severe water stress.
Fig. 2.2 summarises the role of K in plants under drought stress (Wang et al., 2013). One
such mechanism is by active solute accumulation in plant organs subjected to prolonged
stress, referred to as ‘osmotic adjustment’, which maintains turgor in plants, or an increased
water potential gradient from soil to leaf resulting in water uptake (Gebre & Tschaplinski,
2000). Different plant species accumulate different solutes for osmotic adjustment (Gebre &
Tschaplinski, 2000). Potassium is the most common solute in wheat and other species
(Damon et al., 2011). In an experiment by Damon et al. (2011), to study the osmotic
adjustment under drought stress, among 5 wheat genotypes with differing K-use efficiency, K
fertiliser application accounted for 38 % (Wyalkatchem) to 51 % (Nyabing) of leaf osmotic
adjustment with drought stress imposed by withholding water from 35 days after sowing.
Fig. 2.2 Role of K in drought stress
2.2.5.3 Low temperature and frost stress
Under chilling and frost stresses, plant metabolic reactions are inhibited due to cold
osmotic, oxidative and other stresses
lipids and membrane structure are altered. Reactive oxygen species are formed under low
temperature stress because absorbed light energy exceeds the chloroplast capacity to use it in
CO2 fixation (Römheld & Kirkby, 2010)
electron transport, stomatal conductance, and CO
Increasing K supply helps in alleviating low
secondary metabolite transcripts associated with cold tolerance
acts as an osmoticum in maintaining high concentration of K in cell sap thus lowering its
freezing point (Römheld & Kirkby, 2010)
Role of K in drought stress (Wang et al., 2013)
2.2.5.3 Low temperature and frost stress
Under chilling and frost stresses, plant metabolic reactions are inhibited due to cold
osmotic, oxidative and other stresses (Wang et al., 2013). Also the fluidity of membrane
lipids and membrane structure are altered. Reactive oxygen species are formed under low
temperature stress because absorbed light energy exceeds the chloroplast capacity to use it in
(Römheld & Kirkby, 2010). Moreover low temperature impairs photosynthetic
electron transport, stomatal conductance, and CO2 fixation in plants
Increasing K supply helps in alleviating low temperature stress in plants by producing
secondary metabolite transcripts associated with cold tolerance (Wang et al., 2013)
in maintaining high concentration of K in cell sap thus lowering its
(Römheld & Kirkby, 2010).
11
(Wang et al., 2013)
Under chilling and frost stresses, plant metabolic reactions are inhibited due to cold-induced
Also the fluidity of membrane
lipids and membrane structure are altered. Reactive oxygen species are formed under low
temperature stress because absorbed light energy exceeds the chloroplast capacity to use it in
. Moreover low temperature impairs photosynthetic
fixation in plants (Cakmak, 2005).
temperature stress in plants by producing
(Wang et al., 2013), and K
in maintaining high concentration of K in cell sap thus lowering its
12
2.2.5.4 Disease and pest resistance
Potassium fertiliser is widely reported to decrease disease and pest symptoms in many host
plants (Wang et al., 2013). As a mobile regulator of enzyme activity, K is involved in
essentially all cellular functions that influence disease severity (Huber & Arny, 1985). High
K status in plants favours the synthesis of high molecular weight compounds like proteins,
starch and cellulose, thereby depressing the synthesis of low molecular weight compounds
like soluble sugars, organic acids, amino acids and amides in plant tissues. The low molecular
weight compounds are necessary for feeding pathogens and insects. Therefore, K deficient
plants are more vulnerable for pest and disease attack (Marschner, 1995).
The generally inverse relationship of available K in soil to disease severity has made it a
common practice to fertilise with K to reduce certain diseases. Potassium significantly
decreased the incidence of fungal diseases by 70 %, bacteria by 69 %, insects and mites by 63
%, viruses by 41 % and nematodes by 33 % (Wang et al., 2013). It is believed that high levels
of K could directly inhibit the growth and zoospore release of pathogens in crop production
(Sugimoto et al., 2009).
Although there is a large volume of literature on the relationship between K and plant
disease, there is a very little quantitative information available on the concentrations of K in
soil or plant tissues that result in changed disease expression (Huber & Arny, 1985). Higher
levels of K, relative to other nutrients, decreased the severity of yellow disease (Fusarium
oxysporum) of cabbage, Fusarium wilt of tomato, Fusarium wilt of pea, Stewart’s wilt
(Erwinia stewartii) of maize, and downy mildew (Perenospora tabacina) of tobacco (Huber
& Arny, 1985). The application of 20- 30 mM potassium nitrate significantly reduced the
infections caused by Phytophthora sojae (stem rot) in soybean (Sugimoto et al., 2009). High
rates of K application reduced population density of homopterous pests on cereals, legumes
and maize plants (El-Gindy et al., 2009). Also, K fertilisers promotes thicker epidermal walls
which promotes vigorous plant growth by inducing disease resistance (Sugimoto et al., 2009),
and an increase in the thickness of epidermal leaves suppressed the infestation of piercing and
sucking pests (El-Gindy et al., 2009).
2.3 K deficiency in plants
The reason for wide spread K deficiency includes a gradual decline in soil K levels due to
land clearing, increased cropping in marginal K soils, introduction of crops with a high K
requirement, increased demand for K due to improved agronomic practices including
13
increased use of N fertilisers (Edwards, 1997). Potassium deficiency is extensive across
Western Australia (Brennan et al., 2004). Potassium concentration of top soils is usually > 1
% (10 g K/kg) in most of the areas of the world, whereas, the top soils of WA are K-deficient
and concentrations of > 1 % are relatively rare which reflects the highly weathered state of
these soils (Pal, 1999). Soil properties arising from parent material, degree of weathering,
texture, clay mineralogy as well as land use and rainfall pattern influence the development of
K deficiency (Moore, 2004).
When plant K concentration is lower than 10 mg K/g dry weight, most species will show
deficiency symptoms with interveinal chlorosis in older leaves, and with the progression of
deficiency, necrosis and death of tips and margins of old leaves occur in extreme cases
(Gierth & Mäser, 2007). Since K is highly mobile in plants, mild deficiency does not result in
visible symptoms immediately as K is withdrawn from old leaves and retranslocated to
growing tissues. In early stages of K deficiency, there is a reduction in growth rate (without
visible symptoms- called hidden hunger), and later on, chlorosis and necrosis develop in
mature leaves (Römheld & Kirkby, 2010).
2.4 K uptake and accumulation by plants
The availability of soil K to plants is influenced by many factors, which include clay
mineralogy, particle size, water content, acidity, aeration, and organic matter level (Moore,
2004). It is also dependent on the levels of other cations, especially Ca, Mg and Na, in soil
solution (Jalali, 2008). Plant K+ acquisition from soil is dependent on factors influencing root
development such as root structure, root density, rooting depth and root hair length, root
distribution in the soil and the ability of roots to absorb mineral nutrients. Factors like
salinity, drought, soil compaction, Al toxicity in acid soils, and B deficiency can inhibit root
growth and hence lower K uptake from the soil (Römheld & Kirkby, 2010). Potassium
retention in the soil in a plant-available form is achieved by cation exchange. Plant roots take
up K+ from a wide range of external concentrations which typically vary from 0.1 to 10 mM
(Szczerba et al., 2009).
Plants accumulate considerable quantities of K which constitutes between 20 and 100 mg K/g
of plant dry weight (Ashley et al., 2005). The critical K concentration for many crop species
is in the range of 5 to 20 mg K/g, plant dry matter (Zörb et al., 2014). For effective
biochemical functions in plants, K concentrations of 100- 150 mM must be present in
metabolically active compartments like the cytosol, nucleus, stroma of chloroplast, and
14
matrix of mitochondria (Britto & Kronzucker, 2008; White, 2013). In plants, the cytosolic K+
pool appears to be relatively stable, around 100 mM (Gierth & Mäser, 2007), which is
considered optimal for the function of cytosolic functions (Ashley et al., 2005). In contrast,
vacuolar K+ concentrations vary greatly, between 10 and 500 mM (Marschner, 1995;
Szczerba et al., 2009), reflecting K status of the plant (Gierth & Mäser, 2007). When K
supply is sufficient the vacuolar K+ pool is increased, but when K is deficient vacuolar K+
storage is depleted to sustain a constant concentration in the cytoplasm (Gierth & Mäser,
2007). An optimal cytosolic K+ concentration is considered necessary for optimal enzyme
activity and photosynthetic activity (Cuin et al., 2003; Szczerba et al., 2009).
2.5 Forms of K in soil
Potassium occurs in primary minerals, clay minerals and also in crop and microbial residues;
it may be in soluble or insoluble forms. Soils usually have > 2 % (20 g K/kg) of total K but of
this generally < 0.1 % (1 g K/kg) is available to plants (Schulte & Kelling, 2009). There are
dynamic reactions between different forms of K: the four different forms of K and their
relative availabilities are illustrated in figure (Fig. 2.3) and discussed below.
Fig. 2.3 Schematic representation of different forms of soil K (Department of Environment
and Primary Industries, 2014)]
15
2.5.1 Unavailable/ Mineral K
This large reservoir of K is present in the crystalline structure of minerals such as feldspars
and micas and it is slowly available (Malvi, 2011; Schulte & Kelling, 2009). Mineral K
becomes available when primary minerals such as micas (biotite, muscovite) and feldspars
(KAlSi 3O8 orthoclase, microcline) weather or decompose. Soils formed on rocks weathered in
situ (i.e. acid and basic igneous or metamorphic rocks) may have adequate reserves of K.
Also in alluvial soils, feldspars weather more readily than micas and thus are an important
source of K. Feldspars are abundant in acid igneous rocks such as granite which underlie
much of the south-western Australia, but in this landscape they are highly weathered and so
in most soils the only unweathered primary mineral left is quartz due to its high resistance to
weathering (Moore, 2004). The weathering process is far too slow to supply the required K
for field crops in any given year, while trees and long-term perennials are benefited by K
released by weathering (Schulte & Kelling, 2009).
2.5.2 Slowly available/ non-exchangeable/fixed K
Non-exchangeable K (unlike mineral K) is associated with clay minerals, but is not bonded
covalently with soil minerals. It is held between adjacent tetrahedral layers of micas,
vermiculites, and 2: 1 clay minerals such as illite (Moore, 2004; Schulte & Kelling, 2009).
The non-exchangeable K acts as a reserve source of K in the soil, and is released as these
minerals expand when wet and is slowly available to plants (Malvi, 2011). Some is released
to become exchangeable when the levels of exchangeable and soil solution K are decreased
by crop uptake, removal and leaching (Peterburgsky & Yanishevsky, 1961). Soils of south-
western Australia have limited non-exchangeable K due to the low content of 2: 1 clay
minerals (Pal et al., 2001).
2.5.3 Readily available/ Exchangeable K
Exchangeable K is held by the negative electrostatic charges on the surfaces of organic matter
and clay minerals and is in rapid equilibrium with soil solution K (Römheld & Kirkby, 2010).
It is easily exchanged with other cations and readily available to plants (Moore, 2004).
Exchangeable K is the form that is extracted in the routine soil analysis of soil samples to
generate a recommendation for K fertiliser use (Malvi, 2011).
2.5.4 Soil solution K
Soil solution K is found in the thin film of water in pores and around soil particles and is
easily absorbed by plants. The levels of dissolved K in soil solution usually range between
16
0.2 to 10 mM (Schulte & Kelling, 2009). Soluble K is easily available to plants and microbes,
but it is subjected to leaching (Zörb et al., 2014). As the readily available K is absorbed, soil
solution K is replenished from both readily exchangeable K and non-exchangeable K held in
clay particles (Moore, 2004; Römheld & Kirkby, 2010).
2.6 Removal of K from soil
There are several mechanisms by which K is removed from soil, including leaching of K
below the root zone, loss of K by erosion, and removal of K in harvested produce and hay.
The continued export of K in primary produce is regarded as the ‘mining’ of K from soil
reserves (Pal et al., 2001). There is a potential for rapid K depletion in soil, if K removed is
not balanced by regular K fertilisation either with mineral K fertilisers or crop residue
recycling (Römheld & Kirkby, 2010). Leaching of K is dependent on the amount of applied
K, form of K, concentration of other cations in the soil solution, soil organic matter and clay
contents (Kolahchi & Jalali, 2007). Also the amount of rainfall and the porosity of soil will
influence K leaching. For example, in sandy soils, K does not interact strongly with the soil
matrix and is subsequently leached by rainfall or irrigation, because of low clay content and
low sorption capacity (Kolahchi & Jalali, 2007). The forms of K in soil, in order of their
availability for leaching are solution > exchangeable > non-exchangeable > mineral
(Kolahchi & Jalali, 2007).
Large quantities of K are removed from soil with the harvest of plants. It is reported that
globally the annual above-ground plant biomass contains 60 million tonnes of K (Römheld &
Kirkby, 2010). The amounts of K present in common crops and removed at harvests are listed
in Table 2.1.
The vegetative parts of plants contain higher concentrations of K than the grains/reproductive
part (Table 2.1). The removal of animal dung and crop residues from farmland as a source of
animal feed, bio-energy for heating and cooking considerably lowers the soil K status
(Römheld & Kirkby, 2010).
17
Table 2.1 Removal of K through grain and hay harvest of different crops
Crop Grain kg K/t Mature shoots kg K/t
Rice 2 -
Wheat/barley/oats 4 12
Maize 5 3
Sorghum 5 5
Chick pea 8 15
Lupin 8 -
Soybean 16 14
Canola 9 -
Sunflower 8 -
Peanut 7 20
Millet 16 -
Source: Australian Soil Fertility Manual, 2000
2.7 Na functions in plants
In contrast to K, sodium (Na) is only beneficial for certain halophytes and plant species
characterized by C4 and CAM photosynthetic pathways at relatively low concentrations
(Mäser et al., 2002). It may be classified as an essential mineral nutrient for some plant
species in the families of Amaranthaceae, Chenopodiaceae, and Cyperaceae and a supply of
around 100 µM Na+ enhanced the growth and alleviated visual K deficiency symptoms in
these plants (Marschner, 1995). For the role of Na in mineral nutrition of plants, three aspects
have to be considered: a) its essentiality for that plant species, b) the extent to which it can
replace K functions in plants, and c) its additional growth enhancement effect (Marschner,
1995). Non-halophytic plants from non-saline environments, while expressing genetic
variation for salt tolerance within a particular species, are generally effective in excluding
Na+ and preferential absorption of K+ ions (Schachtman & Liu, 1999).
According to Marschner (1995), the application of Na fertilisers has beneficial effects: a) in
natrophilic plant species, b) when soil levels of available K or Na or both are low, and c) in
areas with irregular rainfall or transient drought during the growing season, or both. Sodium
18
caused growth stimulation in plants by its effect on generation of turgor, osmotic adjustment,
cell expansion and water balance of plants (Kronzucker et al., 2013; Wakeel et al., 2011). The
application of Na fertilisers also results in an increase in the leaf area index early in the
growing season and a corresponding increase in light interception, thus improving the water
use efficiency of leaves under moderate stress conditions during the growing season (Durrant
et al., 1978).
In C4 plants, Na increases the efficiency of CO2 utilization between mesophyll and bundle
sheath cells in the photosynthetic pathway, enhances NO3 uptake by the roots and its
assimilation in the leaves, and is also required for chlorophyll synthesis (Subbarao et al.,
2003). Under Na deficiency in C4 plants, the conversion of pyruvate into phosphoenol
pyruvate (PEP), which takes place in mesophyll chloroplasts, is impaired. Furthermore, C3
metabolites like alanine and pyruvate were found to accumulate, whereas C4 metabolites,
PEP, malate, and asparate, decreased under Na deficiency. Also in Na-deficient plants (C4,
CAM, or C3), nitrate reductase activity is very low but can be rectified within 2 days when
Na is supplied (Marschner, 1995).
2.8 Interaction between K and Na
Potassium and Na ions are similar in ionic radius and ionic hydration energies. The hydrated
Na ion (Na+) has a radius of 0.358 nm, and the radius of K ion (K+) is 0.331 nm (Marschner,
1995). Because of the chemical similarity between K and Na, it is assumed that K and Na
compete for common adsorption sites in the roots (Subbarao et al., 2003). Generally an
excess of one cation in the nutrient medium reduces the net uptake of other cations, but the
sum of all cations in the plant tissue often remains constant (Pervez et al., 2006). However, it
has been argued that many plants have high degree of selectivity and there is some evidence
that concentration of cations like Ca, Mg and Na in plants can be reduced as a result of
inhibition by the uptake of K (Mundy, 1983). Potassium substitution and growth stimulation
by Na are of great interest for agronomic production, and better use of fertilisers (Mäser et
al., 2002).
Larson and Pierre (1953) suggested that plants can be classified according to their Na/K
response as follows: a) plants that respond to Na with an adequate supply of K; b) plants that
respond to Na when K is deficient, indicating that Na partly replaces K in its functions; and c)
plants that respond slightly, if at all, to Na under any conditions. Marschner (1995) proposed
four groups according to the differences in their growth response to Na as follows: a) plant
19
species, where a high proportion of K can be replaced by Na and additional growth
stimulation occurs which is not achieved by increased K content of plants, e.g., sugar beet,
table beet, turnip, Swiss chard and many C4 grasses (wheat grass, Rhodes grass etc.); b)
specific growth responses in plants to Na are observed but are much less distinct and a
smaller proportion of K can be replaced without a decrease in growth, e.g., cabbage, radish,
cotton, pea, flax, wheat and spinach; c) substitution can only take place to a very limited
extent and Na has no specific effect on growth, e.g., barley, millet, rice, oat, tomato, potato
and ryegrass; d) no substitution of K by Na is possible, e.g., maize, rye, soybean. However,
this classification should be treated with caution, since the differences among cultivars in the
same species are not taken into account.
2.8.1 High Na
2.8.1.1 Na toxicity effects
In arid and semiarid regions, the presence of excessive Na as NaCl has a detrimental effect on
the growth of most of crop plants (Marschner, 1995). In highly saline soil, plants will take up
Na+ in place of K+. Excessive Na+ interferes with the shoot transport or long distance
transport and cytosolic functions of K+, and greatly inhibits plant growth and development.
Even in the case of halophytes that accumulate larger quantities of Na+ inside the cell, their
cytosolic enzymes are as sensitive to Na+ as those of glycophytes (Malvi, 2011). High Na+
accumulation under hot temperatures may lead to cell wall rupturing and slow programmed
death of plants (Malvi, 2011).
Replacement of a high proportion of K+ by Na+ inhibits the activity of many enzymes that
specifically or more sensitively respond to K+. It was reported that plant growth was inhibited
in many species grown on saline soils due to depression in K+ uptake by competing Na+ ions
(Al-Rawahy et al., 1992). This inhibition is dependent on levels of Na and K; the higher the
Na+/K+ ratio, the greater the damage (Malvi, 2011).
2.8.1.2 Imbalance in K/Na ratios
The ability of plants to maintain a high K+/Na+ ratio in the cytosol plays an important role in
stress tolerance. The influx of Na+ ions through K+ pathways alters the ion ratios in plants.
The cytosolic enzymes in plants are not adapted to high Na+ levels and hence, plants respond
to elevated Na+ concentrations by maintaining low cytosolic Na+ concentrations and high
K+/Na+ concentrations by Na+ extrusion and/or the intracellular compartmentalisation of Na+
(predominantly in plant vacuole) (Blumwald, 2000). In saline soils, high Na+/K+ ratio reduces
20
plant growth and eventually becomes toxic to plants (Schachtman & Liu, 1999). Therefore,
increase in K uptake in saline soils will increase the K+/Na+ soil ratio, and potentially
minimise the salt stress in plants (Wu et al., 1996). It is critical to understand how plant roots
distinguish K+ and Na+ ions in saline soils for proper nutrient management (Schachtman &
Liu, 1999).
2.8.2 Low to moderate Na
Even though high concentrations of Na have a large depressive effect on plant yield, K and
Na share various physiological functions (Almeida et al., 2010). Sodium can substitute
biophysical functions of K, provided that the plants have the ability to take up Na, translocate
it to the shoot, and compartmentalise it in their vacuoles (Subbarao et al., 2003). There are
studies on beneficial effects of Na in various plant species that focussed on partial to near-
complete replacement of K by Na. Substantial positive effects on plant growth have been
reported in plants, particularly at deficit K supply and in plants suffering K+ deprivation
(Kronzucker et al., 2013). The functions of K replaced by Na and studies where Na was
found to be beneficial are reviewed briefly in the following sections.
2.9 Functions of K replaced by Na
Sodium improves the water balance of plants when the water supply is limited via effects on
stomatal conductance. Under drought stress, when there is a sudden decrease in plant
available water, the stomata of the plants supplied with Na close more rapidly than the plants
supplied with K alone, and after stress release, exhibit a delay in opening (Mäser et al., 2002).
It is claimed that in natrophilic species, Na+ replaces K+ in its role in stomatal opening, as for
example, in sugar beet, Na+ substituted K+ in stomatal functions (Marschner, 1995). The
addition of Na to the culture medium lessened the effects of K deficiency on photosynthetic
or respiratory CO2 exchange (Terry & Ulrich, 1973), and increased leaf net photosynthesis of
Theobroma cacao (Gattward et al., 2012). A list of the key functions of K substituted and not
substituted by Na is provided in Table 2.2.
21
Table 2.2 Functions of K replaced and not replaced by Na
Functions of K replaced by Na Source
Growth stimulation (Wakeel et al., 2011)
Improved water balance via stomatal conductance
(Marschner, 1995)
Maintenance of ionic balance (Subbarao et al., 2003)
Regulation of osmotic pressure (Marschner, 1995)
Vacuolar functions (Ali et al., 2006; Mäser et al., 2002)
Photosynthesis/CO2 exchange (Gattward et al., 2012; Terry & Ulrich, 1973)
Functions of K not replaced by Na Source
Enzyme activity (Subbarao et al., 2003)
Cytoplasmic functions (Benito et al., 2014)
Protein synthesis (Kronzucker et al., 2013)
Oxidative phosphorylation (Kronzucker et al., 2013)
Biochemically, enzyme activation requires relatively low concentrations of K (about 10-50
mM) for maximum activity, and this K requirement may be further reduced by the
substitution of Na and other monovalent cations like Cs and Rb (Subbarao et al., 2003). In K
deficient soils, Na assumes the role of K in maintaining ionic balance, electroneutrality and
regulating osmotic pressure (Marschner, 1995). Sodium can alleviate or even eliminate K
deficiencies in some species such as barley, sugar beet, lettuce, cotton, turnip, pangola grass,
Italian ryegrass and Rhodes grass by partly replacing the role of K in the plant (Marschner,
1995; Mundy, 1983). Some crops like marigold, spinach, and sugar beet showed a yield
increase to added Na even in the presence of adequate amounts of K (Idowu & Aduayi,
2007). It was found in an experiment that high yields occurred in ryegrass at optimal and also
suboptimal K concentrations when Na was included in the nutrient solution with the highest
yield for plant tops containing 4-8 % K without Na, and 1-4 % K with Na (Mundy, 1983).
There was poor growth, chlorosis and necrosis in Atriplex vesicaria when the basic nutrient
solution had less than 0.1 µM Na+, despite a high K+ concentration (Marschner, 1995).
Sodium ion can replace K+ to a certain degree in the vacuole, particularly in osmotic
functions. It is reported that in red beet Na+ can replace K+ in vacuolar functions, thus
22
offsetting 95 % of the plant’s K requirement (Subbarao et al., 2003). According to Marschner
(1995), when Na contents in the leaves are high, the K content required for optimal growth
decreased from 3.5 to 0.8 % of the leaf dry weight in Italian ryegrass, from 2.7 to 0.5 % in
Rhodes grass, and from 4.3 to 1.0 % in lettuce. The grain yield of rice grown at 25 µM K
more than doubled in the presence of 43 mM Na. Addition of Na also increased dry weight of
Arabidopsis at 10 µM K. Sodium ion can exert beneficial effects on certain plant species
even when K+ supply is not limiting. In sugar beet, replacement of 5 mM K in the nutrient
substrate with 2.5 mM each of K and Na increased plant dry weight and sucrose content in
the storage root. Also, Na may improve the plant’s water balance in sugar beet (Mäser et al.,
2002).
2.10 Plant responses to Na at low K
The presence of low to moderate Na may stimulate plant growth under K depleted conditions.
The studies in different plant species where Na was found to have beneficial effects are
summarised in Table 2.3 together with the K and Na concentrations used and key results from
the study. There are numerous studies that have reported beneficial effects of Na on growth
and yield, plant physiology and on visible improvement with less pronounced K deficiency
symptoms. Among the species examined, the Chenopodiaceae family including crops like
spinach, beet and sugar beet had received detailed attention, and studies in different crop
species have been listed in a recent review by Kronzucker et al. (2013).
In some plants, supplementation of Na in reduced amounts was found to eliminate K
deficiency symptoms (Marschner, 1995; Wakeel et al., 2011). This may offset the
requirement of K fertiliser and therefore may help in profitable cropping. Wheat is a major
cereal grown in saline, semi-arid regions of Western Australia. But there is little research on
potential replacement of K by Na in wheat. Box and Schachtman (2000) investigated whether
Na can benefit wheat growth under low K by stimulating K+ uptake through the Na+
energised HKT1 symporter and they found that low concentrations of Na+ stimulated wheat
growth at low external K in only one of their experiments when light levels were low. By
contrast, Ma et al. (2011) reported no growth stimulation in wheat with Na addition at low K
supply even though barley growth was stimulated under the same conditions. Rubio et al.
(1995) showed an increase in K+ uptake by wheat with 1 mM Na+ addition.
There are differences in accumulation of K+ and Na+ among different species. Wheat shows
distinctive behaviour relative to other species like barley with very high K+ selectivity. In the
23
study of Ma et al. (2011), shoot K+ /Na+ in wheat cv. Wyalkatchem was 10-15 times higher
than the ratio in shoots of two barley cultivars grown under the same conditions. Further
detailed research on K and Na interactions in wheat cultivars are essential since the findings
could have practical implications in nutrient management.
24
Table 2.3 Sodium response at low K
Crop species K level Na level What improved with Na?
Type and duration of experiment
Key results Reference
1. Cacao tree (Theobroma cacao)
2.5 and 4.0 mM K.
Six replacement ratios of K+ by Na+ (0, 10, 20, 30, 40, 50% replacement, mol/mol)
Photosynthetic rate, WUE, and mineral nutrition
Soil culture in glasshouse and harvested at 180 DAT
Results show that Na+ can partially replace K+, with significant beneficial effects on photosynthesis, WUE and mineral nutrition.
(Gattward et al., 2012)
2. Rice (Oryza sativa)
1, 2, 5, 10, 50, 100 and 200 ppm (as KCl) or
0.02, 0.05, 0.13, 0.26, 1.28, 2.56 and 5.13 mM K
1000 ppm (as NaCl) or 43.5 mM Na
Leaf turgidity, shoot dry wt (panicle wt. and straw)
Solution culture and maintained until harvest of rice.
Shoot dry weight increased with addition of 1000 ppm (43.5 mM) Na in 1, 2, 5 and 10 ppm (0.02, 0.05, 0.13 and 0.26 mM, respectively) K treatments.
Application of Na+ altered the leaf habit, from ‘flaccid’ to ‘erect’, in K deficient plants.
High % of unfilled grains in low K without Na treatments.
Relationship between shoot dry weight and K content showed that, within the deficiency range, higher dry weights were obtained at the same K content in the presence of Na.
(Yoshida & Castaneda, 1969)
25
3. Eucalyptus (Eucalyptus grandis)
1.5 (58kg K/ha), 3.0 (116kg K/ha), 4.5 kmol K/ha (174 kg K/ha) as KCl
3.0 kmol K/ha (116kg K/ha) applied as K2SO4
3.0 kmol Na/ha
(68 kg Na/ha) and 1.5 kmol K/ha + 1.5 kmol Na/ha (58 kg K/ha as KCl and 34 kg Na/ha as NaCl). Treatments were compared with control with no K, Na application
Growth (height and basal area)/ above- ground biomass
Field study for 4 years from planting
Application of 3.0 kmol Na/ha increased tree height and basal area by 14 % and 32 %, respectively, in comparison with control. Combined K and Na fertilization (K 1.5 + Na 1.5) led to a growth in height and basal area that was intermediary between treatments K 1.5 and K 4.5.
(Almeida et al., 2010)
4. Tomato (Lycopersicon esculentum)
Growth experiment
0.5 and 4.5 mM K+ (as KCl)
1, 5, 15 and 30 mM Na+ (as NaCl)
Plant growth and root growth stimulation.
Solution culture and harvested on day 28
In K+ -deficient plants, external NaCl concentrations of 1 and 5 mM were optimal, restoring growth to levels not significantly different from the maximal (4.5 mM) level.
The presence of Na+ increased ability to direct K+ to growing leaves which is crucial for maximization of growth under K+ -deprivation.
(Walker et al., 2000)
26
5. Tomato (Lycopersicon lycopersicum)
0, 32, 64 and 128 mg/kg (as KCl)
0, 2, 4, 8, 16 and 32 mg/kg soil (as NaCl)
Number of leaves, Number of flowers, and fruit yield
Soil culture experiment
The interaction of 2-4 mg Na/kg soil with 32-64 mg K/kg soil resulted in vigorous and healthy plant growth with bright green leaves, broad, succulent and well-spaced foliage.
The results indicated an efficient nutrient combination of Na and K at Na: K ratio of 1:8 to 1:32, which resulted in balanced tomato nutrition when compared with plants that received high doses of K.
(Idowu & Aduayi, 2007)
6. Tomato
(Lycopersicon esculentum and L. pennelIii)
K-free solution and 5 mM K
5 mM and 100 mM NaCl
Plant dry weight Solution culture experiment
2 varieties: Lycopersicon esculentum Mill. cv.M82 and its wild salt-tolerant relative species L. pennellii
Addition of 5 mM Na+ to K-free medium increased plant dry weight in both species.
L. pennellii plants (wild sp.) were more efficient in substitution of K+ function by Na +, as there was a greater increase of dry weight in the wild species; a higher retranslocation of K+ from old to young leaves was noticed, and consequently a higher K-efficiency (dry weight/K+) ratio.
In wild species, Na+ can be
(Tahal et al., 2000)
27
used as a cheap osmoticum in the vacuole and, possibly, as a partial substitute for K+ in some of its functions.
7. Wheat
(Triticum aestivum)
growth experiment
a) 20, 100 and 2000 µM KCl
b) 20 µM KCl
c) 20, 100 and 2000 µM KCl
0 and 500 µM NaCl
Plant dry weight Solution culture and harvested at 32 days (exp- a), 42 days (exp- b) and 40 days (exp- c)
Sodium stimulated the wheat growth significantly only at low (20 µM) external K+ in one of the long term experiments, but not in two other experiments.
(Box & Schachtman, 2000)
8. Wheat
(Triticum aestivum)
Wheat root high-affinity K uptake transporter HKT1 was shown to function as a high-affinity K+-Na+ cotransporter. High-affinity K+ uptake was activated by micromolar Na+ concentrations, however, at physiologically detrimental concentrations of Na+, K+ accumulation mediated by HKT1 was blocked and low-affinity Na+ uptake occurred (approximately 16 mM Na+), which correlated to Na+ toxicity in plants.
(Rubio et al., 1995)
28
9. Barley (Hordeum vulgare L.)
30 mg K/kg and 75 mg K/kg (all plants were grown in for 4 weeks to create mild K deficiency)
0, 100 and 300 mg Na/kg
Leaf development, tiller numbers, shoot dry weight
Soil culture experiment in glasshouse for 7 weeks
Moderate Na treatment enhanced shoot growth by 42 % in the K deficient plants.
(Ma et al., 2011)
10. Red beet (Beta vulgaris)
5.0, 1.25, 0.25, and 0.10 mM
50 mM Na replaced K in osmotic functions without negatively affecting the plant water status, or growth
Nutrient film technique for 42 days with two cultivars
Leaf relative water content and osmotic potential were significantly higher for both cultivars with Na at low-K treatments.
(Subbarao et al., 2000)
11. Cotton
(Gossypium hirsutum)
a) glasshouse experiment- 6 mM K (as KNO3) K: Na ratios- 1:0, 2/3:0, 1/3:0, 2/3:1/3, 1/3:2/3, 0:1
(Na as NaNO3 and NaH2PO4)
b) field experiment- 5 combinations of KCl and NaCl (mg/kg): 1) K 80, Na 35; 2) K 115, Na 35; 3) K 80, Na 65; 4) K 80, Na 90; 5) K 115, Na 65
Dry matter accumulation and cotton seed yield
a) Sand culture in GH watered with modified Hoagland solution, harvested @ 30 days
b)field experiment
Replacing 1/3rd K with Na increased cotton seedling development; replacing 2/3rd or completely with Na- reduced germination rate, restrained cotton seedling growth and nutrition uptake.
Highest seed cotton yield was obtained when K and Na were added at rates of 115 and 65 mg kg-1, respectively in the top 20 cm of soil.
(Zhang et al., 2006)
29
12. Cotton
(Gossypium hirsutum)
3 combinations of K and Na
1) 3:1 (2.25 mM K: 0.75 mM Na),
2)1:1 (1.5 mM K: 1.5 mM Na)
3) 1:3 (0.75 mM K:
2.25 mM Na)
Main aim of this study was to look at genotypic difference.
Solution culture with 4 genotypes, harvested at 35 and 42 days after transplanting
The growth was better when K and Na were added in ratio of 3:1. There was significant effect on biomass production, K- use efficiency and K: Na ratios among genotypes.
(Ali et al., 2009)
13. Cotton
(Gossypium hirsutum)
Different levels of
K and Na with K: Na ratios of 3.5: 1 (control), 3.75: 1, 4: 1, 4.25: 1, 4.5: 1, 2.8: 1, 3: 1, 3.2: 1, 3.4: 1, 3.6: 1. K+ Na in kg ha-1 with K: Na ratios were as: 210+60 (3.5: 1) i.e. control, 225+60 (3.75: 1), 240+60 (4:1), 255+60
(4.25: 1), 270+60 (4.5: 1), 210+75 (2.8: 1), 225+75 (3: 1), 240+75 (3.2: 1), 255+75 (3.4: 1) and 270+75 (3.6: 1)
Number of bolls/plant and seed cotton yield
Field study.
Varieties differing in K-use efficiency used.
Harvest at 80 DAS to measure ionic ratios
Maximum seed cotton yield was obtained at K: Na ratio of 3.4:1 followed by 3.6:1 in both cotton varieties.
Enhanced number of bolls/plant was produced with at ratio 4.25:1 followed by 3.4:1. Variety difference: NIBGE-2 showed relative high Na substitution capacity for K and yielded better than MNH-786. The variation in K/Na selectivity of xylem transport from roots to the leaves proved to be one important cause of inter-specific differences in cotton varieties.
(Ali et al., 2013)
30
2.11 K and Na transporters
Epidermal and cortical cells of roots are involved in K+ uptake from soil solution. Plants use
low and high affinity transporters to take up K+ from extracellular medium (Britto &
Kronzucker, 2008). The pathways of Na+ entry into plants are not definitively known, despite
years of intensive research, however, evidence mostly supports a primary role of K+
transporters and transporters from the HKT family for Na+ influx into plant roots
(Kronzucker & Britto, 2011). High external Na+ concentrations may upset K+ equilibrium by
increased efflux, reduced influx and decreased cytoplasmic K+ concentrations (Wakeel,
2013).
Potassium transporters and channels are located at the plasma membrane of root cells (Wang
& Wu, 2010), and a large number of genes encoding for plant K+ transport have been
identified in many species (Very & Sentenac, 2003). For example, a total of 71 K+ channels
and transporters have been identified in Arabidopsis sp. so far (Wang & Wu, 2010). External
K+ concentration influences the activities of K+ channels and transporters (Wang & Wu,
2010). Potassium acquisition from low external concentrations is usually considered to be an
energy-demanding process, while that from high K concentrations in solution is energetically
passive (Britto & Kronzucker, 2008).
Low-affinity K+ transport system works at high external K+ concentrations (> 1 mM K). The
three important low-affinity K+ channels are: 1. Inward rectifying K+ channels (KIRC), such
as AKT1 that show high K+/Na+ selectivity and activate K+ influx upon plasma membrane
hyperpolarization; 2. Outward rectifying K+ channels (KORC) which play a role in Na+
influx into plant cells, and these channels unlike KIRC, open upon plasma membrane
depolarization; 3.Voltage-independent cation channels (VIC) in plant plasma membranes that
have relatively high Na+/K+ selectivity (Blumwald, 2000; Szczerba et al., 2009).
The high-affinity K+ uptake (HAK) mechanism operates at low external K+ concentrations (<
1 mM K) and the high affinity K+ transporter (HKT1) is highly selective for K+ but could also
be a low-affinity Na+ transporter or Na+/K+ symporter (Britto & Kronzucker, 2008; Szczerba
et al., 2009).
2.12 Genotypic variation in Na substitution of K
It is well known that varieties within the same species differ widely in their ability to take up
and utilize mineral nutrients. The extent to which Na+ can replace K+ varies among different
plant species, different cultivars of the same species, and even between different leaves of the
31
same plant with younger leaves relying more on K than older ones (Mäser et al., 2002). Also,
the extent of substitution differs between individual organs and between cell compartments,
being very large in the vacuoles, but very limited in the cytoplasm (Marschner, 1995).
Variation in K uptake and utilization was noticed among varieties of sweet potato (George et
al., 2002), cotton (Ali et al., 2006), and wheat (Damon & Rengel, 2007). Genotypic
differences are also reported in terms of salt tolerance and Na substitution of K functions (Ali
et al., 2006; Mäser et al., 2002). Ali et al. (2006) studied 30 cotton genotypes in hydroponics
and found that the genotypes differed significantly in growth responses, K uptake, K use
efficiency and the extent K substitution by Na, and they confirmed that screening of
genotypes is an effective approach for enhancing growth and yield under K deficient
conditions. There is evidence that genotypes can vary in Na substitution of K functions in
plants illustrating that Na resistance is a quantitative trait (Ali et al., 2006; Mäser et al.,
2002). In addition there are variations in Na/salt tolerance among genotypes. There is lack of
research on Na substitution of K functions among wheat cultivars.
The variation in Na absorption and the extent of K substitution by Na in plant species may
explain their differential response to Na and K applications. The increased capacity to
substitute Na+ for K+ was suggested by Rengel and Damon (2008) to be one of the possible
mechanisms underlying K utilization efficiency. However, in their previous studies to rank
wheat genotypes according to K-use efficiency there was no examination of the role of low to
moderate Na supply Na on plant K nutrition. Also the salt tolerance among wheat genotypes
differing in K use efficiency has not been studied.
2.13 Salinity and duplex soils
Soil salinity in arid and semi arid areas is a major constraint affecting crop productivity in
many parts of the world (Alhagdow et al., 1999). Sodic soils and duplex soils are also major
soil constraint affecting crop production in Australia. Duplex soils are described as the soils
that have an abrupt textural difference between the surface soil horizons and the subsurface,
and they exhibit great diversity in their properties, particularly genesis and mode of
development (Chittleborough, 1992) and they are classified under order ‘Sodosols’ according
to the Australian soil classification (Isbell, 2002). Duplex soils account for about 12 % of the
land area of Australia (Dracup et al., 1992) and, are widespread in Western Australia,
accounting for half to 2/3rd of the cultivated area in south-Western Australia (Belford, 2005).
The distribution of nutrients with depth in duplex soils is different from that of uniform soil
32
profiles. The duplex soils of western Australia often have low topsoil K but varying
concentrations of K in the heavier textured subsoils due to differences in clay content and
mineralogy (Wong et al., 2000). For example, the duplex soils of the western wheatbelt (with
clay mineralogy- kaolinite, sesquioxides) have uniformly low Colwell K concentration in
sandy horizon A and clay horizon B. In contrast, duplex soils in eastern and south-eastern
wheatbelt usually hold higher concentration of potassium in the clay horizon B compared to
the sandy A horizon (McArthur, 1991; Wong et al., 2000). The clayey subsoils are sodic
when about 10 % of exchangeable ions are Na and this may interfere with crop production
(Belford, 2005). In case of duplex soils, sampling only the top soil gives an incomplete
estimate of nutrient profile and misjudgement in nutrient application.
The distributions of Na and K down the profile may vary in duplex soils, but the implications
of this for crop K nutrition have not been examined. While topsoils are commonly low in Na
and K, variable levels of Na and K can occur in sub-soil. It is not known for example if Na in
the sub-soil is able to alleviate the effects of low topsoil K. Whether Na substitution is
effective in alleviating K deficiency may depend on the plant species, the levels of Na and K
in the sub-soil, the length of time it takes for roots to reach the sub-soil (which depends on
depth of the sub-soil) and the root length density in the sub-soil. In sodic duplex soils, Na+
may assume the role of K+ in some of the physiological functions as discussed above, but the
variable distribution of both K and Na with depth in the root zone adds uncertainty to the
effectiveness of Na as a replacement for K requirements in such soils.
2.14 Research scope, aim and research questions
The spread of high-yielding varieties and increases in cropping intensity to fulfil increasing
food demands globally are depleting soil K (Wakeel et al., 2011). Application of K fertilisers
to increase crop productivity is relatively expensive especially for resource-poor farmers in
developing countries. The partial substitution of K+ by Na+ in physiological processes of
plants can have substantial implications for K management and could reduce costs of crop
production if its value to crop grain yield was better understood.
The main aim of this research is to study the role of Na and cultivar effects on K nutrition of
wheat in the context of drought-prone environments and soils that are moderately saline
and/or sodic. To understand the interaction between K and Na in wheat, the present study was
conducted with the following research questions:
• Is Na beneficial to wheat and if so what levels of soil Na are beneficial?
33
• Can Na substitute for K requirements in wheat when available soil K is deficient?
• Do wheat genotypic differences in K-use efficiency alter the extent of K substitution
by Na?
• Is there a relationship between K responses and sub-soil Na?
• Is there an impact of Na supply on wheat K nutrition when K uptake from the topsoil
is restricted by water deficit conditions?
• What happens when K and Na are present at different root sections? Does K
replacement by Na depend on both these cations being present in the same part of the
root system?
• Is there an increase in wheat K+ and/or Na+ uptake with addition of external Na at
deficient K supply, and are there any differences due to K-use efficiency of wheat
cultivars and their uptake?
2.15 Conclusion
The literature review above explains in detail the role of K in plants, K deficiency effects,
forms of K in soil, importance of Na, interactions between K and Na, functions of K that can
be replaced by Na, and then relates the proposed research to sodicity and duplex soil
conditions prevailing in Western Australia. The beneficial effect of Na ion has been
established for some C4 plants, while for many higher plants Na can have beneficial effects
under specific circumstances without being an essential element. The present thesis aims to
identify whether Na is beneficial to wheat growth and the circumstances under which this
occurs. In addition to varying K and Na supply and placement in the root zone, variation in K
use efficiency of wheat cultivars is used to explore the research questions. The research
questions will be tested with a series of pot and solution culture experiments described in the
following chapters (Chapter 3 to 7).
34
CHAPTER 3
Wheat responses to sodium vary with potassium use efficiency of cultivars1
3.1 Introduction
Key physiological roles for K are in stomatal regulation and in photosynthesis (Römheld &
Kirkby, 2010). Sodium can substitute for non-specific biophysical functions of K+, especially
where plants have the ability to take up, translocate, and compartmentalise Na in their
vacuoles where it can replace functions of K in maintaining cell turgor (Gattward et al., 2012;
Subbarao et al., 2003). In K-deficient soils, Na can play the role of K in maintaining ionic
balance (Subbarao et al., 2003), regulating osmotic pressure (Marschner, 1995), provide
partial K-substitution in protein synthesis (Flowers & Dalmond, 1992), contribute to vacuolar
functions (Mäser et al., 2002), and improve water balance via regulation of stomatal
conductance (Gattward et al., 2012; Marschner, 1995). Under K deficiency, the addition of
Na replaced K in stomatal functions of sugar beet and reduced the effects of K deficiency on
photosynthetic or respiratory CO2 exchange (Terry & Ulrich, 1973), and in net photosynthetic
rate of Theobroma cacao (Gattward et al., 2012). Also under water deficit, stomata of sugar
beet leaves supplied with Na closed more rapidly but exhibited a delay in opening compared
to supply of K only (Mäser et al., 2002).
Sodium is reported to alleviate effects of K deficiency on plant growth in sugar beet, lettuce,
cotton, ryegrass, spinach, marigold, tomato (Idowu & Aduayi, 2007; Marschner, 1995;
Mundy, 1983; Pi et al., 2014; Tahal et al., 2000), and barley (Ma et al., 2011). However, in
wheat which maintains a high selectivity of K+ uptake relative to Na+ uptake, there are few
reports of Na partially substituting for K. Box and Schachtman (2000) investigated whether
Na supply can benefit wheat growth under low K by stimulating K+ uptake through the Na+
energised HKT1 symporter and found that low concentrations of Na+ did not increase K+
uptake to a large extent and while Na+ stimulated wheat growth at low external K it was only
when light levels were low. By contrast, Marschner (1995) classified wheat as having
moderate response to low Na at low K. Hence further investigation is needed to clarify the
response of wheat to low Na levels especially under low K.
Varieties of the same species can vary in K accumulation and utilization, e.g. sweet potato
(George et al., 2002), cotton (Ali et al., 2006), and wheat (Damon & Rengel, 2007).
1 This Chapter is a slightly modified version of Krishnasamy et al. (2014). Wheat responses to sodium vary with potassium use efficiency of cultivars. Frontiers in Plant Science, 5: 631. doi: 10.3389/fpls.2014.00631.
35
Genotypic differences in cotton (Ali et al., 2006) and sugar beet (Marschner et al., 1981) are
also reported in terms of Na substitution of K functions. Ali et al. (2006) studied 30 cotton
genotypes in hydroponics and found that the genotypes differed significantly in growth
responses, K uptake, K-use efficiency and the extent of K substitution by Na. However, there
is a shortage of information about how cultivar variation in K-use efficiency alters effects of
low to moderate soil Na on plant K nutrition. Understanding K and Na interactions among
wheat cultivars that vary in K-use efficiency would improve management of K fertiliser in
sodic and K-deficient soils.
We hypothesised that if high K efficiency in wheat was related to higher K uptake, K
efficient cultivars would exhibit not only reduced salinity effects but also a reduced response
to Na-substitution of K in plants. Alternatively, if greater Na substitution of K was the main
mechanisms for greater K-use efficiency such cultivars could be more susceptible to salinity
and demonstrate greater response to low to moderate Na levels in low soil. We examined the
effect of Na levels on K uptake, the K+/Na+ ratios, leaf gas exchange, and plant growth of
wheat cultivars differing in K-use efficiency. Supply of NaCl ranged from low to moderate
levels, designed for substitution of K by Na at low K supply, ranging to toxic levels for wheat
at high Na.
3.2 Materials and methods
Wheat (Triticum aestivum L.) cvv Wyalkatchem, Cranbrook, Gutha, and Gamenya were
grown in a naturally-lit glasshouse at Murdoch University, Perth (32°04′S, 115°50′E) from
late winter to mid spring. The average minimum and maximum temperatures during the
experiment were 8.4 and 26 ˚C, respectively. Cultivars Wyalkatchem and Cranbrook are K-
efficient, whereas Gutha and Gamenya are K-inefficient in terms of K uptake and use
(Damon & Rengel, 2007). The sandy soil (classified as ‘Chromosols’ according to the
Australian soil classification) was collected from an unfertilised field, 150 km northeast to
Perth, and had the following properties: pH 4.9 (0.01 M CaCl2), EC1:5 0.03 dS/m, 7 mg NH4-
N/kg and 9 mg NO3-N/kg (Searle, 1984), <15 mg K/kg and 29 mg P/kg (Colwell, 1963) and
organic C 0.17% (Walkley & Black, 1934).
Sieved soils (< 2 mm) were well mixed with basal nutrients and individual treatments of K
and Na, and filled into undrained plastic pots (diameter 190 mm, depth 190 mm) at 6 kg/pot.
Basal nutrients were applied at the following rates (mg/kg): 103 (NH4)2HPO4, 237
Ca(NO3)2.4H2O, 80 MgSO4.7H2O, 18 FeSO4.7H2O, 14 MnSO4.H2O, 9 ZnSO4.7H2O, 8.3
36
CuSO4.5H2O, 0.33 H3BO3, 0.3 CoSO4.7H2O, 0.33 Na2MoO4.2H2O. Seeds were washed with
5% (w/w) hypochlorite solution for 1 minute, thoroughly rinsed and soaked in de-ionised
(DI) water for 2 hours, and then placed in a refrigerator at 4 ˚C overnight. The sprouted seeds
were transferred to trays containing 0.05 mM CaCl2 solution, and covered for 2 days. Five
germinated seeds per pot were transplanted, and 10 days later the seedlings were thinned to 3
plants per pot. During the experiment, the pot soils were watered daily to field capacity (15 %
w/w) with DI water. The plants were supplied with 0.5 mM urea solution fortnightly to
maintain adequate N supply. The pots were re-arranged every week to reduce positional
effects on plant growth.
3.2.1 Potassium and sodium treatments
Two soil K levels were applied: 40 mg K/kg (low) and 100 mg K/kg (adequate) based on
earlier trials (Ma et al., 2011). Muriate of potash (KCl) was used as it is the dominant K
fertiliser (Moore, 2004). Each soil K level also included 5 Na levels: nil, 25, 50, 100 and 200
mg Na/kg supplied as NaCl. Equivalent Na concentrations in soil solution were 0, 7.25, 14.5,
29.1, and 58.2 mM, respectively, while ECe (electrical conductivity of saturated soil extract)
values for Na treatments at 40 K were 0.85, 1.26, 2.66, 5.18 and 10.9 dS/m and at 100 K were
1.23, 1.82, 3.78, 7.28 and 13.7 dS/m. Therefore, the experiment comprised a factorial
combination of 4 wheat cultivars, 2 K levels, and 5 Na levels. All the treatments were
replicated three times. At potting, individual treatments with various K and Na levels were
mixed thoroughly with basal nutrients using a rotary mixer.
3.2.2 Measurements
Plants were grown for 8 weeks and during that period the number of leaves and tillers was
recorded weekly. Leaf net photosynthesis, transpiration and stomatal conductance were
measured using the LCpro+ advanced photosynthesis system (ADC Bioscientific, UK) at 7
weeks after transplanting. The measurements were made in fully expanded young leaves at
ambient relative humidity of 50 %, leaf temperature of 25 ˚C, reference CO2 of 380
µmol/mol, and photosynthetically active radiation of 1500 µmol/m2·s1.
At harvest, the shoot was cut at the soil surface, and the fresh weight was recorded
immediately. Roots were collected after washing in tap water and rinsing in de-ionised (DI)
water. The shoot and root samples were dried in a forced-draught oven at 60˚C for 48 hours
and dry weight was recorded. About 0.2 g of each milled sample was weighed into centrifuge
tubes and digested in 5 mL 70 % (w/w) HNO3 at 75 ˚C for 10 min, and then at 109 ˚C for 15
37
min. After the samples were cooled, 1 mL of 30 % (w/w) H2O2 was added and further
digested at 109 ˚C for 15 min. The digestion was made in a micro-wave oven (CEM Mars 5,
CEM Corp., USA) based on method of Huang et al. (2004) for cation analysis. The digested
samples were diluted with milli-Q water and concentrations of K, Na, Ca and Mg were
determined by inductively coupled plasma-atomic emission spectroscopy (VISTA
Simultaneous ICP-AES, Varian). The K+/Na+ ratios in shoots and roots were calculated based
on their content.
A supplementary study was conducted to determine whether the extractable cation levels,
particularly K, were influenced by different soil Na levels. Two kilograms of soils were
thoroughly mixed with basal nutrients and two K levels (40, 100 mg K/kg). Each soil K level
was treated with 2 levels of Na: nil, 50 mg Na/kg. The pots were watered with DI water to
field capacity, and allowed to equilibrate for a week, while mixing daily. The soil samples
were then analysed for bicarbonate-extractable (Colwell) K (Colwell, 1963) and
exchangeable cations. Soils were extracted at a ratio of 1: 10 with 0.1 M NH4Cl for 2 hours
and exchangeable cation concentrations were determined by ICP (Rayment & Lyons, 2010).
3.2.3 Statistical analysis
Statistical analyses were conducted using the SPSS statistical package (IBM SPSS statistics,
vs 18). Three-way analysis of variance was conducted to assess the effects of soil K and Na
supply, genotype and their interactions. Tukey’s HSD was computed at P ≤ 0.05 to test for
differences among the treatment means.
3.3 Results
3.3.1 Plant growth
Shoot growth
Low K supply (40 mg K/kg) induced K-deficiency symptoms after 6 weeks (Fig. 3.1 and 3.2)
and significantly reduced shoot dry weight at 8 weeks, but the reduction was greater in K-
inefficient cultivars Gutha and Gamenya (32 % lower) than K-efficient cultivars
Wyalkatchem and Cranbrook (17-18 % lower). When K supply was low, the addition of low
to moderate Na (25, 50 mg Na/kg) alleviated K-deficiency symptoms in old leaves (Fig. 3.1
and 3.2) but had no significant effects on shoot dry weight (Fig. 3.3). Similarly, at adequate K
supply, addition of 25 - 50 mg Na/kg had no effect on shoot dry weight. High soil Na levels
(100, 200 mg Na/kg) reduced shoot dry weight especially in K-inefficient cultivars (Fig. 3.3).
When compared with nil Na, high Na reduced shoot dry weight by 44 % in Gamenya, 38 %
38
in Gutha, 31 % in Wyalkatchem and 22 % in Cranbrook. The interactions between K, Na and
cultivars on shoot dry weight were significant (P ≤0.05) (Table 3.1).
Fig. 3.1 Wyalkatchem (K-efficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg)
under soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks.
Fig. 3.2 Gutha (K-inefficient cultivar) at low (40 mg K/kg) and high K (100 mg K/kg) under
soil Na concentrations of nil, 25, 50, 100 and 200 mg Na/kg at 7 weeks.
39
Fig. 3.3 Shoot dry weight (g/plant) (upper sub-figures), and tillers/plant (lower sub-figures)
(n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open
circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.1
for analysis of variance results.
Gutha
0 50 100 150 2000
2
4
6
8
10 Gamenya
Soil Na levels (mg/ kg)
0 50 100 150 200
Wyalkatchem
No.
of t
ille
rs
0
2
4
6
8
10Cranbrook
Gutha
0
2
4
6
8 Gamenya
WyalkatchemS
hoot
Dry
we
ight
(g)
0
2
4
6
8
10Cranbrook
K*Na*cv P= 0.02HSD= 1.40
K*Na*cv P= 0.008HSD= 1.60
40
Adequate soil K produced the same number or more tillers than low K at all soil Na levels,
except in Gamenya that had fewer tillers at 100 mg Na/kg. Compared with Wyalkatchem,
fewer tillers per plant were produced in cvv Gutha and Gamenya (Fig. 3.3). Plants treated
with low to moderate soil Na (25, 50 mg Na/kg) had similar tiller number as those of nil Na
plants. However, high Na reduced tillers significantly in all four cultivars (P ≤0.05; Table
3.1).
Table 3.1 Statistical summary of plant growth and leaf gas exchange in four wheat cultivars
(Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two K levels (40, 100 mg
K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks.
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Soil K Soil Na
Cultivar K×Na K×cv Na×cv K×Na×cv
Shoot dry weight *** *** * ** *** *** *
Tiller number *** *** *** * *** *** **
Root dry weight *** *** *** n.s * *** n.s
Root: shoot ratio ** *** *** n.s * *** n.s
Photosynthesis *** *** *** *** n.s * ***
Stomatal conductance
*** *** ** * n.s n.s *
Root growth
Root dry weight of all four cultivars was greater at adequate K than at low K supply,
regardless of soil Na levels (Fig. 3.4). Low to moderate soil Na had positive effects on root
dry weight in all four cultivars when soil K was low, and even at adequate K supply low soil
Na was beneficial to root dry weight except in Gamenya (Fig. 3.4). The Na-induced root
stimulation was greater in K-efficient cultivars. High soil Na levels suppressed root dry
weight in all four cultivars at both soil K levels, with greater reduction of root dry weight in
K-inefficient cultivars (55 % in Gutha and 66 % in Gamenya) than in K-efficient cultivars
(33 % in Wyalkatchem, 50 % in Cranbrook). In general, low K plants had lower root: shoot
ratios compared with K adequate plants, except Cranbrook at low Na (Fig. 3.4), however, the
interactions between K and Na for root: shoot ratio was not significant (Table 3.1).
41
Gutha
0 50 100 150 2000.0
0.2
0.4
0.6
0.8 Gamenya
Soil Na levels (mg/ kg)
0 50 100 150 200
Wyalkatchem
root
: sh
oot r
atio
0.0
0.2
0.4
0.6
0.8Cranbrook
Gutha
0
1
2
3Gamenya
Wyalkatchem
Roo
t Dry
we
ight
(g)
0
1
2
3
4Cranbrook
K*Na*cv P= 0.09HSD= 0.82
K*Na P=n.sK*Na*cv P= n.s
Fig. 3.4 Root dry weight (g/plant) (upper sub-figures) and root: shoot ratio (n=3) (lower sub-
figures) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg
(open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See
Table 3.1 for analysis of variance results.
42
Gutha
0 50 100 150 2000
100
200
300
400 Gamenya
Soil Na levels (mg/ kg)
0 50 100 150 200
Wyalkatchem
Sto
mat
al c
ondu
ctan
ce (
mm
ol H 2
O /m
2 s)
0
100
200
300
400Cranbrook
Gutha
0
5
10
15
20
25
Wyalkatchem
Leaf
ne
t pho
tosy
nthe
sis
(µm
ol C
O 2/m
2 s)
0
5
10
15
20
25
30
Gamenya
CranbrookK*Na*cv P= 0.001HSD= 5.2
K*Na*cv P= 0.04HSD= 167
Fig. 3.5 Leaf photosynthesis (upper sub-figures) and stomatal conductance (lower sub-
figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg
K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks.
See Table 3.1 for analysis of variance results.
43
3.3.2 Leaf gas exchange
At 4 weeks, low K depressed net photosynthesis of the youngest fully expanded leaves but
there was no effect of low to moderate Na on gas exchange. At 8 weeks, consistent with the
shoot dry weight responses, net photosynthesis of the youngest fully expanded leaves was
higher in plants with adequate K than low K supply, except in Gutha at 50 mg Na/kg (Fig.
3.5). The addition of 25 and 50 mg Na/kg increased leaf photosynthesis in all cultivars at low
soil K and also in the K-inefficient cultivars at adequate K supply, whereas higher soil Na
suppressed leaf photosynthetic rate relative to addition of 25 and 50 mg Na/kg. The increase
in leaf net photosynthesis induced by 25 to 50 mg Na/kg at low K was almost equal to that at
100 mg K/kg and nil Na. There were significant interactions between soil K, Na and cultivars
for leaf photosynthesis (P ≤0.05) (Table 3.1). Similarly, stomatal conductance of low K
plants increased with the addition of 25 mg Na/kg in all cultivars (Fig. 3.5). Higher soil Na
reduced stomatal conductance in the K-efficient cultivars but was less so in the K-inefficient
cultivars. At low K supply, the addition of low to moderate soil Na increased transpiration
rate of K- efficient cultivars by 54 %, whereas in K-inefficient cultivars the increase was only
by 35 % relative to nil Na (data not presented).
3.3.3 K and Na concentrations in shoots and roots
Potassium concentration in leaves and stems of all four cultivars was significantly higher
with adequate K than low K supply when soil Na levels ranged from nil to moderate, whereas
spikes had similar K concentrations irrespective of K and Na treatments (see Appendix 1.2
for K and Na concentrations in leaves, spikes and stem). At low K supply, plants with nil Na
treatment had the lowest shoot K concentration in all cultivars (Fig. 3.6). Low to moderate Na
supply increased shoot K concentrations of the four cultivars on average by 25 % relative nil
Na at low K supply (Table 3.2). High soil Na also increased shoot K concentrations with both
low and adequate soil K supply in all cultivars, but probably due to a concentration effect as a
result of growth suppression.
Although shoot K content was much greater in the adequate K soil than in the low K soil, it
showed little difference or declined across soil Na levels at the high level of soil K (Fig. 3.7).
At low soil K supply (40 mg K/kg), shoot K contents increased significantly with low to
moderate soil Na addition in K-efficient cultivars but not in K-inefficient cultivars (Fig. 3.7).
There were significant interactions of soil K, Na supply and cultivars on shoot K contents
(P≤0.05) (Table 3.2).
44
Fig. 3.6 K concentration (mg/g, dry weight) in shoot (upper sub-figures) and root (lower sub-
figures) (n=3) of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg
K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks.
See Table 3.2 for analysis of variance results.
Gutha
0 50 100 150 2000
2
4
6
8
10 Gamenya
Soil Na levels (mg/kg)
0 50 100 150 200
Wyalkatchem
Roo
t K c
once
ntra
tion
(m
g/g,
dry
we
ight
)
0
2
4
6
8
10Cranbrook
Gutha
0
10
20
30
Wyalkatchem
Sho
ot K
con
cent
ratio
n (
mg/
g, d
ry w
eig
ht)
0
10
20
30
40
Gamenya
Cranbrook K*Na P=0.000K*Na*cv P= n.sHSD= 6.2
K*Na*cv P= 0.01HSD= 3.04
45
Fig. 3.7 K content (mg/plant) in shoot (upper sub-figures) and root (lower sub-figures) (n=3)
of four wheat cultivars, treated with 40 mg K/kg (closed circle) and 100 mg K/kg (open
circle), and five soil Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table 3.2
for analysis of variance results.
Gutha
0 50 100 150 2000
50
100
150
200Gamenya
Soil Na levels (mg/kg)
0 50 100 150 200
Wyalkatchem
Sho
ot K
con
tent
(m
g/pl
ant)
0
50
100
150
200
K 40 K 100
Cranbrook
Gutha
0 50 100 150 2000
5
10
15
20 Gamenya
Soil Na levels (mg/kg)
0 50 100 150 200
Wyalkatchem
Roo
t K c
onte
nt (
mg/
plan
t)
0
5
10
15
20 Cranbrook
K*Na*cv P= 0.03HSD= 18.4
K*Na*cv P= 0.00HSD= 4.0
46
Wheat roots accumulated considerably less K than shoots. Root K concentration and content
of all cultivars was significantly higher at adequate K supply than with low K supply (Fig. 3.6
and Fig. 3.7). At low K supply, soil Na addition had no significant effect on root K content
(Fig. 3.7). At adequate K supply, there was decrease in root K content with addition of soil
Na, except in Gutha at 25 mg Na/kg which showed a significant increase, and the decrease
due to Na was more obvious in K-inefficient cv. Gamenya. The three way interaction
between soil K and Na levels and cultivars was significant (P≤0.05, Table 3.2) for root K
concentrations and contents.
Table 3.2 Statistical summary of K and Na concentrations and contents in four wheat
cultivars (Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two levels of soil K
(40, 100 mg K/kg) and five levels of Na (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks.
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Soil K Soil Na Cultivar K×Na K×cv Na×cv K×Na×cv
K conc. shoot *** *** n.s ** n.s n.s n.s
K conc. Root *** *** *** *** *** *** **
Shoot K content *** *** *** *** *** * **
Root K content *** *** *** *** *** *** ***
Na conc. shoot ** *** n.s n.s n.s n.s n.s
Na conc. root ** *** *** n.s *** *** ***
Shoot Na content n.s *** n.s n.s n.s n.s n.s
Root Na content *** *** *** *** n.s *** n.s
Shoot K+/Na+ *** *** ** *** * n.s n.s
Root K+/Na+ *** *** n.s *** n.s n.s n.s
In all cultivars, shoot Na concentrations were closely associated with soil Na levels (Table
3.3). Old leaves and stem concentrated at least four times more Na than young leaves.
Sodium concentration in spikes was least influenced by soil Na irrespective of soil K and
cultivars, and there were only negligible concentrations of Na measured in spikes (data in
Appendix 1.1). Soil K levels did not influence shoot Na concentration and content. There
47
were no significant interactions observed between K and Na supply for shoot Na
concentrations and contents, nor among the cultivars (Table 3.2).
In contrast to K, Na concentration and content in roots were much higher than in shoots
(Table 3.3). Root Na concentrations rose with increase in soil Na levels. Similar to shoot, soil
K levels had no influence on root Na concentration. However, root Na content was higher in
plants grown at adequate K except at nil Na. The interactions between K and Na and cultivars
were significant for root Na concentrations but not for Na contents (Table 3.2).
Shoot K+/Na+ ratios noticeably decreased with increase in soil Na levels in all four cultivars
regardless of soil K levels (Table 3.4). At low soil K, Gamenya and Wyalkatchem had the
lowest and highest shoot K+/Na+ ratios, respectively. However, at adequate K, Cranbrook had
highest shoot K+/Na+ ratio at most of the soil Na levels. Roots had considerably lower K+/Na+
ratios than shoots. They decreased further with increasing soil Na levels but there was no
particular trend observed among the cultivars. The interaction between K and Na supply on
shoot and root K+/Na+ ratio was significant but not among the cultivars (Table 3.2).
48
Table 3.3 Shoot and root Na concentrations (mg/g, dry weight) and contents (mg/plant) of
four wheat cultivars treated with two K levels (40, 100 mg K/kg) and five Na levels (0, 25,
50, 100, 200 mg Na/kg) for 8 weeks (n=3). See Table 3.2 for statistical summary of main
effects and interactions of the treatments.
Measured parameters
cvv Wyalkatchem Cranbrook Gutha Gamenya
Na K40 K100 K40 K100 K40 K100 K40 K100
Shoot Na concentration
HSD0.05= 1.5
Nil Na 0.09 0.15 0.11 0.08 0.12 0.13 0.11 0.12
Na25 0.18 0.23 0.33 0.18 0.26 0.33 0.29 0.33
Na50 0.61 0.49 0.82 0.42 0.82 0.54 1.07 0.49
Na100 1.16 1.51 2.11 1.27 1.48 2.36 2.55 1.61
Na200 2.76 2.72 3.71 2.81 4.00 2.89 3.34 2.71
Root Na concentration
HSD0.05= 3.57
Nil Na 2.74 1.56 1.76 1.32 3.07 1.32 2.92 1.32
Na25 4.23 4.01 4.9 4.94 7.73 6.23 5.22 4.62
Na50 7.72 8.47 7.14 8.79 9.57 7.48 9.21 8.93
Na100 10.1 10.03 12.1 12.3 10.6 12.8 10.9 8.65
Na200 16.5 12.9 15.4 18.5 16.5 14.6 18.4 15.6
Shoot Na content
HSD0.05= 6.2
Nil Na 0.42 0.91 0.52 0.49 0.52 0.99 0.47 0.83
Na25 0.90 1.30 1.53 1.11 1.28 2.41 1.38 2.10
Na50 3.05 2.92 4.51 2.33 3.82 3.93 4.91 3.21
Na100 5.31 7.94 9.48 6.94 5.43 10.9 10.5 8.93
Na200 8.77 10.9 12.6 13.5 12.1 13.1 8.04 11.0
Root Na content
HSD0.05= 6.3
Nil Na 4.13 3.18 3.44 3.22 2.68 2.62 2.89 2.95
Na25 9.86 11.6 12.8 14.9 8.87 13.5 6.34 7.62
Na50 15.9 24.1 17.9 22.4 11.0 14.5 9.50 15.2
Na100 16.9 22.0 15.7 22.3 7.60 14.1 9.50 12.6
Na200 14.1 19.4 16.0 21.6 9.19 10.6 6.79 10.9
49
Table 3.4 Shoot and root K/Na ratios of four wheat cultivars treated with two K levels (40,
100 mg K/kg) and five Na levels (0, 25, 50, 100, 200 mg Na/kg) for 8 weeks (n=3). See
Table 3.2 for statistical summary of main effects and interactions of the treatments.
Measured parameter
cvv Wyalkatchem Cranbrook Gutha Gamenya
Treatment K40 K100 K40 K100 K40 K100 K40 K100
Shoot K+/Na+
HSD0.05= 81
Nil Na 123 196 115 279 90.5 177 104 196
Na25 86.7 100 46.1 137 54.5 68.3 44.7 68.4
Na50 24.5 48.9 17.1 56.5 17.7 39.3 12.9 47.2
Na100 12.9 17.9 8.05 21.9 10.3 13.4 5.73 17.8
Na200 6.63 10.04 5.93 9.71 4.55 8.78 4.98 10.4
Root K+/Na+
HSD0.05= 2.5
Nil Na 0.36 7.03 0.67 5.49 0.72 5.56 0.72 7.53
Na25 0.45 1.67 0.14 0.94 0.35 1.34 0.38 1.38
Na50 0.19 0.81 0.04 0.57 0.11 0.69 0.24 0.72
Na100 0.15 0.77 0.11 0.45 0.23 0.69 0.27 0.70
Na200 0.17 0.78 0.12 0.39 0.21 0.62 0.25 0.62
Leaf Ca and Mg concentrations measured were lower in K adequate plants when compared
with K deficient plants. Leaf Ca concentrations in both soil K levels decreased with increase
in soil Na in all four cultivars. The interaction between K and Na levels among the genotypes
was not significant for leaf Ca and Mg concentrations (see Appendix 1.2).
3.3.4 Soil exchangeable cations after K and Na addition
The soil incubation experiment did not show any significant effects of Na addition on
exchangeable soil K levels or exchangeable Ca, Mg and Al levels. Colwell-extractable K and
exchangeable K measured were slightly higher when soil had Na applied, compared with nil
soil Na, but the increase was not statistically significant (Table 3.5).
50
Table 3.5 Concentrations of exchangeable cations in non-planted soils (n=3) with or without
50 mg Na/kg at two K levels (40, 100 mg K/kg) after one week of incubation. Means with
different letters differ at P≤0.05.
Measured parameters
Soil treatment
K 40 K 40 K 100 K 100
Nil Na 50 Na Nil Na 50 Na
Colwell K (mg/kg) 48.3b 51.0b 96.0a 102a
Exc. K (cmol/kg) 0.13a 0.14a 0.24b 0.27b
Exc. Na (cmol/kg) 0.02b 0.19a 0.02b 0.18a
Exc. Ca (cmol/kg) 0.68a 0.71a 0.44b 0.61ab
Exc. Mg (cmol/kg) 0.09a 0.10a 0.10a 0.10a
Exc. Al (cmol/kg) 0.02a 0.03a 0.03a 0.03a
3.4 Discussion
Wheat response to soil NaCl supply in the present study varied with soil Na and K levels: 1)
root growth was stimulated by low to moderate soil Na levels with low soil K; 2) shoot and
root dry weight were suppressed with high Na regardless of soil K levels. However, the Na
effect varied with K-use efficiency of wheat cultivars with K-efficient cultivars being more
responsive to root dry weight stimulation by low to moderate Na under K deficiency along
with greater increases in shoot K uptake and stomatal conductance than K-inefficient
cultivars. Genotypic differences in K-use efficiency also influenced Na uptake and salt
tolerance: K-efficient cultivars were more tolerant of high salt levels than K-inefficient
cultivars.
The growth stimulation at low to moderate Na (25, 50 mg/kg) supply under K deficiency was
clearly evident in wheat roots but in shoots only through the alleviation of old leaf K
deficiency symptoms. The shoot dry weight and fresh weights were not significantly affected
by low to moderate Na in K deficient plants. However, Na at 100-200 mg/kg negatively
affected both root and shoot dry weight of wheat with both low and adequate soil K due to
salt toxicity. The present results in wheat were in contrast to salt-tolerant barley where the
addition of 100 mg Na/kg to K-deficient soil stimulated significant shoot growth increase but
51
not in root growth (Ma et al., 2011). Moreover, Ma et al. (2011) found, similar to the present
study, no significant benefit after 20 days of 100 mg Na/kg supply on K deficient (30 mg
K/kg) wheat growth. Indeed, 100 mg Na/kg (equivalent to about 30 mM Na in soil solution)
for 8 weeks had negative effects on wheat growth in the current study. Clearly there were
contrasting effects of low to moderate Na on wheat and barley. Barley responded positively
to moderate Na (100 mg Na/kg) supplied to K-deficient plants, but the response was
restricted to the shoots and not the roots. Wheat on the other hand only responded to Na at
lower levels (25-50 mg Na/kg but not 100 mg Na/kg) with the strong response in roots but
not in shoots.
Differences in accumulation of K+ and Na+ between barley and wheat may explain their
contrasting responses to low to moderate Na supply. Wheat roots accumulated significantly
higher Na than in shoots. Indeed the low to moderate level of Na that stimulated root growth,
increased root Na from 3 mg Na/kg (at nil Na) to 9.5 and 14 mg Na/kg at 25 and 50 mg
Na/kg respectively, while shoot Na only increased from 0.48 mg Na/kg to 1.3 and 4 mg
Na/kg, at 25 and 50 mg Na/kg respectively. In the study of Ma et al. (2011), shoot K+ /Na+ in
wheat cv Wyalkatchem was 10-15 times higher than the ratio in shoots of two barley cultivars
grown under the same conditions. By contrast, there were no differences among barley
cultivars and wheat in K+ /Na+ ratio in roots. Hence, the high accumulation of Na in barley
shoots produced a K+ /Na+ ratio of 0.5 in barley cv. CM72 but 10 in wheat. Under these
conditions, the partial substitution of K by Na is feasible in barley since there is sufficient
Na+ to provide equivalent osmotic effects to those of K+. In barley the accumulation of Na+
by low-K plants was limited to shoots as was the shoot growth response. By contrast, the low
shoot Na+ concentration in wheat shoots relative to K+, provides too little Na for the
replacement of K functions in the shoot to be feasible. For example in the low K plants, shoot
K concentration was 13.9 mg K/g, dry weight (equivalent to 89.5 mM K in tissue water) at 25
mg Na/kg of soil, whereas, depending on cultivars there was only 0.18-0.33 mg Na/g, dry
weight (mean tissue water Na concentration of 2.8 mM). Hence, there may be other processes
in wheat that led to growth stimulation at 25-50 mg Na/kg.
In both the present study, and that of Ma et al. (2011), low to moderate Na supply to low-K
wheat plants increased shoot K concentrations and this should have stimulated growth. The
average shoot K concentrations in low and adequate soil K treatments were 14 g/kg and 24
g/kg, which are in the deficient and sufficient ranges, respectively, for wheat growth at the
boot to heading stage (Reuter et al., 1997). The increases in photosynthesis rate and stomatal
52
conductance also evident with low to moderate Na supply to K-deficient are both expected
responses in the shoot to increased K concentration (Zhao et al., 2001). The increase in root
growth is another expected plant response to increased shoot K concentration because
increased photosynthesis results in greater assimilate supply to roots and increases root: shoot
ratio of cereals (e.g. (Degl'Innocenti et al., 2009; Ma et al., 2011; Ma et al., 2013)). Hence a
possible explanation for the Na stimulation of growth in wheat is that Na increases K supply
to the shoot which in turn stimulates photosynthesis and the greater supply of assimilate
allows for increased root growth. With only a single harvest it is not possible to definitively
piece together this chain of events. However, clearly the evidence in support of the first
response, the increase in K uptake leading to greater shoot K is pivotal.
Increased shoot K content could arise from several mechanisms. Firstly, increase of root Na
concentration at low to moderate Na may release vacuolar K+ that is made available for
cytoplasmic functions in the root cells or for translocation to the shoot (Walker et al., 2000).
The increase in root Na concentration at 25 -50 mg Na/kg of soil was substantial, while root
K contents remained unchanged. The effects of 25-50 mg Na/kg of soil on root K
concentrations varied among cultivars. By contrast, shoot K content increased by about 40 %
with the supply of 25-50 mg Na/kg of soil. Hence the low to moderate Na supply appeared to
favour K partitioning to the shoot of wheat.
A second possible mechanism for increased K uptake is Na activation of K+ symporters in
roots. At low external Na+ and K+ concentrations, high-affinity K+ uptake transporters
function as Na+-K+ symporters, as demonstrated by Na+-stimulated K+ uptake and K+-
stimulated Na+ uptake, however, at high external Na+ concentrations, some of these
transporters become Na+ uniporters, no longer transporting K+ (Benito et al., 2014; Rubio et
al., 1995). However, in an experiment by Box and Schachtman (2000), there was no evidence
of enhanced K+ uptake in wheat due to Na supply, even though there was an increase in
wheat growth due to external Na+ i.e., according to them the positive effect of Na at low soil
K can be largely attributed to substitution of Na+ in wheat K functions and direct effect of
Na+ on growth. Box and Schachtman (2000) investigated the Na+ activated (activation of K+
symporters) K+ uptake only under low light conditions in wheat and concluded that it was
functionally a minor process for K+ uptake by wheat, indicating there may be effects of Na on
transporters not identified by them. Other mechanism for increased K+ uptake could be by a
low-affinity K+ uptake system (such as AKT) which at moderate salinity (20 mM NaCl in
53
barley) hyperpolarized the plasma membrane and increased K+ uptake via inward-rectifying
hyperpolarized-activated K+ channels (Chen et al., 2005; Shabala & Cuin, 2008).
A part but not the entire increase of K content could be attributed to the increase in
extractable soil K by soil Na supply. In the incubation experiment, soil exchangeable and
Colwell K showed a non-significant increase with addition of Na. However, for a pot with 6
kg of soil, the change in Colwell K was equivalent to around 18 mg in the 50 mg Na/kg
treatment and could have provided 6 mg of extra K+ to each plant in the 3-plant pots, which
would account for part of the increased shoot K content in the Na-added plants. From the
present results, it would be premature to conclude that Na stimulation of wheat growth in K-
deficient plants is unrelated to the increased K availability in soil. Interestingly, previous
studies on Na stimulation of plant growth in K-deficient plants did not consider increased K
uptake from soil as an explanation for the response: they focussed on Na substitution of K
functions.
As explained above, Na substitution of K in shoots of wheat in the present experiment was
unlikely because the shoot Na concentrations were too low to provide any significant
replacement of the osmotic effects of K in vacuoles or in other functions of K+. By contrast,
the increase in root Na+ was more than sufficient to replace osmotic functions of K+ in roots.
The increase in root Na concentration may stimulate root elongation of K-deficient plants
(Ali et al., 2009) by turgor effects on cell expansion. Whether an increase in root elongation
could contribute to increase root K uptake is unclear and there is no direct evidence in the
present study to address this question. Such an effect is more likely to be expressed in soil
where root elongation has a major role in determining nutrient uptake by providing access to
additional nutrient supply (Barber & Silberbush, 1984). It is unlikely the Na substitution of K
in roots would directly increase root growth because their dry matter increase would be
limited by inadequate assimilate supply to roots under low K supply. Hence, it is proposed
that the stimulation of root growth by low to moderate Na is mediated in shoots, probably by
increased photosynthesis leading to greater assimilate supply to roots. The increase in root
growth in turn could allow for increased K uptake by roots.
Potassium efficient cultivars were more salt tolerant than K-inefficient cultivars in the order:
Cranbrook> Wyalkatchem> Gutha> Gamenya, in terms of shoot dry weight (Genc et al.,
2007). However, K-efficient cultivars mostly had similar K+/Na+ ratio as K-inefficient
cultivars. This is consistent with an earlier study where K+/Na+ ratio did not explain the
variation in salt tolerance among wheat cultivars (Genc et al., 2007). In contrast, the ability of
54
plants to maintain a high K+/Na+ ratio was positively correlated with salt tolerance in other
studies (Chen et al., 2007; Cuin et al., 2009; Shabala & Cuin, 2008; Wu et al., 1996). Cuin et
al. (2008) emphasized that Na+ exclusion is not a sufficient tool for salt tolerance but the
ability of roots to retain K+ correlated better with salt tolerance in wheat. Moreover, a recent
study in wheat suggests that salt-tolerant cultivars have an enhanced ability to sequester Na+
into vacuoles of root cells, whereas in sensitive cultivars large quantities of Na+ are located in
the root cell cytosol (Cuin et al., 2011). In this study, cv. Cranbrook was least effective in
retaining root K under increasing Na supply among the cultivars. Cultivars may differ in the
extent of Na translocation to shoots. The substitution of K+ by Na+ in cereals is likely to be
influenced not only by plant K status, but also by the potential of the cultivar to accumulate
significant Na concentrations in their shoots, as emphasised for the salt tolerant barley cv.
CM72 (Ma et al., 2011), or in roots as with wheat in the present study.
In the present study, the K-use efficiency of wheat cultivars studied across a range of Na
levels from no added Na up to toxic levels was consistent with the ranking of cultivars for K-
use efficiency by Damon and Rengel (2007). However, there has been little information
reported on the role of Na supply in K-use efficiency in wheat. According to this study, K-
efficient cultivars Wyalkatchem and Cranbrook had higher response to low to moderate Na
supply relative to K-inefficient cultivars Gutha and Gamenya. In contrast to the suggestion by
Rengel and Damon (2008) that increased capacity to substitute Na+ for K+ may be a
mechanism underlying K-use efficiency in wheat, we found that Na stimulated greater K
uptake in K-efficient cultivars. The main mechanism identified by Damon and Rengel (2007)
for K efficiency in wheat cultivars like Wyalkatchem was greater utilization efficiency of
shoot K rather than greater K uptake. According to them K efficiency is a measure of
genotypic tolerance in low K soils and can be quantified as K efficiency ratio (ratio of growth
at deficient and adequate K supply). In the present study, there was greater K uptake by K-
efficient cultivars or greater K content in shoots with low to moderate Na supply. The
stimulation of photosynthesis, stomatal conductance and transpiration efficiency and root dry
weight were greater in the K-efficient cultivars. This is consistent with greater utilization
efficiency of shoot K in the K-efficient cultivars leading to greater photosynthesis and hence
roots dry weight response to shoot K. Given this explanation the weak responses in shoot dry
weight to low to moderate Na are surprising. There was alleviation of K deficiency symptoms
in old leaves by low to moderate Na. However, since the symptoms only appeared at 6 weeks
55
after sowing and the shoots were harvested at 8 weeks, it is possible that the shoot response
lagged behind that of roots and given more time would have been more substantial.
The stimulation of root growth to a greater extent than shoot growth in wheat by low to
moderate Na in low K plants may have greater significance when the crop is under stress in
the field than in the present well-watered pot experiment. There should be a direct benefit
from an increased root mass under drought stress particularly in K-deficient wheat for which
depressed root growth is a characteristic symptom (Ma et al., 2011; Ma et al., 2013).
3.5 Conclusion
In this experiment, wheat cultivars differing in K-use efficiency varied in response to soil K
and Na supply. When supplied with low to moderate Na under K deficiency, positive
responses in K uptake, leaf photosynthesis, stomatal conductance and root dry weight were
observed in all four cultivars, particularly in K-efficient cultivars. In contrast to previous
findings, we conclude that low to moderate Na stimulated increase in shoot K uptake by
wheat, which particularly in K-efficient cultivars promoted photosynthesis and root growth
and further access to soil K. In the present study, the shoot Na concentrations at low to
moderate Na supply to soil were too low to feasibly substitute for biophysical functions of K
in the shoot. Four mechanisms are proposed to explain the increased K uptake in shoots of
wheat by low to moderate Na supply, but further studies are needed to clarify the relative
contribution of each mechanism to the growth stimulation.
56
CHAPTER 4
SPLIT-ROOT EXPERIMENT
Moderate sodium increased K uptake, leaf gas exchange and plant growth of wheat cv.
Wyalkatchem grown in a K-deficient split-root system
4.1 Introduction
In saline soils, excessive NaCl has a detrimental effect on nutrition of most crops (Römheld
& Kirkby, 2010), and plant physiological functions are inhibited due to depression in
potassium (K+) uptake by competing sodium (Na+) ions (Blumwald et al., 2000). Potassium
and Na ions are similar in ionic radius and ionic hydration energies (Marschner, 1995), and
because of this chemical similarity, it is assumed that both these ions compete for common
adsorption sites in the roots (Subbarao et al., 2003). Potassium is required in high
concentrations for plant growth and development, whereas Na is beneficial to many
glycophytes as well as to certain halophytes in relatively low concentrations (Greenway &
Munns, 1980; Mäser et al., 2002).
Despite the fact that high Na is detrimental to plant growth and K nutrition, low to moderate
Na was reported to have beneficial effects in some plant species, especially when K is present
at suboptimal concentrations (Ma et al., 2011). Plants can utilize Na+ ions in several key
cellular processes as long as the concentrations remain less than osmotically challenging
levels (Kronzucker et al., 2013). Sodium can help maintain cell turgor, ionic balance, regulate
osmotic pressure, and improve water relations via stomatal conductance (Subbarao et al.,
2003). It has been shown that under K deficiency, low external Na+ could substitute as an
enzyme activator or as a vacuolar solute by releasing vacuolar K+ to fulfil its functions in
cytoplasm (Walker et al., 2000).
The beneficial effects of Na on some natrophilic plant species are well documented
(Marschner, 1995), but there is only limited research available on cereal crops especially in
soil-based systems (Ma et al., 2011; Miyamoto et al., 2012). Growth stimulation due to added
Na at deficient soil K supply was observed in rice (Miyamoto et al., 2012; Yoshida &
Castaneda, 1969), barley (Ma et al., 2011) through solution/sand culture studies under
controlled conditions or in field studies. Studies on wheat reported insignificant or limited
effects on growth due to low to moderate Na supply under K deficiency in a soil-based
system (Ma et al., 2011) but significant growth stimulation when low concentrations of Na
57
(500 µM Na+) were added to K-deficient nutrient solution (20 µM external K) (Box &
Schachtman, 2000).
An earlier pot experiment with four wheat cultivars (Chapter 3) suggested that addition of
low to moderate Na (25 to 50 mg Na/kg) to low K soil (40 mg K/kg) had beneficial effects on
plant growth (especially roots), leaf gas exchange and plant K uptake. The implications of
varying K and Na distribution in a soil profile for wheat K nutrition, and the extent of Na
substitution in K functions have not been examined previously. The main objective of this
experiment was to study whether the beneficial effect of Na depends on both K and Na being
present at same part of the root system in wheat. A split-root experiment was conducted,
where the root system was divided into two halves and treated with different combinations of
K and Na supply, to investigate how shoot and root growth, and concentrations and uptake of
K and Na in wheat were affected by varying combinations of K and Na between the
compartments.
4.2 Materials and methods
Wheat (Triticum aestivum L.) cv. Wyalkatchem was grown in a naturally-lit glasshouse at
Murdoch University, Perth (32°04′S, 115°50′E) from mid winter to early spring. Soil
(classified as ‘Chromosols’) was collected from an unfertilised paddock east of Dowerin, and
had the following properties: pH (CaCl2) 4.9; EC1:5 0.03 dS/m; 7 mg NH4-N/kg and 8.7 mg
NO3-N/kg (Searle, 1984); < 15 mg K/kg and 29 mg P/kg (Colwell, 1963); organic C 0.17%
(Walkley & Black, 1934). A split root system was used by joining two undrained plastic pots
(length and breadth 110 mm, depth 140 mm) together, filling each with sieved soils (< 2 mm)
with a mixture of basal nutrients and individual treatments of K and Na into the two adjoining
compartments containing 2 kg soils/compartment. Two notches were made in the adjoining
walls to facilitate planting of two plants per split root system. The level of soil was kept 2 cm
below the top edge of the compartments to avoid soil contamination between the two
compartments. Basal nutrients were applied at the following rates (mg/kg): 237
Ca(NO3)2.4H2O, 103 (NH4)2HPO4, 80 MgSO4.7H2O, 18 FeSO4.7H2O, 14 MnSO4.H2O, 9
ZnSO4.7H2O, 8.3 CuSO4.5H2O, 0.33 H3BO3, 0.3 CoSO4.7H2O, 0.33 Na2MoO4.2H2O. Wheat
seeds were first sown in potting mix in germination trays and after 10 days, two uniform
seedlings were transplanted into each split-root system with the seminal roots being evenly
divided between the two compartments. The pots were watered to field capacity (15% w/w)
with de-ionised water throughout the experiment and were re-arranged every week to reduce
positional effects on the plant growth.
58
4.2.1 Potassium and sodium treatments
Two levels of soil K were used in this experiment: 40 mg K/kg (low) and 100 mg K/kg (high)
using KCl. Each soil K level was combined with three soil Na levels applied to one or two of
the compartments: 0, 50 and 200 mg Na/kg in a total of 11 split-root treatments, as listed in
Table 4.1. When potting, K and Na treatments were thoroughly mixed with basal nutrients in
a rotary mixer, and each split-root treatment was replicated four times.
Table 4.1 Split- root treatments experimental design
Treatment Compartment/ Side A (mg/kg) Compartment/ Side B (mg/kg)
soil K soil Na soil K soil Na
1 40 0 40 0
2 40 0 40 50
3 40 50 40 50
4 40 0 40 200
5 40 200 40 200
6 40 0 100 0
7 100 0 100 0
8 100 0 100 50
9 100 50 100 50
10 100 0 100 200
11 100 200 100 200
4.2.2 Measurements
The plants were harvested 6 weeks after being transplanted into the split-root system. The
development of leaves and tillers was recorded weekly throughout the experiment. Before
harvest, leaf net photosynthesis, stomatal conductance and transpiration rate were measured
using an LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The
measurements were made in fully expanded young leaves at ambient relative humidity of 50
%, leaf temperature of 25˚C, reference CO2 of 380 µmol/mol, and photosynthetically active
radiation of 1500 µmol/m2·s1.
59
At harvest, the shoots were cut at the soil surface and shoot fresh weight was recorded
immediately. Roots were collected from individual compartments of the split-root system
after being washed in tap water and rinsed with DI water. The shoot and root samples were
dried in a forced-draught oven at 60˚C for 48 hours and the dry weights were recorded. The
dried samples were milled for K and Na analysis. Samples of 50- 200 mg were weighed into
50 mL centrifuge tubes and digested in 5 mL of 70% (w/w) HNO3 at 75˚C for 10 min, and
then at 109˚C for 15 min, and after samples were cooled, 1 mL of 30% (w/w) H2O2 was
added and further digested at 109˚C for 15 min. The digestion was made in a micro-wave
(CEM Mars 5, manufactured by CEM Corp., USA) for cation analysis (Huang et al., 2004).
The samples were then diluted with milli-Q water and the concentrations of K and Na were
determined by flame photometer (Model 410, Sherwood Scientific Ltd, UK).
4.2.3 Statistical analysis
Statistical analyses were conducted using the statistical program SPSS 18.0. One-way
analysis of variance was conducted to assess the effects of split-root treatments. Tukey’s
HSD was computed at P ≤ 0.05 for comparing the means of the 11 split-root treatments and
the specific responses between the two compartments were compared within each split-root
treatment.
4.3 Results
4.3.1 Plant growth
Low soil K (40 mg K/kg) in combination with nil soil Na in both compartments had the
lowest shoot growth. Severely restricted growth was also observed in treatments which had
low soil K and high Na (200 mg Na/kg) either in one or both compartments (Table 4.2). Low
soil K in combination with moderate Na (50 mg Na/kg) in one compartment significantly
increased shoot growth and tiller production relative to low K in combination with nil Na. At
low soil K, the addition of moderate Na to either one or both compartments increased the
shoot dry weight on average by 8.2 and 11 times, respectively, compared to low K treatment
without Na in both compartments. Interestingly, at low K supply, the presence of moderate
Na in both compartments resulted in significantly higher shoot dry weight than adequate K
combined with nil Na in one compartment only (Table 4.2). The effect of adequate K without
Na in both compartments was similar to low K with moderate Na in both compartments. At
high soil K supply (100 mg K/kg), the presence of moderate Na in both compartments
60
reduced shoot dry weight, with further reduction in shoot growth and tiller production at high
Na (Table 4.2).
Table 4.2 Shoot dry weight (g), and number of tillers per plant of wheat cv. Wyalkatchem
treated with two levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg
Na/kg) combined in 11 different split-root systems. Means (n=4) in a column with different
letters differ at P≤0.05.
Treatment Side A (mg/kg) Side B (mg/kg) Shoot dry weight
(g/plant) Tillers
soil K, Na soil K, Na
1 40, 0 40, 0 0.10f 1.00e
2 40, 0 40, 50 0.86abc 4.87bc
3 40, 50 40, 50 1.16a 6.00ab
4 40, 0 40, 200 0.15f 1.75de
5 40, 200 40, 200 0.28ef 1.75de
6 40, 0 100, 0 0.61cde 3.62bcde
7 100, 0 100, 0 1.05ab 8.00a
8 100, 0 100, 50 0.95abc 6.12ab
9 100, 50 100, 50 0.75bcd 4.75bc
10 100, 0 100, 200 0.70bcd 4.12bcd
11 100, 200 100, 200 0.40def 2.87cde
Root growth showed similar response to K and Na treatments as shoot growth. Low soil K in
combination with moderate soil Na in both compartments resulted in the highest root dry
weight among all the treatments, followed by high soil K with moderate Na in both
compartments (Table 4.3). Low K supply when combined with moderate Na in one
compartment stimulated root growth significantly relative to root growth in the adjoining
compartment with nil soil Na. At nil soil Na, root growth was significantly greater in the
adequate K compartment than the adjoining low K compartment (Treatment 6; Table 4.3),
but root growth was largely uniform between the two compartments when K supply was the
61
same. Unlike shoot growth, the presence of moderate Na in both compartments stimulated
root growth at high soil K. However, the presence of high Na (200 mg Na/kg) in both
compartments reduced root growth regardless of soil K levels.
Root: shoot ratios did not show a particular pattern among the treatments (Table 4.3). The
ratio was higher when there was moderate soil Na in both compartments regardless of soil K
levels. Presence of high soil Na in both compartments also increased root: shoot ratios,
mainly due to poor shoot growth.
Table 4.3 Total root dry weight (g) per plant and their root: shoot ratios of wheat cv.
Wyalkatchem in the split- root systems consisting of two K levels (40 and 100 mg K/kg) and
three Na levels (0, 50 and 200 mg Na/kg). One-way analysis of variance was conducted to
assess the effects of split-root treatments. Tukey’s HSD was computed at P ≤ 0.05 for
comparing the differences in total root dry weight and root: shoot ratios between the 11 split-
root treatments and the specific root responses between the two compartments were
compared within each split-root treatment. Means (n=4) with different letters differ at
P≤0.05.
Treatment Side A (mg/kg) Side B (mg/kg) Root dry weight (g) Root: Shoot
soil K, Na soil K, Na Side A Side B Total
1 40, 0 40, 0 0.02x 0.02x 0.04e 0.41bc
2 40, 0 40, 50 0.10y 0.19x 0.29cd 0.34bc
3 40, 50 40, 50 0.34x 0.32x 0.66a 0.57abc
4 40, 0 40, 200 0.03x 0.01y 0.04e 0.30c
5 40, 200 40, 200 0.06x 0.11x 0.17de 0.59abc
6 40, 0 100, 0 0.06y 0.11x 0.18de 0.30c
7 100, 0 100, 0 0.16x 0.17x 0.33cd 0.32c
8 100, 0 100, 50 0.26x 0.23x 0.49abc 0.51abc
9 100, 50 100, 50 0.26x 0.31x 0.57ab 0.77a
10 100, 0 100, 200 0.20x 0.15x 0.35bcd 0.50abc
11 100, 200 100, 200 0.12x 0.12x 0.25de 0.64ab
62
4.3.2 Leaf gas exchange
Low K in combination with nil soil Na in both compartments had the lowest leaf net
photosynthesis among the treatments (Table 4.4). Leaf net photosynthesis increased when
moderate Na was added at least in one compartment with low K supply and the increase was
almost similar to nil Na with adequate soil K (100 mg K/kg) in one compartment. Regardless
of soil K levels, an addition of high Na (200 mg Na/kg) in both compartments reduced
photosynthesis rate considerably. The response of stomatal conductance (Gs) in the leaves
showed similar trends as net photosynthesis rate (Pn). Stomatal conductance and transpiration
rate of wheat supplied with adequate soil K were higher than low soil K plants. The addition
of moderate Na in one compartment of low soil K split-root system increased transpiration
rate significantly compared to low K treatment without soil Na.
Table 4.4 Leaf net photosynthesis rate (Pn), stomatal conductance (Gs) and transpiration (E)
of wheat cv. Wyalkatchem treated with two levels of K (40 and 100 mg K/kg) and three
levels of Na (0, 50 and 200 mg Na/kg) combined in 11 different split-root systems. Means
(n=4) with different letters differ at P≤0.05.
Treatment Side A (mg/kg)
Side B (mg/kg)
Pn Gs E
soil K, Na soil K, Na µmol CO2/m2.s mmolH2O/m2.s mmolH2O/m2.s
1 40, 0 40, 0 4.49d 59.3c 1.15b
2 40, 0 40, 50 14.6ab 169ab 3.14a
3 40, 50 40, 50 16.3a 167ab 3.14a
4 40, 0 40, 200 11.2bc 198a 2.74ab
5 40, 200 40, 200 6.09d 96.9bc 2.11ab
6 40, 0 100, 0 15.2ab 219ab 3.47a
7 100, 0 100, 0 17.1a 224a 3.70a
8 100, 0 100, 50 16.7a 220a 3.40a
9 100, 50 100, 50 16.6a 218a 3.25a
10 100, 0 100, 200 12.0bc 177ab 2.57ab
11 100, 200 100, 200 8.37cd 154ab 2.58ab
63
4.3.3 K and Na concentrations and accumulation
Shoot K concentrations did not correlate well with soil K supply alone. At low soil K, plants
with nil soil Na in both compartments had highest shoot K concentration, followed by low K
with high soil Na in one or both compartments (Table 4.5). With adequate soil K supply,
moderate Na significantly reduced shoot K concentration, but high Na even in one
compartment increased shoot K concentration mainly due to a concentration- effect.
In contrast to shoot K concentrations, root K concentrations correlated better with soil K
levels. Root K concentrations were higher in adequate soil K treatments than low K. At low
soil K, root K concentration was lowered in wheat grown with high soil Na in one
compartment and nil Na in both compartments (Table 4.5). The root K concentrations were
similar between the two compartments when there was low soil K in both compartments
irrespective of soil Na levels. At low soil K, addition of moderate Na in one compartments
increased root K concentration in the adjoining compartment as well. However, the
compartment with adequate K and nil Na had significantly higher root K concentration than
adjoining compartment of low K without Na or adequate K with high Na (Table 4.5).
Plants grown in adequate K without Na in both compartments accumulated a significantly
higher amount of K than other treatments (Fig. 4.1). At low soil K, addition of moderate Na
to one or both compartments increased plant K content significantly compared to low K
treatments with nil or high soil Na. At adequate K level, addition of moderate Na decreased K
accumulation with further reductions at high soil Na.
Shoot and root Na concentrations in wheat increased with increase in soil Na concentrations
(Table 4.6). Low K in combination with 200 mg Na/kg in both compartments had the highest
shoot and root Na concentrations. Adequate K in combination with nil Na in both
compartments had the lowest shoot and root Na concentrations. The addition of moderate and
high Na, irrespective of soil K levels increased the shoot Na concentrations. There was
significant difference in root Na concentrations between the compartments of the split-root
system, and the roots grown in the compartment with high soil Na had higher Na
concentration than the adjoining compartment (Table 4.6).
64
Table 4.5 Shoot and root K concentrations (mg K/g) of wheat cv. Wyalkatchem in the split-
root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50 and
200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the differences in
shoot K concentrations between the 11 split-root treatments and the specific root K
concentrations between the two compartments were compared within each split-root
treatment. Means (n=4) with different letters in a column differ at P≤0.05.
Treatment Side A (mg/kg)
Side B (mg/kg)
Shoot K
(mg K/g, dry wt) Root K (mg K/g, dry wt)
Soil K, Na Soil K, Na Side A Side B
1 40, 0 40, 0 51.8a 2.40x 3.12x
2 40, 0 40, 50 28.4d 11.8x 11.5x
3 40, 50 40, 50 16.1e 10.8x 11.9x
4 40, 0 40, 200 45.8ab 3.61x 1.42x
5 40, 200 40, 200 34.8cd 7.00x 7.78x
6 40, 0 100, 0 41.4bc 16.4y 22.8x
7 100, 0 100, 0 40.3bc 22.9x 19.5x
8 100, 0 100, 50 32.0d 24.6x 22.4x
9 100, 50 100, 50 30.3d 22.5x 21.7x
10 100, 0 100, 200 34.1cd 20.8x 16.8y
11 100, 200 100, 200 43.7ab 16.6x 15.4x
65
Potassium uptake per plantK
up
take
(m
g/p
lant
)
0
20
40
60
80
100
120
140
K uptake
side A: K- 40 40 40 40 40 40 100 100 100 100 100 Na- 0 0 50 0 200 0 0 0 50 0 200
side B: K- 40 40 40 40 40 100 100 100 100 100 100 Na- 0 50 50 200 200 0 0 50 50 200 200
P=0.000HSD0.05=43
Fig. 4.1 Potassium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two
levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined
in 11 different split-root systems. (±SE, n=4).
66
Table 4.6 Shoot and root Na concentrations (mg Na/g) of wheat cv. Wyalkatchem in the
split- root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0,
50 and 200 mg Na/kg). Tukey’s HSD was computed at P ≤ 0.05 for comparing the
differences in shoot Na concentrations between the 11 split-root treatments and the specific
root Na concentrations between the two compartments were compared within each split-root
treatment. Means (n=4) with different letters differ at P≤0.05.
Treatment Side A (mg/kg)
Side B (mg/kg)
Shoot Na
(mg Na/g, dry wt) Root Na (mg Na/g, dry
wt)
Soil K, Na Soil K, Na Side A Side B
1 40, 0 40, 0 1.29c 0.502x 0.50x
2 40, 0 40, 50 1.96c 5.15y 8.58x
3 40, 50 40, 50 2.39c 10.6x 10.5x
4 40, 0 40, 200 17.3ab 5.16y 24.4x
5 40, 200 40, 200 19.4a 17.82x 15.7x
6 40, 0 100, 0 0.15c 1.03x 0.69x
7 100, 0 100, 0 0.08c 0.42x 0.38x
8 100, 0 100, 50 1.69c 2.69y 9.80x
9 100, 50 100, 50 4.74c 11.1x 10.5x
10 100, 0 100, 200 5.12c 3.40y 14.0x
11 100, 200 100, 200 12.7b 12.03x 12.6x
Wheat accumulated the highest Na when grown in the treatment with low K and moderate Na
(50 mg Na/kg) in both compartments, followed by adequate soil K and 50 mg Na/kg in both
compartments (Fig. 4.2). There was no significant difference in plant Na content between the
treatments with low and adequate soil K.
67
Sodium uptake per plantN
a up
take
(m
g/p
lant
)
0
20
40
60
80
100Na uptake
side A: K- 40 40 40 40 40 40 100 100 100 100 100 Na- 0 0 50 0 200 0 0 0 50 0 200
side B: K- 40 40 40 40 40 100 100 100 100 100 100 Na- 0 50 50 200 200 0 0 50 50 200 200
P=0.000HSD0.05=12.6
Fig. 4.2 Sodium uptake/ plant (shoot+ root) of wheat cv. Wyalkatchem treated with two
levels of K (40 and 100 mg K/kg) and three levels of Na (0, 50 and 200 mg Na/kg) combined
in 11 different split-root systems. (±SE, n=4).
The K+/Na+ ratios of whole plants decreased with increase in soil Na concentrations. The
treatments with nil Na added to either of the compartments had high K+/Na+ ratios. Wheat
grown in adequate K without Na in both compartments had significantly higher K+/Na+ ratio
than the other treatments (Table 4.7). With addition of 200 mg Na/kg, there was more than 50
fold reduction in K+/Na+ ratio.
68
Table 4.7 K/Na ratios of wheat (whole plant) cv. Wyalkatchem were compared between the
split-root systems consisting of two K levels (40 and 100 mg K/kg) and three Na levels (0, 50
and 200 mg Na/kg). Means (n=4) with different letters differ at P≤0.05.
Treatment Side A (mg/kg) Side B (mg/kg) K/Na ratio
soil K, Na soil K, Na
1 40, 0 40, 0 26.3bc
2 40, 0 40, 50 3.33c
3 40, 50 40, 50 1.66c
4 40, 0 40, 200 1.09c
5 40, 200 40, 200 0.98c
6 40, 0 100, 0 53.0b
7 100, 0 100, 0 109a
8 100, 0 100, 50 5.70c
9 100, 50 100, 50 2.80c
10 100, 0 100, 200 3.30c
11 100, 200 100, 200 2.03c
4.4 Discussion
Sodium can be beneficial or toxic, depending on plant species, cultivar and levels of Na+ and
K+ in the root medium. The effects of Na toxicity in saline soils have received much more
attention than the benefit from low to moderate Na. In some C4 plant species, Na is
considered as a ‘functional-nutrient’ (Subbarao et al., 2003) where it can stimulate
photosynthesis and Na+-coupled trans-membrane transport (Murata & Sekiya, 1992). In non-
C4 plants, Na can have beneficial effects in some plant species, even though it is not essential
for growth (Gattward et al., 2012; Terry & Ulrich, 1973), and there is evidence that Na+ can
replace some K+ functions under K deficiency (Ali et al., 2006; Subbarao et al., 2003; Walker
et al., 2000). The previous pot experiment showed Na was beneficial to wheat cultivars
grown under K deficient conditions (Chapter 3). The role of K+ in minimising the effects of
Na+ toxicity has been extensively researched in wheat (El-Lethy et al., 2013; Zheng et al.,
69
2008). However, the possibility of Na+ in substituting for K+ functions in wheat grown under
K deficiency has not been thoroughly examined. This study demonstrated moderate Na (50
mg/kg)- induced increased plant K uptake, increased leaf gas exchange, and increased growth
of K deficient wheat cv. Wyalkatchem in a split-root set up. Relative to the stimulation of
growth in wheat cv. Wyalkatchem in Chapter 3, the growth response here was much more
striking and included a strong shoot response in contrast to the effects reported in Chapter 3.
In this experiment, plants grown under low soil K without Na in both compartments showed
severe K deficiency symptoms with old leaf necrosis, stunting and drooping of plants (Gierth
& Mäser, 2007). With moderate NaCl addition, the leaves were erect, healthier and did not
show any deficiency symptoms (Fig. 4.3). The observations suggest that Na can be a partial
replacement for K responsible for maintaining turgidity in wheat cells particularly when K
supply is limited. Similar beneficial effect due to added Na was noticed in rice grown at low
K concentrations (Yoshida & Castaneda, 1969). In this experiment, shoot Na concentrations
with 50 mg Na/kg addition to one or both low K compartments did not exceed 2.5 mg Na/kg
suggesting there was not enough shoot Na to replace K functions while root Na
concentrations increased significantly with moderate Na. However, shoot Na content
increased significantly from 0.14 mg Na/plant at nil soil Na to 1.8 mg Na/plant and 3.0 mg
Na/plant with addition of 50 mg Na/kg to one and both the compartments, respectively at low
soil K (40 mg K/kg).
The response of wheat in tiller production and shoot growth to addition of moderate Na (50
mg Na/kg) in either one or both compartments of the split-root system was comparable to that
at 100 mg K/kg supply. This finding suggests that Na addition to K deficient medium
stimulated shoot growth in wheat regardless of the presence of K and Na in same or different
parts of the root system.
70
Fig. 4.3 Wheat (cv. Wyalkatchem) at six weeks after transplanting grown in a split-root
system treated with 40 mg K/kg and nil, or 50 mg Na/kg. The image shows the growth
difference with and without Na addition (50 mg Na/kg).
At low soil K, addition of moderate Na even to one of the compartments stimulated root
growth and also boosted root growth in the adjoining compartment with nil Na. Interestingly,
moderate Na addition to both compartments stimulated root growth at adequate soil K by 70
% when compared with nil Na treatment, and root: shoot ratio was also significantly higher.
Moderate salt (50 mg Na/kg) was beneficial to root development in wheat even when there
was adequate soil K. The stimulation of root growth by 50 mg Na/kg was in contrast to a
recent study by Ma et al. (2011) who reported adverse effects of soil 100 mg Na/kg on root
growth in wheat (cv. Wyalkatchem) at either deficient or adequate K supply. Soil Na
concentration at which Na stimulated wheat root growth in this experiment was in range of
Na levels (25 to 50 mg Na/kg) found to stimulate root growth in the earlier (Chapter 3)
experiment. Interestingly, NaCl induced growth stimulation in the split-root system of this
study was considerably higher than that previously measured in wheat (Box & Schachtman,
2000; Ma et al., 2011). In the previous pot experiment, the shoot dry weight showed no
71
significant increase while root dry weight of K-efficient cultivars in particular increased
significantly with low to moderate Na added to K-deficient soils (Chapter 3). Although,
similar low K treatment of 40 mg K/kg was used in both the experiments, and root to soil
mass ratio was almost the same (1: 2.1 in pot and 1: 2.0 in split-root), the K deficiency
symptoms were seen earlier (3- 4 weeks after transplanting) and severe in the split-root
experiment with K-efficient Wyalkatchem, and hence there appeared to be a stronger shoot
and root growth stimulation due to Na.
There was a significant increase in leaf gas exchange in the presence of moderate Na in one
or both compartments at low soil K supply, compared with nil Na plants which showed
obvious K deficiency symptoms, e.g. yellow and droopy leaves. At low K, moderate Na
increased net photosynthesis, stomatal conductance and transpiration rate in leaves by more
than three times, and the results was comparable to that by adequate K. Researchers suggest
that Na is beneficial in non-specific ionic roles in cell vacuoles, as an osmoticum at deficient
K (Marschner, 1995; Subbarao et al., 2003). Moreover, the presence of moderate Na at one or
both compartments at low K would be beneficial to physiological properties like stomatal
conductance which could improve plant water relations and growth (Ma et al., 2011).
However, non-specific ionic roles of Na in cell vacuoles would not explain increased net
photosynthesis with added Na at low K supply.
High shoot K concentrations at low K in combination with nil Na at both compartments and
high Na were mainly due to the ‘concentration- effect’ as a result of growth reduction since in
these treatments plant K accumulation was considerably lower than other treatments. Shoot
dry weight at low soil K supply was inversely correlated to shoot K concentration (Fig. 4.4).
Wheat grown in low K and nil Na compartments had adequate shoot K, however, there was
very low K in roots (~0.3 %) which would have restricted root growth and hence access to
nutrients and water due to poorly developed root systems.
72
Fig. 4.4 Correlation between shoot dry weight/plant (g) harvested at 6 weeks after
transplanting and the shoot K concentration (mg K/g, dry weight) measured in low soil K (40
mg K/kg) split-root treatments.
The results showed that K uptake at low soil K was significantly stimulated by addition of
moderate Na (50 mg Na/kg equivalent to ~15 mM Na in soil solution) either to one or both
compartments. A similar increase in K uptake due to Na was noticed by other researchers
(Idowu & Aduayi, 2007; Walker et al., 2000; Zhang et al., 2006). In tomato, however, shoot
K uptake was increased at much lower soil Na concentration of 4 to 16 mg Na/kg, and it
decreased at 32 mg Na/kg (Idowu & Aduayi, 2007), and in solution culture experiment, there
was increase in plant K uptake with 1 or 5 mM Na addition (Walker et al., 2000) and in
cotton field experiment, there was increase in K uptake from soil when topsoil Na
concentration was 65 mg Na/kg (Zhang et al., 2006). Increased K uptake in wheat cv.
Wyalkatchem was found to be consistent with previous experiment where there was
significant increase in shoot K uptake in K-efficient cultivars Wyalkatchem and Cranbrook
with low to moderate Na addition (Chapter 3).
The increase in K uptake in this study could be due to two main possibilities: firstly, an
increase in plant-availability of soil K due to added soil Na and secondly, increased K uptake
due to K uptake transporters. A soil incubation study with the present soil showed the change
in exchangeable K was not significant (Chapter 3). For a split-pot with total 4 kg of soil, an
y = -26.498x + 48.923R=-0.89
R² = 0.7896
0
10
20
30
40
50
60
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Sho
ot K
con
cent
ratio
n (m
g/g)
Shoot dry weight/plant (g)
Correlation made in low K (40 mg K/kg) and three Na (0, 50 and 200 mg Na/kg) split-root treatments
Shoot dry weight vs Shoot K concentrations
73
increase of 12 mg Colwell K with 50 mg Na/kg could provide an extra 6 mg K per plant
which could at best explain only a part of increase in K uptake due to soil Na.
One of the high affinity K+ uptake mechanisms is a Na+- energized high- affinity K+ symport
HKT1 (Schachtman & Liu, 1999), and it may play an important role in K+ acquisition when
external Na+ concentrations are low (Box & Schachtman, 2000; Rubio et al., 1995). The
capacity to take up Na+ in soils at low K supply may be an evolutionary advantage that plants
have developed, and molecular approaches have identified high-affinity K+ and Na+ transport
systems for various species including rice, barley and wheat (Rodriguez-Navarro & Rubio,
2006). It was claimed that high-affinity K+ uptake was activated at micromolar Na+
concentrations, while at physiologically detrimental concentrations of Na+, K+ uptake
mediated by HKT1 was blocked and low-affinity Na+ uptake occurred, which correlated to
Na+ toxicity in plants (Rubio et al., 1995). However, recent evidence suggests that HKT
select Na+ over K+ and functions as a Na+ uniporter (Benito et al., 2014). The role of low-
affinity K+ uptake mechanism is also been suggested to increase K+ uptake at low external
Na+ concentrations, however detailed studies to directly demonstrate this mechanism are
lacking (Shabala & Cuin, 2008).
Wheat roots concentrated more Na than shoots in this experiment with Na addition which
was consistent with an earlier experiment (Chapter 3). In the split-root experiment, there was
translocation of Na to the nil Na compartment from adjoining compartments with Na, as the
roots in nil Na had significantly higher Na concentration when there was Na in adjoining
compartment than roots grown in nil Na in both compartments (Table 4.6). However, the
roots grown in the Na compartment had significantly higher Na concentration than those in
the adjoining compartment without Na. Since Na is classified as a mobile element in plant
phloem (Marschner, 1995), the presence of Na+ could activate K+ uptake channels and
transporters even in the adjoining compartments without Na. In plants, the pathways of Na
entry into plants are not widely researched (Kronzucker & Britto, 2011) and highly Na+-
selective channels have not been found (Schachtman & Liu, 1999). It is believed that because
of thermodynamics and interactions between K and Na uptake, it is possible that Na+ enters
the cell cytoplasm through K+ channels, and transport proteins like HKT1, KUP or HAK,
NSC (Non-selective cation channels), LCT (low-affinity cation transporter) transports both
K+ and Na+ into the plants (Kronzucker & Britto, 2011; Schachtman & Liu, 1999).
In this experiment, there was inhibition of wheat growth at low soil K without soil Na. The
beneficial role of Na+ in cv. Wyalkatchem was evident with significant increase in plant K+
74
uptake, leaf gas exchange measurements and plant growth. Although, moderate soil Na
addition did not increase shoot Na concentration to levels that could replace K+ functions,
there was significant increase in wheat K+ uptake due to added Na+. This increased shoot K
uptake may explain the increased leaf net photosynthesis rate and stomatal conductance with
Na addition and could promote carbon assimilate supply to roots which in turn could
stimulate root growth, and access to more soil K, water and other nutrients from soil.
Moreover, the addition of moderate rates of Na+ at deficient soil K supply has stimulatory
effects on roots and shoots, irrespective of whether K and Na are present in the same or
different parts of the roots.
Further detailed investigation in solution culture is warranted on whether the presence of low
external concentrations of Na+ increases K+ uptake in wheat under K deficiency. Such
information would be of greater use to understand K and Na interactions and uptake
particularly because the effect of Na on exchangeable K availability in growing media would
be eliminated and using a tracer like Rb+ in nutrient solution could help test effects of
external Na on K+ uptake more accurately.
4.5 Conclusion
Moderate Na level (50 mg Na/kg) was beneficial for wheat cv. Wyalkatchem grown under K
deficiency (40 mg K/kg) and Na stimulation of growth by low-K plants occurred regardless
of the supply of K and Na in the same or different parts of the root system. Moderate Na
eliminated the effects of low soil K supply on shoot and root growth and produced dry matter
similar to adequate K supply. There was significant increase in leaf photosynthesis, stomatal
conductance and plant K uptake due to moderate Na addition, but probably mediated by
increased shoot K uptake. A detailed solution culture based study using tracers can help to
validate Na+-induced K+ uptake in wheat cultivars more directly (Chapters 6 and 7).
75
CHAPTER 5
COLUMN EXPERIMENT
Potassium response of wheat grown in columns with drying topsoil and varying subsoil
K and Na levels
5.1 Introduction
Large agricultural areas of the world are reported to be K deficient and the problem is
exacerbated by continued removal of grain and straw from crop fields and this is limiting
cereal production (Ma et al., 2011). Potassium concentrations measured in topsoils of south-
west Western Australia (SWA) showed 55 % of samples had Colwell K < 100 mg/kg, 26 %
had < 40 mg/kg and 11 % had < 20 mg/kg (McArthur, 1991). Moreover, there is an
increasing demand for K fertiliser usage due to the expansion of intensive agriculture
(Römheld & Kirkby, 2010; Zörb et al., 2014). The annual growth rate of the global demand
for K fertilisers is estimated to be 3.8 % for the period 2011- 2014 and, the cost for K
fertilisers is increasing as the reserves are concentrated in a few countries (Miyamoto et al.,
2012). It is therefore important to increase the efficiency of K fertiliser use by the crops
without decreasing crop yields.
Sodium is not an essential element for all higher plants, however, application of Na+ was
found to be beneficial in some cellular functions when K is present at suboptimal
concentrations (Ma et al., 2011; Miyamoto et al., 2012; Subbarao et al., 2003). It is believed
that Na+ could substitute for K+ in its role as an enzyme activator in vacuoles, and its
accumulation could release K+ to fulfil other more specific biochemical functions, reducing
K+ requirements (Rodriguez-Navarro & Rubio, 2006; Walker et al., 2000). The extent of K
substitution and growth stimulation by Na are of great interest for crop production and K
fertiliser management (Mäser et al., 2002). The use of Na by plants may be particularly
important in saline-sodic soils where K+ and Na+ compete for uptake (Box & Schachtman,
2000). My previous glasshouse experiments (Chapter 3 and 4) suggest that Na at low to
moderate concentrations are beneficial to wheat root growth under K deficient conditions and
alleviated leaf symptoms of K deficiency.
Duplex/ texture-contrast soils account for about 12 % of the land area of Australia (Dracup et
al., 1992) and are a major soil constraint for crop production. The nutrient distribution in
duplex soils varies with depth in contrast to that of uniform soil profiles. The duplex soils of
western Australia (WA) have varying concentrations of K with depth due to differences in
76
clay content and mineralogy (Wong et al., 2000). For many duplex soils in WA, both surface
and subsoil layers are low in extractable K, as the dominant clay mineral is kaolinite, which
naturally has a low K content (Brennan & Bell, 2013).
In the no-till farming systems, K can be stratified within the fertilised topsoil. It is common
also for profiles with low K to contain significant exchangeable Na in the subsoil (Q. Ma and
R. Bell, unpublished data). In SWA, varying levels of subsoil K and Na from that of topsoil
are common in the duplex soil, but the influence of such heterogeneity on crop K nutrition
has not been examined. Topsoil drying/ drought are also common occurrences during the
growing season in rain-fed conditions of SWA. The presence of shallow and localised K may
limit plant K uptake in such water-limited environments since the topsoil is prone to drying,
because water deficit reduces not only root growth but also K diffusion. Understanding K
response under such conditions can help to manage K application strategies and can increase
grain productivity in rainfed cropping systems.
A column experiment was conducted to investigate the effect of subsoil Na and K on K
substitution by Na and crop K nutrition when K uptake from the topsoil is restricted by water
deficit. In this study, wheat was grown in soil columns with different K levels in the topsoil
but low to moderate Na supply in the subsoil. The potential interaction of subsoil Na and K
was assessed under the condition of dry topsoil. Potassium deficiency has detrimental effects
on wheat root development when compared to shoots (e.g. Ma et al. (2011)). In previous
experiments, addition of moderate levels of Na (25 and 50 mg K/kg) stimulated root growth
significantly under K deficient conditions (Chapters 3), and the beneficial effect was evident
even when K and Na were present at different parts of the root system (Chapter 4). It was
hypothesised based on previous experiments (Chapters 3 and 4) that subsoil Na may make a
positive contribution to growth (root growth) when plants have limited K supply. The
hypothesis was investigated under well-watered and topsoil dry conditions.
5.2 Materials and methods
Wheat (Triticum aestivum L.) cv. Wyalkatchem was grown in a naturally-lit glasshouse at
Murdoch University, Perth, (32°04′S, 115°50′E) from mid winter to early spring. Soil was
collected from an unfertilised paddock at Northam (classified as Chromosols), and had the
following properties: pH (CaCl2) 5.2; EC1:5 0.03 dS/m; 3.56 mg NH4-N/kg and 4.8 mg NO3-
N/kg (Searle, 1984); 20 mg K/kg, 2.5 mg S/kg and 1.2 mg Al/kg; 0.37 % organic C (Walkley
& Black, 1934). The air-dried soil was thoroughly mixed with basal nutrients and treatments
77
of K and Na and then filled into specially structured PVC columns (about 10 kg/column).
Each column (80 cm depth and 10 cm diameter) was formed of two halves of a PVC cylinder
that was split vertically and held together with adhesive tape. Basal nutrients were applied at
the following rates (mg/kg): 237 Ca(NO3)2.4H2O, 103 (NH4)2HPO4, 80 MgSO4.7H2O, 18
FeSO4.7H2O, 14 MnSO4.H2O, 9 ZnSO4.7H2O, 8.3 CuSO4.5H2O, 0.33 H3BO3, 0.3
CoSO4.7H2O, 0.33 Na2MoO4.2H2O.
5.2.1 Treatments
Two levels of K (40, 120 mg K/kg) in the topsoil were combined with three levels of subsoil
Na (0, 50 and 200 mg Na/kg). Half of the columns were well-watered throughout, but the
topsoil of the remaining columns was dried by withholding watering from 5 weeks after
sowing. All treatments were replicated three times, totalling 72 columns (Table 5.1). In the
columns, topsoil (0- 15 cm of the column) had a mixture of basal nutrients and two K
treatments (40 and 120 mg K/kg) with top 3- 4 cm left free for watering. The buffer layer
(15- 20 cm) was filled with basal nutrients, and the subsoil (20- 80 cm) with basal nutrients,
three Na treatments, two levels of K in topsoil dry columns (40 and 120 mg K/kg) or one
level of K in topsoil wet columns (40 mg K/kg). A plastic tube (1 cm diameter) with holes
made at 20, 25, 30, 35 and 40 cm depth and closed at the bottom was inserted into the
columns to water the subsoil only (Fig. 5.1). This was mainly to prevent wetting of topsoil
from subsoil watering alone and to prevent leaching of nutrients, particularly K, from the
topsoil. During the course of experiment (including initial 5 weeks), the subsoil was always
watered using the plastic tube inserted and care was taken to add just enough water to wet
topsoil and over watering was avoided to minimise leaching to subsoil.
Six seeds were sown per column, and after emergence the seedlings were thinned to three per
column and one week later the seedlings were further thinned to two per column. The plants
were harvested at either 5 or 11 weeks after sowing. Among the 72 columns, 18 columns
were harvested at 5 weeks after sowing before topsoil drying. The remainder of columns
were split into topsoil wet (18) and topsoil dry (36) columns (Table 5.1). Topsoil wet
columns were continuously watered with DI water to field capacity (14 % w/w), while topsoil
dry columns had water withheld from 5 weeks after sowing. The subsoils of all columns
received DI water to FC through the inserted tubes. Final harvest was made at 11 weeks after
sowing.
78
Table 5.1 Experiment design showing topsoil watering, topsoil K (mg K/kg) and subsoil K
(mg K/kg) and Na (mg Na/kg) treatments harvested at 5 and 11 weeks after sowing.
Topsoil watering and harvest Topsoil K
mg K/kg
Subsoil K
mg K/kg
Subsoil Na
mg Na/kg
No. of columns
Well-watered
(harvest at 5 weeks after sowing) 40, 120
40
0, 50 and 200
18
Well-watered
(harvest at 11 weeks after sowing)
40, 120
40
0, 50 and 200
18
Dry
(watering withheld from 5 weeks after sowing to harvest at 11 weeks
after sowing)
40, 120
40 and 120
0, 50 and 200
36
Fig. 5.1 Column experiment of wheat cv. Wyalkatchem at 3 weeks after sowing (left).
Column set-up with plastic tubes used for subsoil watering, commencing at 5 weeks after
sowing (right)
79
5.2.2 Measurements
Plant phenology was recorded weekly throughout the experiment. Leaf net photosynthesis,
stomatal conductance and transpiration rate were measured at 5, 7 and 10 weeks after sowing,
using the LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The
corresponding growth stages at measurements were Z 16, 27 (5 weeks), Z 19, 29, 36 (7
weeks) and Z 19, 29, 53 (10 weeks) identified using Zadoks growth scale for cereals. The
measurements were made on fully expanded young leaves at ambient relative humidity of 50
%, leaf temperature of 25˚C, reference CO2 of 380 µmol/mol, and photosynthetically active
radiation of 1500 µmol/m2·s. Shoot fresh weight was recorded immediately after harvest, and
the shoot were separated into young leaves (top 2-3 leaves in each tiller), old leaves (basal 3-
5 on main tiller), rest of the shoot and ears. The growth stage at the time of harvest was Z 19,
29, 61. Then the column was split open to observe root development at various depths in the
column by separating roots at 0- 20, 20- 40 and 40- 80 cm. The roots were collected on a 2
mm sieve after washing in tap water and rinsing with DI water. The shoot and root samples
were dried in an oven for 48 hours at 60˚C and dry weights were recorded. The samples were
then milled for K and Na analysis following methods described in the previous chapter
(Chapter 4).
5.2.3 Statistical analysis
Statistical analyses were conducted using SPSS 18.0. Two-way analysis of variance was
conducted to assess the effects of soil K and Na supply and their interactions at 5 weeks and
11 weeks harvest. Repeated measures ANOVA was used to analyse leaf gas exchange
parameters as there were three measurements made in columns at 5, 7 and 10 weeks after
sowing. Tukey’s HSD was computed at P ≤ 0.05 for pair-wise comparison of means.
5.3 Results
5.3.1 Plant growth
Initial harvest- 5 weeks after sowing
Shoot dry weight at the initial harvest (5 weeks) was unaffected by treatments except for a
significant reduction with addition of high Na in the subsoil (200 mg Na/kg) when low soil K
was applied throughout the soil profile. There was no significant change in shoot dry weight
with the addition of 50 mg Na/kg in the subsoil (P > 0.05; Table 5.2). More tillers were
produced with high topsoil K and nil subsoil Na. There was a significant reduction (P ≤ 0.05)
in tiller number with high subsoil Na and low subsoil K (Fig. 5.2 and 5.3). The potassium
80
effect was significant for shoot dry weight and tiller numbers harvested at 5 weeks after
sowing (P ≤ 0.05), while the interaction between K and subsoil Na was not significant for
shoot dry weight (Table 5.2).
Soil K
40/40(W) 120/40(W)
Tille
rs/ p
lant
0
2
4
6
0 Na 50 Na 200 Na
Shoot Dry weight and tillers- harvest @ 5 WASsh
oot d
ry w
t/ pl
ant (
g)
0.0
0.2
0.4
0.6
0.8
1.0
Fig. 5.2 Shoot dry weight (g) and tiller number per plant at 5 weeks after sowing (±SE, n=3).
For treatment descriptions refer to Table 5.1. See Table 5.2 for statistical analysis.
81
Fig. 5.3 Columns were supplied with low K (40 mg K/kg) in the whole profile with varying
subsoil Na levels: a) nil Na, b) 50 mg Na/kg, and c) 200 mg Na/kg. Shoot growth and
tillering was depressed by 200 mg Na/kg at 5 weeks after sowing.
Table 5.2 Statistical summary of plant growth at 5 and 11 weeks after sowing treated with
two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200 mg
Na/kg). For treatment details refer to Table 5.1.
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
5 weeks after sowing 11 weeks after sowing
Parameters Na K Na*K Na Treatment
(combination of K and water)
Na*treatment
Shoot dry wt/plant
* ** n.s *** ** n.s
No of tillers/plant *** *** * *** ** n.s
Root dry wt/plant n.s * n.s *** *** *
Root: shoot ratio n.s n.s n.s *** n.s n.s
The main effect of K treatment was significant for root dry weight harvested at 5 weeks after
sowing (Table 5.2) but presence of subsoil Na did not had an effect on root dry weight (Fig.
a b c
82
5.4; Table 5.2). In the column, the top section (0-20 cm) had more root dry weight than other
sections. Root: shoot ratios ranged from 0.37 to 0.4 with no significant difference among the
treatments (Appendix 2).
0 Na
0.00
0.05
0.10
0.15
0.20
0.25
0.30
50 Na
40/40(W) 120/40(W)
Roo
t dry
wei
ght/
plan
t(g)
0.00
0.05
0.10
0.15
0.20
0.25
200 Na
Soil K
40/40(W) 120/40(W)
0.00
0.05
0.10
0.15
0.20
0.25
40- 60 cm20- 40 cm0- 20 cm
Fig. 5.4 Root dry weight (g/plant) of wheat cv. Wyalkatchem in different sections of column
(0- 20, 20- 40 and 40- 60 cm) at 5 weeks after sowing. For treatment descriptions refer to
Table 5.1. See Table 5.2 for statistical analysis.
Final harvest- 11 weeks after sowing
Shoot dry weight measured 11 weeks after sowing did not show significant difference
between 40 and 120 mg K/kg treatments, and ranged from 12.2 to 13.7 g/plant (averaged
across Na levels and water treatments). However, low soil K throughout the profile in
combination with dry topsoil resulted in the lowest shoot biomass among all the treatments.
The plants grown in high subsoil K had higher biomass than high soil K in topsoil only when
topsoil was kept dry. In this experiment, the combination of K and water treatments had a
significant effect but its interaction with subsoil Na was not significant for shoot dry weight
83
at 11 weeks harvest (P > 0.05) (Table 5.2). The addition of moderate subsoil Na to low K
profile did not change shoot dry weight significantly either in topsoil dry and wet treatments.
Tiller production increased when there was moderate subsoil Na with low soil K in the whole
profile regardless of wet or dry topsoil (Fig. 5.5). However, high subsoil Na decreased the
number of tillers in all treatments. There was no difference in tillering between well-watered
and dry topsoil profiles (Table 5.2).
Total root dry weight was relatively high when there was high soil K throughout the profile
and reduced in the dry topsoil columns in presence of low soil K throughout the soil profile
(Fig. 5.6). There was significant reduction in root dry weight when there was high subsoil Na
(200 mg Na/kg) with low soil K throughout the profile. Among the sections, the top 0- 20 cm
of the soil column had almost 65 % of the total root distribution. The root dry matter in 40-
80 cm section of the column decreased due to topsoil drying with low soil K throughout the
profile, especially with addition of subsoil Na. The presence of high subsoil Na reduced root
dry weight when there was 120 mg K/kg either in topsoil or subsoil of the soil profile with
significant interaction between Na and treatments (P ≤ 0.05) (Table 5.2). Root: shoot ratios in
the nil and moderate subsoil Na columns were almost the same irrespective of difference in
soil K levels and watering (Fig. 5.7). However, at high subsoil Na, the ratio was lowered
significantly.
84
Shoot Dry weightsh
oot d
ry w
t/ pl
ant (
g)
0
2
4
6
8
10
12
14
16
whole profile wet Top soil dry, sub soil wet
No. of tillers
Soil K mg/kg
40/40(W) 120/40(W) 40/40 120/40 40/120 120/120
Tille
rs/ p
lant
0
2
4
6
8
0 Na 50 Na 200 Na
Fig. 5.5 Shoot dry weight (g) and tillers per plant at 11 weeks after sowing (±SE, n=3). For
treatment descriptions refer to Table 5.1 and see Table 5.2 for statistical analysis.
85
0 Na
0
2
4
6
8
50 Na
Roo
t dry
wei
ght/
plan
t (g)
0
2
4
6
200 Na
Soil K levels
40/40(W) 120/40(W) 40/40 120/40 40/120 120/120
0
2
4
6
40-80 20-40 0-20
whole profile wet topsoil dry, subsoil wet
Root dry weight
Fig. 5.6 Root dry weight (g/plant) in different sections of column (0- 20, 20- 40 and 40- 60
cm) at 11 weeks after sowing. For treatment descriptions refer to Table 5.1.
86
Root: Shoot
Soil K
40/40(W) 120/40(W) 40/40 120/40 40/120 120/120
root
: sho
ot ra
tio
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0 Na 50 Na 200 Na
whole profile wet topsoil dry, subsoil wet
Fig. 5.7 Root: shoot ratios of wheat at 11 weeks after sowing (±SE, n=3). For treatment
descriptions refer to Table 5.1 and Table 5.2 for statistical analysis.
5.3.2 Leaf gas exchange
Leaf gas exchange parameters were measured at 5, 7 and 10 weeks after sowing (WAS).
Photosynthesis and stomatal conductance at 5 WAS showed a significant reduction in
presence of high subsoil Na (200 mg Na/kg) compared to nil and moderate Na levels in
subsoil (Fig. 5.8). The main effect of K and watering treatments for leaf net photosynthesis
was not significant (P > 0.05), and neither was the interaction of subsoil Na with K and
watering treatments (Table 5.3). However, stomatal conductance showed a significant
interaction (P ≤ 0.05). Stomatal conductance at 5 WAS reduced considerably with dry topsoil
and low soil K throughout the profile with all levels of subsoil Na when compared with well-
watered high K topsoil without Na. Low K and wet soil profile with high subsoil Na had the
low stomatal conductance among all treatments. The interaction of Na with K and watering
treatments was not significant for transpiration (Table 5.3).
87
At 7 WAS, high soil K throughout the profile had significantly higher photosynthesis rate,
stomatal conductance and transpiration compared with low soil K throughout the profile,
especially when the profile had dry topsoil (Fig. 5.9). Addition of high subsoil Na lowered
photosynthesis, especially when there was low K throughout the profile and the interaction
between subsoil Na and treatment was significant for photosynthesis measured at 7 WAS (P
≤ 0.05; Table 5.3). Although the Na and treatment effect was significant for photosynthesis,
there was no interaction for stomatal conductance and transpiration (Table 5.3). Leaf gas
exchange measured at 10 WAS had a similar pattern to that observed at 7 WAS, except there
was significant interaction between subsoil Na and treatment for transpiration rate measured
at 10 WAS (Fig. 5.10 & Table 5.3). Irrespective of the treatments, the rate of photosynthesis,
stomatal conductance, and transpiration decreased considerably in later growth stages.
88
Transpiration -5 weeks
Soil K
40/40(W) 120/40(W) 40/40 120/40 40/120 120/1200
2
4
6
0 Na 50 Na 200 Na
Photosynthesis -5 WAS
Pho
tosy
nthe
sis
µmol
CO
2/m
2 .s
0
5
10
15
20
25
30
Transpiration -5 weeksStomatal conductance -5 WAS
Sto
mat
al c
ondu
ctan
ce
mm
olH
2O/m
2 .s
0
200
400
600
Transpiration -5 WAS
Tran
spira
tion
mm
olH
2O/m
2 .s
whole profile wet Topsoil dry, subsoil wet
Fig. 5.8 Leaf photosynthesis, stomatal conductance, and transpiration at 5 weeks after sowing
(±SE, n=3). For treatment descriptions refer Table 5.1 and for statistical analysis refer Table
5.3.
89
Photosynthesis- 7 WAS
Pho
tosy
nthe
sis
µmol
CO
2/m
2 .s
0
5
10
15
20
25
30
Stomatal conductance- 7 WAS
Sto
mat
al c
ondu
ctan
ce
mm
olH
2O/m
2 .s
0
200
400
600
Transpiration- 7 WAS
Soil K
40/40(W) 120/40(W) 40/40 120/40 40/120 120/120
Tran
spira
tion
mm
olH
2O/m
2 .s
0
2
4
6
0 Na 50 Na 200 Na
whole profile wet Topsoil dry, subsoil wet
Fig. 5.9 Leaf photosynthesis, stomatal conductance, and transpiration at 7 weeks after sowing
(±SE, n=3). For treatment descriptions refer Table 5.1 and Table 5.3 for statistical analysis.
90
Photosynthesis- 10 WAS
Pho
tosy
nthe
sis
µm
ol C
O2/
m2 .s
0
5
10
15
20
25
30
Stomatal conductance- 10 WAS
Sto
mat
al c
ondu
ctan
ce
mm
olH
2O/m
2 .s
0
200
400
600
Transpiration- 10 WAS
Soil K
40/40(W) 120/40(W) 40/40 120/40 40/120 120/120
Tran
spira
tion
mm
olH
2O/m
2 .s
0
2
4
6
0 Na 50 Na 200 Na
whole profile wet topsoil dry, subsoil wet
Fig. 5.10 Leaf photosynthesis, stomatal conductance, and transpiration at 10 weeks after
sowing (±SE, n=3). For treatment descriptions refer Table 5.1 and Table 5.3 for statistical
analysis.
91
Table 5.3 Statistical summary of leaf gas exchange at 5, 7 and 10 weeks after sowing treated
with two levels of soil K (40 and 120 mg K/kg) and three levels of subsoil Na (0, 50 and 200
mg Na/kg). For treatment details refer to Table 5.1.
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Na Treatment
(combination of K and water treatments)
Na*Treatment
Gas exchange- 5 WAS
Photosynthesis * n.s n.s
Stomatal conductance *** *** *
Transpiration n.s * n.s
Gas exchange- 7 WAS
Photosynthesis n.s * **
Stomatal conductance ** *** n.s
Transpiration n.s *** n.s
Gas exchange- 10 WAS
Photosynthesis *** ** *
Stomatal conductance * *** n.s
Transpiration ** *** *
5.3.3 K and Na concentrations
Shoot K concentrations at 5 weeks after sowing were similar (ranging from 37 to 42 mg K/g,
dry weight) among the treatments (Table 5.4).
92
Table 5.4 Shoot K and Na concentrations and accumulation in wheat cv. Wyalkatchem
harvested at 5 weeks after sowing. Means (n=3) with different letters differ at P≤0.05.
Topsoil Soil K (mg/kg) Subsoil Na
(mg/kg)
Concentration (mg/g, plant dry wt)
Accumulation (mg/plant, dry wt basis)
Watering Topsoil K
Subsoil K
Shoot K Shoot Na
Shoot K Shoot Na
Wet 40 40 0 40.3a 0.13c 25.7ab 0.08c
50 39.7a 0.52bc 26.0ab 0.34bc
200 37.2a 1.34a 18.2b 0.65a
Wet 120 40 0 41.9a 0.12c 31.4a 0.09c
50 40.5a 0.39c 26.1ab 0.25bc
200 40.3a 0.89ab 24.1ab 0.53ab
At 5 weeks, wheat grown at low soil K throughout the profile with high subsoil Na
accumulated less K than high topsoil K column with nil Na (Table 5.4). However, addition of
subsoil Na did not have a significant effect on shoot K concentration (P > 0.05, Table 5.5).
Shoot Na concentrations were dependent on subsoil Na levels and the highest concentration
was with high subsoil Na in a low soil K column among the treatments. Similarly, shoot Na
accumulation was much higher with high subsoil Na (200 mg Na/kg) in low K soil profile
than nil and 50 mg Na/kg subsoil Na (Table 5.4).
93
Table 5.5 Statistical summary of shoot K and Na concentrations and content at 5 and 11
weeks after sowing in wheat plants treated with two levels of soil K (40 and 120 mg K/kg)
and three levels of subsoil Na (0, 50 and 200 mg Na/kg). For treatment details refer to Table
5.1. Note only whole shoots and roots were analysed at 5 weeks.
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
5 weeks after sowing 11 weeks after sowing
Parameters Na Treatment
(combination of K and water)
Na × treatment
Na Treatment
(combination of K and water)
Na × treatment
Shoot K concentration
n.s n.s n.s
Shoot Na concentration
* *** n.s
K concentration- Ears
** *** n.s
K concentration- young leaves
*** *** **
K concentration- old leaves
*** *** *
Na concentration- Ears
*** *** *
Na concentration- young leaves
*** *** ***
Na concentration- old leaves
*** *** ***
Shoot K accumulation
* n.s n.s *** *** **
Shoot Na accumulation
*** n.s n.s *** *** ***
Potassium concentration in ears/spikes was significantly decreased by Na (P ≤ 0.05) (Table
5.5 and 5.6). High soil K throughout the profile without subsoil Na had significantly higher K
94
concentration in spikes than low soil K throughout the profile irrespective of topsoil drying
and subsoil Na levels (Table 5.6). Soil K supply increased K concentrations in young and old
leaves. Under topsoil dry conditions, wheat grown in high soil K (120 mg K/kg) throughout
the profile irrespective of subsoil Na, and those grown in high subsoil K with nil and
moderate subsoil Na had significantly higher K concentrations in young and old leaves than
low K throughout the profile (Table 5.6). Potassium concentration in old leaves was
significantly lower when there were low soil K throughout the soil profile at all Na levels
than high K profiles in both topsoil dry and wet conditions. Old leaves had considerably
lower K compared with young leaves particularly at low K supply throughout the profile. The
addition of moderate subsoil Na did not alter K concentrations in any of the treatments.
Wheat grown in low soil K throughout the profile irrespective of subsoil Na accumulated
significantly less shoot K than treatments that had high subsoil K. The interaction between
subsoil Na and treatment was significant for shoot K content in columns harvested at 11
WAS (P ≤ 0.05, Table 5.4), and the shoot K accumulation in treatments with high subsoil K
and nil and 50 mg Na/kg subsoil Na was significantly higher than other treatments (Table
5.7).
Subsoil Na levels largely determined the shoot Na concentration and accumulation (Table 5.7
and 5.8), and the interaction between subsoil Na and treatment was significant for Na
concentration in ears and leaves, and shoot content harvested at 11 WAS (P ≤ 0.05, Table
5.5). Shoot Na accumulation increased significantly with addition of moderate or high subsoil
Na in the low subsoil K treatments (40 mg K/kg) but was significantly lowered by high
subsoil K even with moderate and high subsoil Na (Table 5.7). In contrast to K
concentrations, old leaves concentrated considerably more Na than ears and young leaves.
95
Table 5.6 K concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11
weeks after sowing. Means (n=3) with different letters differ at P≤0.05.
Topsoil Soil K (mg/kg) Subsoil Na (mg/kg)
K concentration (mg/g, plant dry weight)
Watering Topsoil K Subsoil K ears old leaves young leaves
Wet 40 40 0 12.8bcde 2.66e 9.86defg
50 12.4cde 2.64e 9.73defg
200 12.2cde 2.29e 9.22efg
Wet 120 40 0 13.5abcde 9.18cd 13.7cd
50 13.8abcd 9.85cd 13.9cd
200 12.6cde 8.33d 12.5cdef
Dry 40 40 0 12.7bcde 2.58e 9.71defg
50 12.1de 2.75e 8.06g
200 11.8e 2.40e 8.59fg
Dry 120 40 0 12.7bcde 8.49d 12.6cdef
50 13.1bcde 9.79cd 13.5cde
200 12.4cde 8.19d 12.2cdefg
Dry 40 120 0 14.7ab 17.9a 19.4b
50 14.0abcd 18.4a 19.2b
200 14.1abc 12.8bc 16.5bc
Dry 120 120 0 15.1a 19.4a 25.0a
50 14.7ab 18.5a 24.0a
200 13.8abcd 15.7ab 18.9b
96
Table 5.7 Shoot K and Na accumulation in wheat cv. Wyalkatchem harvested at 11 weeks
after sowing. Values are means of 3 replicates. Means (n=3) with different letters differ at
P≤0.05.
Topsoil Soil K (mg/kg) Subsoil Na (mg/kg)
Accumulation (mg/plant, dry wt basis)
Watering Topsoil K Subsoil K Shoot K Shoot Na
Wet 40 40 0 130fgh 3.45e
50 124gh 14.3bcd
200 110h 21.1ab
Wet 120 40 0 175de 2.01e
50 177de 15.9bc
200 150defg 17.2abc
Dry 40 40 0 115gh 5.66e
50 116gh 17.7abc
200 110h 24.5a
Dry 120 40 0 152defg 3.17e
50 164def 13.3cd
200 139efgh 15.2bc
Dry 40 120 0 224bc 0.96e
50 226bc 3.55e
200 174de 7.44de
Dry 120 120 0 266a 0.88e
50 243ab 1.55e
200 188cd 4.40e
97
Table 5.8 Na concentrations in ears and leaves of wheat cv. Wyalkatchem harvested at 11
weeks after sowing. Means (n=3) with different letters differ at P≤0.05.
Topsoil Soil K (mg/kg) Subsoil Na (mg/kg)
Na concentration (mg/g, plant dry weight)
Watering Topsoil K Subsoil K ears old leaves young leaves
Wet 40 40 0 0.08cd 1.13fg 0.06e
50 0.12cd 4.04cd 0.26de
200 0.32ab 6.06b 0.72ab
Wet 120 40 0 0.06d 0.39g 0.08e
50 0.10cd 2.79de 0.39bcde
200 0.24bcd 4.61c 0.63abc
Dry 40 40 0 0.10cd 1.39efg 0.08e
50 0.24bcd 4.94bc 0.47bcd
200 0.43a 7.99a 0.88a
Dry 120 40 0 0.05d 0.91g 0.08e
50 0.08cd 4.11cd 0.20de
200 0.24bcd 5.44bc 0.74ab
Dry 40 120 0 0.08cd 0.07g 0.05e
50 0.10cd 0.55g 0.23de
200 0.10cd 2.38ef 0.27cde
Dry 120 120 0 0.05d 0.07g 0.06e
50 0.08cd 0.14g 0.18de
200 0.15bcd 1.05fg 0.21de
Shoot K+/Na+ ratios were influenced by soil K and Na levels (Table 5.9). There was no
significant difference in ratios between topsoil wet and dry columns. The ratios were
increased by K supply, especially in subsoil, and reduced with the increase in subsoil Na
levels.
98
Table 5.9 Shoot K/Na ratios in wheat cv. Wyalkatchem harvested at 11 weeks after sowing.
Means (n=3) with different letters differ at P≤0.05.
Topsoil Soil K (mg/kg) Subsoil Na (mg/kg) Shoot K/Na
Watering Topsoil K Subsoil K
Wet 40 40 0 39.3efg
50 8.90fg
200 5.50g
Wet 120 40 0 90.0d
50 11.9fg
200 9.00fg
Dry 40 40 0 21.4fg
50 6.80g
200 4.50g
Dry 120 40 0 48.2ef
50 12.5fg
200 9.20fg
Dry 40 120 0 240b
50 65.7de
200 23.4fg
Dry 120 120 0 300a
50 160c
200 44.0efg
99
5.4 Discussion
Potassium and Na co-exist on the soil exchange complex and in soil solution, and they may
exert antagonistic or synergistic effects on absorption and translocation of each other within
plants, particularly under saline and sodic conditions (Hussain et al., 2013). Potassium is an
essential nutrient for plant physiology, and higher plants require large amounts of K which
cannot be totally replaced by Na addition. However the capacity of wheat to respond
positively to NaCl applications might have important consequences in wheat K management
in saline and sodic soils. The addition of low to moderate Na (25 to 50 mg Na/kg) to K-
deficient soil enhanced wheat growth, leaf gas exchange measurements and K uptake in
previous experiments (Chapter 3 and 4). In the current experiment, the effect of varied
subsoil K and Na which commonly occur in duplex soils was examined under both well-
watered and topsoil dry conditions.
Wheat growth stimulation due to moderate subsoil (50 mg Na/kg) Na when soil K supply was
limited throughout the profile was insignificant in this experiment which was in contrast to
the previous experiment where the presence of 50 mg Na/kg in one or both the compartments
in split-root increased the shoot and root dry weight significantly (Chapter 4). The negative
effect of Na was evident at 200 mg Na/kg, particularly when there was low K throughout the
profile. In a comparable study by Ma et al. (2011), the addition of 100 mg Na/kg to a K
deficient soil caused a slight but not significant decrease in shoot and root dry weight of cv.
Wyalkatchem while 300 mg Na/kg addition caused a significant reduction in wheat growth.
Potassium deficiency had greater effect on root growth than shoot growth in previous
experiments for wheat (Chapter 3) and also in barley (Degl'Innocenti et al., 2009; Ma et al.,
2011) where root/shoot ratios were generally higher at the adequate soil K levels than low K
levels. In the current column experiment, there was comparatively poor root development
when columns had low K, and the presence of moderate subsoil Na (50 mg Na/kg) did not
affect roots, whereas, high subsoil Na (200 mg Na/kg) further intensified the K-deficiency
effect on roots. In field studies, drought often increase root length and density especially, at
depths in soil in search of available water (Comas et al., 2013) and increases root/shoot ratios.
However, in this experiment there was no detailed scanning of roots to measure diameter,
density, length etc., and the presence of dry topsoil did not influence root dry weight mainly
because subsoil was watered regularly from 5 WAS which was sufficient for plants and there
was no significant water stress observed in the columns unlike topsoil dry field conditions.
100
Also the effect on root growth or root/shoot ratio depends on which occurs first, either K
deficiency or drought.
In this experiment, moderate subsoil salinity (50 mg Na/kg) did not alter leaf gas exchange
measurements. Similar to growth results discussed, this was in contrast to previous
glasshouse experiments (Chapter 3 and 4) where there was significant increase in the
measurements comparable to adequate K supply. The leaf gas exchange measurements were
not significantly altered in barley cultivars and wheat cv. Wyalkatchem with 100 mg Na/kg
addition to K deficient soil (Ma et al., 2011). High subsoil salinity reduced the leaf net
photosynthesis rate, stomatal conductance and transpiration rate of wheat. Leaf net
photosynthesis rate before 11 weeks after sowing was negatively correlated (R= -0.686) with
shoot Na concentrations (Fig. 5.11). Salinity can have negative effect on photosynthetic
process by an effect on stomatal closure that limits the CO2 diffusion to the chloroplasts
(Degl'Innocenti et al., 2009). Moreover, when utilization of absorbed light energy in CO2
fixation was restricted by salinity, the electron flux to O2 increased, resulting in an
accumulation of reactive oxygen species (ROS) in chloroplasts (Shabala et al., 1998).
Stomatal conductance of wheat grown in wet topsoil conditions was higher than with topsoil
dry. Stomatal conductance decreased with increase in water stress as leaf dehydration can
lead to turgor lose causing passive stomata closure and hence carbon entry, and consequently
the supply of CO2 to fixation site is reduced (Khakwani et al., 2012).
Higher K concentration in young leaves than old leaves in treatments with low soil K
throughout the profile suggests that there was effective translocation of K+ from old to young
leaves. However, there was no significant effect of added subsoil Na on K concentrations.
The presence of moderate subsoil Na (50 mg/kg) did not affect K+ uptake which was in
contrast to the previous split-root experiment where K+ uptake at low soil K was significantly
increased due to added Na+. Similar to results of this study, the presence of Na+ did not
increase K+ uptake of rice (Yoshida & Castaneda, 1969), wheat (Box & Schachtman, 2000),
and tomato (Walker et al., 2000) grown under solution culture in various other experiments
although an increase in dry weight due to added NaCl at deficient K levels was noticed. At
high subsoil Na (200 mg/kg), an antagonistic relationship developed between K and Na, and
Na+ inhibited plant K+ uptake.
101
Fig. 5.11 Correlation between leaf net photosynthesis rates and shoot Na concentrations (mg
Na/g, dry weight) at final harvest at 11 weeks after sowing.
Compared to the previous experiments, the columns of this experiment contained more soil
(10 kg/column) and hence more available K for plants. The available K per column with two
wheat plants at low K (40 mg/kg) soil treatments was 400 mg. The supply of 200 mg K/plant
was 2.5 times greater than in the previous pot and split-root experiments (available K/plant=
80 mg) (Chapters 3 and 4). This may explain why the K deficiency symptoms at low K
treatments were absent as were substitution effects of Na at low K in this experiment.
Sodium-enhanced plant growth was earlier considered accidental or under weak
physiological regulation, however, recent studies on characterisation of high-affinity Na+
uptake in several plants when K+ is exhausted strongly suggest there is physiologically
programmed role of Na+ in some plants under insufficient K+ supply (Subbarao et al., 2003;
Wakeel et al., 2011). In this study to explore the possibility of K substitution of Na in wheat,
the beneficial effects of low to moderate Na was considerably greater in previous
experiments when growth was limited due to deficient K supply and wheat showed moderate
to severe K deficiency symptoms. This makes it clear that the synergistic or antagonistic
effect between K and Na depends on the amount of K and Na present in the soil (Ali et al.,
2013). In the presence of sufficient K already in the soil, the addition of subsoil Na had little
effect on wheat growth or the substitution of K functions.
R² = 0.4702
y = -2.638x + 17.696
8
10
12
14
16
18
20
22
0 0.5 1 1.5 2 2.5
Pho
tosy
nthe
sis
(µm
ol C
O 2/m
2 .s)
Shoot Na concentration (mg/g, plant dry weight)
Photosynthesis vs Shoot Na concentration
102
5.5 Conclusion
The presence of moderate levels of subsoil Na (50 mg Na/kg) did not have a significant effect
on growth, leaf gas exchange and K accumulation of wheat cv. Wyalkatchem grown in
columns with low profile K supply under both topsoil dry and wet conditions (P=0.05). The
findings of this experiment differed from the previous pot and split-root experiments (Chapter
3 and 4) probably due to larger soil volume and thus relatively more available K per plant
which largely prevented K deficiency. This emphasises that the beneficial role of Na in K
nutrition may occur only when wheat is grown under K-limited conditions with apparent K
deficiency symptoms. It can be concluded that the extent of Na stimulation of growth by
wheat was determined by the amount of soil available K as well as Na.
103
CHAPTER 6
SHORT-TERM SOLUTION CULTURE EXPERIMENT
Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium
(Na+) supply using rubidium (Rb+) tracer in solution culture experiments: short-term
responses
6.1 Introduction
Low Na was found to be beneficial for wheat shoot and root growth when soil K was
deficient (Chapter 4), and the response varied between K-efficient and K-inefficient cultivars
(Chapter 3). Under low K supply (example 40 mg K/kg), four wheat genotypes differing in
K-use efficiency responded to low to moderate Na (25 to 50 mg Na/kg) by increasing leaf net
photosynthesis, root growth and shoot K+ uptake especially in K-efficient cultivars compared
with nil Na treatment (Chapter 3). Also, in the split-root experiment with cv. Wyalkatchem
(Chapter 5), the presence of Na in just one of the two compartments was able to increase
plant growth and K uptake significantly under low K supply.
Generally the Na+ stimulation of plant growth in low K plants has been attributed to the
uptake of Na which then substitutes for non-specific functions of K in cells. However, in
wheat the shoot Na concentrations were too low to substitute for K in plants. By contrast,
with 25 or 50 mg Na/kg of soil, there was an increase in shoot K concentration and content
(Chapters 3 and 4). This raises questions whether the presence of Na+ as an energising ion
may result in increased K+ acquisition (Box & Schachtman, 2000) or the enhanced K+ uptake
is due to added Na that releases soil K and makes it available to plants. The presence of Na
may also stimulate root growth (Ali et al., 2009) which in turn increases K uptake.
In the split-root experiment, leaf K concentration decreased but root K concentration
increased with addition of moderate Na (50 mg Na/kg) to one or both the root compartments
at low soil K. Box and Schachtman (2000) reported that the beneficial effect of Na on wheat
grown in low external K was due to the direct effect of Na+ on growth in one of their
experiments, but not in two other experiments. They also found that Na+ did not stimulate
Rb+ uptake, but K+ stimulated Na+ uptake in short-term tracer flux experiments. A further
investigation on whether low concentrations of Na+ increase K+ uptake in wheat with low K
supply would help understand K and Na interactions particularly at low soil concentrations of
K and Na.
104
My preliminary soil incubation experiment (an experiment in Chapter 3) showed that adding
Na had a slight but non-significant effect on exchangeable soil K levels. This suggests that
enhanced K availability in soil by Na supply cannot explain the entire increased K uptake in
the experiments reported in Chapters 3 and 4. However, by testing the effect of Na on K
uptake in nutrient solution, any Na-induced increase in K availability in the soil medium can
be avoided. It was hypothesised that low concentrations of external Na+ would increase K+
uptake in wheat under deficient soil K conditions through the effect of Na+ on K+ transporters
(Box & Schachtman, 2000; Rubio et al., 1995) or the effect of Na+ in increasing membrane
hyperpolarisation (Shabala & Cuin, 2008). Short-term tracer flux experiments were
conducted using rubidium (Rb+) as a tracer for K+ uptake to determine whether low
concentrations of Na+ stimulated Rb+ uptake in wheat. Rubidium has almost an identical
hydrated ion radius as K+ and these two ions behave similarly with regard to plant absorption
(Drobner & Tyler, 1998). In this experiment, two wheat genotypes differing in K-use
efficiency (Damon & Rengel, 2007) were used to further probe the effects of Na+ on rate of
K+ uptake.
6.2 Materials and methods
6.2.1 Plant culture
Wheat (Triticum aestivum L.) cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient)
were grown in a naturally-lit glasshouse at Murdoch University, Perth, Western Australia
(32°04′S, 115°50′E). Two short-term experiments with different K and Na levels were
conducted during winter (Experiment 1) and early spring (Experiment 2) in a nutrient
solution culture. Wheat seeds were surface sterilised by washing with 5 % sodium
hypochlorite solution for 1 minute and then thoroughly rinsed with DI water. The seeds were
soaked in DI water for 2 hours and germinated on paper towels moistened with 0.05 mM
CaCl2 in the dark for 2 days at 25˚C. The germinated seedlings were then transplanted to 4 L
pots with ¼ strength modified Hoagland’s solution at six seedlings per pot. The seedlings
were held in plastic lids of the pots supported by polystyrene foam. In the pots, the nutrient
solution was continuously bubbled with compressed air (see Appendix 3 for experimental
setup). The nutrient solution was changed every 3 days throughout the experiment. The
experiments were a factorial combination of two wheat cultivars, two K levels and three Na
levels (see below) and each treatment was replicated four times.
105
6.2.2 Basal nutrient solution
A modified Hoagland’s solution (equivalent to ½ strength) was used in the experiment,
including 2 mM Ca(NO3)2.H2O, 0.5 mM NH4H2PO4, 1 mM MgSO4.7H2O, 200 µM Fe-citrate
(FeC6H5O7.5H2O), 9.2 µM H3BO3, 1 µM MnCl2.4H2O, 0.4 µM ZnSO4.7H2O, 0.2 µM
(NH4)6Mo7O24.4H2O, and 0.4 µM CuSO4.5H2O. The pH of the nutrient solution was
maintained at 5.5 throughout the experiment and was adjusted using either 1M HNO3 or 0.5
M Ca(OH)2. All chemicals used were of analytical grade.
6.2.3 Potassium and sodium treatments
Two short-term experiments with various K and Na concentrations were conducted in K+
uptake studies. In the first experiment, two K concentrations at 0.2 mM (low) and 2 mM K
(adequate) as KCl and three Na concentrations at 0, 10 and 20 mM Na as NaCl were used. In
the second experiment, two K concentrations of 0.05 mM (low) and 2 mM K (adequate) and
three Na concentrations of 0, 2 and 10 mM Na were used. The plants were initially grown in
low K (0.2 mM in first experiment or 0.05 mM K in second experiment) plus basal nutrient
solution without Na for two weeks, followed by a harvest of two plants per pot to analyse
initial ion concentrations of the shoot and root. Then half of the pots were continuously
supplied with low K, while the remaining pots were supplemented with extra K to raise the
concentrations to 2 mM K (adequate). Both the low and adequate K pots were treated with
the three Na levels. Rubidium chloride was added as a tracer (0.5 mM Rb+) together with the
K and Na treatments, and the plants were harvested after 48 hours to measure the uptake of
K, Na and Rb ions in root and shoot.
6.2.4 Measurements
There were two harvests: initial harvest before treatment addition (at 15 days after
transplanting) and final harvest (at 17 days after transplanting in first experiment, and 19 days
after transplanting in second experiment). The final harvests in both experiments were made
48 hours after the K and Na treatments and Rb addition. Plant phenology (leaf numbers, tiller
numbers) was recorded throughout the experiment. Leaf net photosynthesis, stomatal
conductance and transpiration rate were measured before and 1.5 days after treatment
application using the LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The
measurements were made in fully expanded young leaves at ambient relative humidity of 50
%, leaf temperature of 25˚C, reference CO2 of 380 µmol/mol, and photosynthetically active
radiation of 1500 µmol/m2·s.
106
At harvest, shoot fresh and dry weights, and root dry weight were determined. The roots of
two plants per pot were measured for root length, surface area, average diameter, root volume
using a root scanner controlled by WinRhizo software (WinRhizo Pro 2007, Regent
Instruments Quebec, Canada). The shoot and root samples were dried in a forced-draught
oven at 60˚C for 48 hours and their dry weights were recorded. The samples were then
milled, digested as described previously (Chapter 3) and concentrations of K, Na and Rb
were measured using inductively-coupled plasma-atomic emission spectroscopy.
6.2.5 Statistical analysis
Statistical analyses were conducted using the statistical program SPSS 18.0. Three-way
analysis of variance was conducted to assess the effects of soil K and Na supply and their
interactions with cultivars. Tukey’s HSD was computed at P ≤ 0.05 for pair-wise comparison
of means.
6.3 Results
6.3.1 Experiment 1
6.3.1.1 Plant growth
Pre-treatment shoot dry weight and root dry weight were 0.03 and 0.02 g/plant, respectively
in cv. Wyalkatchem, and 0.11 and 0.05 g/plant, respectively in cv. Gutha. Forty eight hours
after K, Na treatments and Rb addition there was no significant treatment effect on shoot or
root biomass in both cvv Wyalkatchem and Gutha (Appendix 4).
6.3.1.2 Leaf gas exchange
Pre-treatment measurements did not show any difference in leaf gas exchange among
cultivars (Appendix 4). Consistent with the responses of plant growth parameters, leaf
photosynthesis did not change after short-term treatments (measurements taken 42 hours after
treatment addition) (Fig. 6.1). By comparison, stomatal conductance and transpiration were
significantly higher in Wyalkatchem than in Gutha after short-term treatment. However, there
was no significant of K and Na treatments or the interaction between them in terms of
stomatal conductance and transpiration (Table 6.1).
107
Table 6.1 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient)
and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2
and 2 mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for a further 48
hours (Experiment 1).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Solution K
Solution Na
Cultivar K×Na K×cv Na×cv K×Na×cv
Photosynthesis n.s n.s n.s * n.s n.s n.s
Stomatal conductance
n.s n.s ** n.s n.s n.s n.s
Transpiration n.s n.s *** n.s n.s n.s n.s
108
0.2 mM 2 mM
Tra
nspi
ratio
n (m
mol
H2O
/m2 .s
)
0
2
4
6
K concentration (mM)
0.2 mM 2 mM
0 Na 10 Na 20 Na
Sto
mat
al c
ondu
ctan
ce (
mm
olH 2O
/m2 .s
)
0
100
200
300
400
Wyalkatchem
Leaf
ne
t ph
otos
ynth
esi
s (µ
mol
CO 2/m
2 .s)
0
5
10
15
20
25
Gutha
Fig. 6.1 Leaf net photosynthesis, stomatal conductance, and transpiration of cultivars
Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for
two weeks, followed by two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM)
(±SE, n=4) and measured 42 hours after the treatments (Experiment 1).
6.3.1.3 K, Na and Rb concentrations in shoot and root
Before the K and Na treatments and Rb addition, the shoots contained 46.6 mg K/g, 0.19 mg
Na/g in cv. Wyalkatchem and 48.2 mg K/g, 0.15 mg Na/g in cv. Gutha on a dry-weight basis.
Rubidium concentration was below the detectable limit (< 0.02 mg/g) in both cultivars. Forty
eight hours after K, Na treatments and Rb addition, shoot K concentration in both cultivars
was higher in 2 mM K than 0.2 mM K treatment without Na, but was similar between the two
K treatments with Na supply (Table 6.2). The interaction between K and Na was significant
109
for shoot K concentration (P ≤ 0.05), and three way interactions between K, Na and cultivars
was not significant (Table 6.3). Sodium addition did not have a significant effect on shoot K
concentrations 48 hours after treatment addition. Shoot Na concentration of both cultivars
increased with addition of Na but more strongly in low K treatments (Table 6.2). There was
nearly two fold decrease in shoot Rb concentrations with high K supply when compared with
low K supply.
Table 6.2 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-
efficient) and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K
levels (0.2 and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a
further 48 hours (harvested 17 days after transplanting) (Experiment 1). Means (n=4) with
different letters differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Ion concentrations in shoot (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.2 0 43.8ef 0.26d 5.79ab 48.7b-e 0.19d 7.18a
0.2 10 44.4def 1.57bc 5.91ab 49.2b-e 1.12cd 7.62a
0.2 20 39.3f 3.48a 4.90abc 46.4def 2.26b 6.56a
2.0 0 52.9a-d 0.20d 2.88bc 58.2a 0.18d 3.09bc
2.0 10 50.7a-e 1.13cd 2.42c 55.7abc 1.46bc 2.45c
2.0 20 47.3c-f 1.76bc 2.00c 56.5ab 1.24bcd 3.11bc
Ion concentrations in root (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.2 0 23.1bcd 0.77d 12.4a 23.1bcd 0.77d 15.8a
0.2 10 27.0abc 4.48c 12.8a 20.8cd 4.92c 15.1a
0.2 20 20.4cd 8.49ab 10.2ab 17.8d 9.93a 14.7a
2.0 0 31.5a 0.86d 5.79bc 33.2a 0.54d 5.50bc
2.0 10 30.7ab 5.38c 4.96bc 32.3a 6.39bc 5.05bc
2.0 20 29.5ab 6.93bc 3.51c 30.4ab 7.00bc 4.96bc
110
Prior to the treatments, the roots contained 33 mg K/g, 0.43 mg Na/g in cv. Wyalkatchem,
and 33 mg K/g and 0.31 mg Na/g in Gutha. Rubidium concentrations were below the
detectable level. The response of post-treatment root K, Na and Rb concentrations showed
similar trends to those of shoot concentrations (Tables 6.3, 6.4). However, roots had lower K
concentration and higher Na and Rb concentrations than shoot (Table 6.2).
Table 6.3 Statistical summary of shoot and root K, Na, Rb concentrations and contents in
cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.2 mM
K) for 2 weeks and two K levels (0.2 and 2 mM K), three Na levels (0, 10 and 20 mM Na)
and Rb tracer (0.5 mM) for 48 hours (Experiment 1; n=4).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Solution K Solution Na Cultivar K×Na K×cv Na×cv K×Na×cv
Shoot K conc. * n.s *** * n.s n.s n.s
Shoot Na conc. n.s n.s *** n.s n.s n.s n.s
Shoot Rb conc. * n.s *** n.s n.s n.s n.s
Root K conc. n.s n.s n.s * n.s n.s n.s
Root Na conc. n.s n.s ** n.s n.s n.s n.s
Root Rb conc. n.s n.s *** n.s n.s n.s n.s
Shoot K content * n.s *** * n.s n.s n.s
Shoot Na content n.s n.s *** n.s n.s n.s n.s
Shoot Rb content * n.s *** n.s n.s n.s n.s
Root K content n.s n.s n.s * n.s n.s n.s
Root Na content n.s n.s ** n.s n.s n.s n.s
Root Rb content n.s n.s *** n.s n.s n.s n.s
6.3.1.4 K, Na and Rb contents in shoot and root
Pre-treatment shoot K and Na contents were 1.67 and 0.007 mg/plant in cv. Wyalkatchem,
5.13 and 0.016 mg/plant in cv. Gutha. The root K and Na contents were 0.64 and 0.008
mg/plant in cv. Wyalkatchem, 1.71 and 0.01 mg/plant in cv. Gutha.
Forty eight hours after treatment addition, shoot K content was not significantly different
among the treatments in both cultivars (Table 6.4). Gutha had considerably higher shoot K
111
content than Wyalkatchem because of greater biomass. Shoot Na content was dependent on
solution Na concentrations. Similar to shoot K content, shoot Rb did not show significant
difference among treatments. Root K, Na and Rb contents followed similar response as shoot
contents. Roots accumulated less K than in shoot, especially cv. Gutha which had a low root:
shoot ratio. Plant K uptake prior to treatment addition was 2.3 mg in Wyalkatchem and 6.84
mg in Gutha. Forty eight hours after Na addition, plant K uptake was increased by 39 to 53 %
in Wyalkatchem, and 23 to 46 % in Gutha (Table 6.5).
Table 6.4 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2
mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours
(harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters
differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Ion content in shoot (mg/plant) (n=4)
K Na Rb K Na Rb
0.2 0 3.01c 0.02e 0.39bc 9.1ab 0.03e 1.34a
0.2 10 3.47c 0.12cde 0.45bc 9.18ab 0.21bcd 1.41a
0.2 20 2.95c 0.25b 0.36bc 8.24b 0.40a 1.16a
2.0 0 3.88c 0.01e 0.21bc 10.5a 0.03e 0.55b
2.0 10 3.57c 0.08e 0.17bc 9.17ab 0.23b 0.40bc
2.0 20 3.18c 0.11de 0.13c 10.2a 0.23bc 0.56b
Ion content in root (mg/plant) (n=4)
K Na Rb K Na Rb
0.2 0 0.84c 0.03de 0.45b 1.77ab 0.06de 1.22a
0.2 10 1.08bc 0.17cde 0.50b 1.54bc 0.37bc 1.13a
0.2 20 0.84c 0.34bc 0.41b 1.32bc 0.73a 1.09a
2.0 0 1.09bc 0.03e 0.19b 2.29a 0.03de 0.38b
2.0 10 1.11bc 0.19cde 0.18b 2.29a 0.45b 0.36b
2.0 20 1.01c 0.23cd 0.12b 2.30a 0.53ab 0.37b
112
Table 6.5 The whole plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient)
and Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2
and 2 mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours
(harvested 17 days after transplanting) (Experiment 1). Means (n=4) with different letters
differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Plant content (mg/plant) (n=4)
K Na Rb K Na Rb
0.2 0 3.86d 0.05e 0.84b 10.9bc 0.09de 2.56a
0.2 10 4.55d 0.29de 0.95b 10.7bc 0.58bc 2.54a
0.2 20 3.79d 0.59bc 0.77b 9.56c 1.13a 2.25a
2.0 0 4.97d 0.04e 0.40b 12.8a 0.07e 0.93b
2.0 10 4.68d 0.27de 0.35b 11.5abc 0.68b 0.76b
2.0 20 4.20d 0.34cd 0.25b 12.5ab 0.75b 0.93b
6.3.2 Experiment- 2
6.3.2.1 Plant growth
Pre-treatment shoot dry weight and root dry weight were 0.2 and 0.08 g/plant, respectively in
cv. Wyalkatchem, and 0.27 and 0.10 g/plant, respectively in cv. Gutha. The growth response
to K and Na treatments for 48 hours was mostly consistent with Experiment 1. There was no
significant difference among the treatments (Appendix 4).
6.3.2.2 Leaf gas exchange
Photosynthesis measured 2 days after the K and Na treatments showed no significant
difference among the treatments and between two varieties (Fig. 6.2; Table 6.6).
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Table 6.6 Statistical summary of leaf gas exchange in cultivars Wyalkatchem (K-efficient)
and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels
(0.05 and 2 mM K), two Na levels (0, 2 and 10 mM Na) and Rb (0.5 mM) (n=4) added for 48
hours (Experiment 2).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Solution K
Solution Na
cultivar K×Na K×cv Na×cv K×Na×cv
Photosynthesis n.s * n.s n.s ** n.s n.s
Stomatal conductance n.s n.s n.s n.s n.s n.s n.s
Transpiration n.s n.s n.s n.s n.s n.s n.s
114
0.05 mM 2 mM
Tran
spir
atio
n (m
mol
H2O
/m2 .s
)
0
2
4
6
0 Na 2 Na 10 Na
K concentration (mM)
0.05 mM 2 mM
Sto
mat
al c
ondu
ctan
ce (
mm
olH 2O
/m2 .s
)
0
100
200
300
400
Wy alkatchem
Leaf
ne
t ph
otos
ynth
esi
s (µ
mol
CO 2/m
2 .s)
0
5
10
15
20
25
Gutha
Fig. 6.2 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars
Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for
two weeks, followed by two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM)
(±SE, n=4) and measured 42 hours after the treatments (Experiment 2).
6.3.2.3 K, Na and Rb concentrations in shoot and root
Before the K and Na treatments and Rb addition, the shoots contained 18 mg K/g, 0.11 mg
Na/g in cv. Wyalkatchem and 17 mg K/g, 0.12 mg Na/g in cv. Gutha on a dry-weight basis.
Rubidium concentration was below detectable limits (<0.02 mg/g) in both cultivars.
After treatment with high K (2 mM) for 2 days, shoot K concentrations increased
significantly compared with continuous low K treatment in both Wyalkatchem and Gutha,
and the interaction between K and Na was significant for shoot K concentration, and there
115
was a relatively weak interaction between K, Na and cultivar (P= 0.09) (Table 6.7). Addition
of 2 and 10 mM Na increased shoot Na concentrations in both cultivars compared with nil Na
supply, and the three way interaction was significant for shoot Na concentrations (P ≤0.05;
Table 6.7). Shoot Rb concentration in high K treatments was significantly lower than low K
treated plants. The addition of 2 mM Na to low K (0.2 mM K) pots increased shoot Rb
concentration of Wyalkatchem but not Gutha (Table 6.8), and the interaction between K and
Na was significant (Table 6.7). However, in high K treatment, Na addition had no effect on
shoot Rb concentrations in both cultivars.
Table 6.7 Statistical summary of shoot and root K, Na, Rb concentrations and contents in
cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM
K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb
tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting) (Experiment 2;
n=4). *P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Solution K
Solution Na
Cultivar K×Na K×cv Na×cv K×Na×cv
Shoot K conc. *** *** n.s *** * n.s n.s
Shoot Na conc. *** *** n.s ** *** ** ***
Shoot Rb conc. *** *** n.s *** * n.s n.s
Root K conc. *** *** * *** n.s n.s n.s
Root Na conc. n.s *** ** n.s n.s ** n.s
Root Rb conc. *** ** * n.s n.s ** n.s
Shoot K content *** *** *** *** *** * n.s
Shoot Na content *** *** *** n.s *** ** ***
Shoot Rb content *** n.s *** * *** n.s n.s
Root K content *** ** *** ** ** * *
Root Na content n.s *** *** n.s n.s *** n.s
Root Rb content *** n.s *** n.s ** n.s n.s
116
Table 6.8 Shoot and root K, Na, and Rb concentrations in cultivars Wyalkatchem (K-
efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K
levels (0.05 and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a
further 48 hours (harvested 19 days after transplanting) (Experiment 2). Means (n=4) with
different letters differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Shoot ion concentration (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.05 0 16.9d 0.35f 29.2c 16.5d 0.44f 30.8bc
0.05 2 18.4d 1.02de 33.9a 17.7d 1.22cd 33.4ab
0.05 10 17.0d 2.17a 30.1c 17.5d 1.41bc 31.8abc
2 0 50.9ab 0.32f 4.95d 54.8a 0.29f 4.49d
2 2 47.8b 0.80e 5.40d 50.3b 1.12cde 4.08d
2 10 42.8c 1.08cde 4.29d 42.6c 1.62b 3.64d
Root ion concentration (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.05 0 4.68d 0.50d 21.0b 4.80d 0.42d 23.2ab
0.05 2 5.34d 3.94c 24.3a 5.02d 4.04c 25.4a
0.05 10 5.21d 5.31ab 23.2ab 4.11d 5.65ab 23.2ab
2 0 30.1a 0.38d 6.17c 30.5a 0.32d 6.66c
2 2 26.8b 3.97c 5.84c 25.5b 4.05c 6.48c
2 10 24.2bc 5.04b 4.65c 21.4c 5.81a 5.42c
Prior to the K and Na treatments, the roots had 9.12 mg K/g, 0.79 mg Na/g in cv.
Wyalkatchem, and 8.06 mg K/g and 0.76 mg Na/g in Gutha. Rubidium concentrations were
below detection level (<0.02 mg/g) for both cultivars.
Root K, Na and Rb concentrations showed similar response pattern as the shoot
concentrations (Table 6.8). While roots had much lower K concentration than shoots, root K
117
concentrations in both Wyalkatchem and Gutha were significantly higher at high K than low
K supply. Root Na concentrations were related to the solution Na levels, and the roots had
higher Na concentration than shoots at 2 and 10 mM Na. Similar to shoot Rb concentrations,
root Rb concentrations were significantly higher in low K treatment than high K treatment
(Table 6.8). The addition of 2 mM Na to low K treatment (0.05 mM) increased root Rb
concentration of Wyalkatchem significantly (Table 6.8).
6.3.2.4 K, Na and Rb contents in shoot and root
Pre-treatment shoot K and Na contents were 3.62 and 0.02 mg per plant in cv. Wyalkatchem,
4.64 and 0.03 mg in cv. Gutha. The root K and Na contents were 0.71 and 0.06 mg in cv.
Wyalkatchem, 0.84 and 0.08 mg in cv. Gutha.
High K treatments had higher shoot K content than low K treatments in both cultivars. At
high K supply, the shoots of Gutha accumulated more K than Wyalkatchem because of
greater biomass (Table 6.9). Shoot Na content increased with increasing Na levels. At low K
supply, Rb content in shoots of Wyalkatchem increased significantly with 2 mM Na addition
when compared with nil Na treatment, however, the increase was not significant in Gutha.
Roots accumulated much lower K than shoots. Also at low K treatment, Rb content in roots
was nearly 3 fold less than shoot Rb content. Whole plant Rb content of Wyalkatchem was
significantly increased with addition of 2 mM Na to 0.05 mM K treatment (Table 6.10).
118
Table 6.9 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05
and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours
(harvested 19 days after transplanting) (Experiment 2). Means (n=4) with different letters
differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Shoot ion content (mg/plant) (n=4)
K Na Rb K Na Rb
0.05 0 5.76e 0.12g 9.93c 8.14de 0.22efg 15.2a
0.05 2 6.25de 0.35ef 11.5b 8.70d 0.60bcd 16.4a
0.05 10 6.25de 0.80a 11.1bc 8.55de 0.69abc 15.6a
2 0 18.1c 0.11g 1.76d 27.9a 0.15fg 2.31d
2 2 16.8c 0.28efg 1.88d 25.2a 0.58cd 2.06d
2 10 16.3c 0.41de 1.62d 21.9b 0.84a 1.87d
Root ion content (mg/plant) (n=4)
K Na Rb K Na Rb
0.05 0 0.68c 0.07c 3.05c 0.91c 0.08c 4.42ab
0.05 2 0.82c 0.62b 3.82abc 0.90c 0.73b 4.60a
0.05 10 0.79c 0.80b 3.50bc 0.81c 1.10a 4.57a
2 0 4.22b 0.05c 0.87d 6.46a 0.07c 1.41d
2 2 4.38b 0.64b 0.94d 4.66b 0.74b 1.18d
2 10 3.82b 0.79b 0.73d 4.55b 1.24a 1.15d
119
Table 6.10 Plant K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and Gutha
(K-inefficient) in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with
low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na levels (0, 2 and
10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days after transplanting)
(Experiment 2). Means (n=4) with different letters differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Plant ion content (mg/plant) (n=4)
K Na Rb K Na Rb
0.05 0 6.44d 0.19f 12.9c 9.05d 0.29f 19.6a
0.05 2 7.08d 0.97e 15.4b 9.61d 1.33cd 21.0a
0.05 10 7.04d 1.60bc 14.5bc 9.36d 1.79ab 20.2a
2 0 22.3c 0.17f 2.63d 34.4a 0.22f 3.71d
2 2 21.2c 0.93e 2.83d 29.9b 1.31cd 3.24d
2 10 20.1c 1.2de 2.35d 26.5b 2.07a 3.03d
6.4 Discussion
In both short-term experiments, the duration of K and Na treatments was 48 h, which was too
short to cause any significant growth changes in terms of shoot and root weight, and leaf gas
exchange. The big difference in biomass production of both cultivars between two
experiments was due to difference in growth conditions, especially temperature. However, 48
hours after the treatments were applied the difference in shoot growth rate between the two
harvests of Gutha was higher than Wyalkatchem, while, the root growth did not show much
difference among the cultivars.
In Experiment 1, with 0.2 mM K pre-treatment shoot K concentrations were 46.6 and 48.2
mg K/g dry weight in Wyalkatchem and Gutha, respectively. The K concentrations are in
sufficient range for wheat growth (Reuter et al., 1997). With sufficient K concentrations in
shoot already, there would be no need for Na substitution of K functions in plants, regardless
of the levels of Na concentrations applied in Experiment 1. However, previous study by
Rubio et al. (1995) in wheat showed an increased K+ uptake (measured as Rb+ uptake) due to
Na addition at the much lower external Na concentration of 1 mM Na. Therefore, in the
120
follow-up short-term experiment low K treatment was lowered from 0.2 mM K to 0.05 mM
K, and the Na concentrations also lowered from 10 and 20 mM Na to 2 and 10 mM Na,
respectively. Experiment 2 had pre-treatment shoot K concentrations of 18 and 17 mg K/g,
dry weight in Wyalkatchem and Gutha, respectively, which was very much lower than
Experiment 1 and with in the deficient range for wheat growth (Reuter et al., 1997).
Shoot K and Rb concentrations in both cultivars showed no change with Na addition in
Experiment 1. However, shoot and root Rb concentrations increased significantly in cv.
Wyalkatchem treated with 0.05 mM K and 2 mM Na in Experiment 2. Rubidium content in
Wyalkatchem also increased significantly with 2 mM Na addition in Experiment 2, while K
content did not increase significantly. Since Rb+ is often used as an analogue for K+ uptake
(Drobner & Tyler, 1998), the short-term Rb+ increase suggested the capacity for increased K+
uptake in Wyalkatchem with 2 mM Na+ addition to low K solution in this study. Similarly an
increased K+ uptake in wheat due to Na+ via high-affinity K+ uptake activated by added Na+
was observed by Rubio et al. (1995). However, the finding is in contrast with previous K+
uptake study using Rb+ as a tracer in wheat, which reported no stimulation in K+ uptake in
presence of low Na+ concentrations while Na+ stimulated growth at low external K (Box &
Schachtman, 2000).
Plant roots have low and high affinity K+ uptake mechanisms to take up K+ from the
extracellular medium (Britto & Kronzucker, 2008; Szczerba et al., 2009). The external K+
concentration directly influences the activities of K+ channels and transporters and also
regulates the two K+ uptake mechanisms to maintain a steady state flux of K+ (Wang & Wu,
2010). In this experiment, K+ acquisition from adequate K supply (2 mM) would be an
energetically passive process, while that from low external K concentrations (0.05 mM) is
usually considered to be an energy-demanding process (Britto & Kronzucker, 2008), and at
least two high-affinity K+ uptake transporters, KUP/HAK (K uptake transporter/ high affinity
K+ transporter) and HKT1 (High affinity K+ transporter) are shown to be induced by K+
starvation (Szczerba et al., 2009). One of the high affinity K+ uptake mechanisms is a Na+-
energized high-affinity K+ symport HKT1 (Schachtman & Liu, 1999), and it may play an
important role in K+ acquisition when external Na+ concentrations are low (Box &
Schachtman, 2000; Rubio et al., 1995).
The HKT transporter mediates high-affinity K+ uptake and high or low-affinity Na+ uptake
depending on external Na+ and K+ concentrations (Benito et al., 2014). At low external Na+
and K+ concentrations, some transporters function as Na+-K+ symporters, as demonstrated by
121
Na+- stimulated K+ uptake and K+- stimulated Na+ uptake, however, at high external Na+
concentrations, some of these transporters become Na+ uniporters, no longer transporting K+
(Benito et al., 2014; Rubio et al., 1995). Furthermore, recent studies suggest that HKT
transporters discriminate less between K+ and Na+ or even select for Na+ over K+ (Benito et
al., 2014).
Another possible effect of Na+ on transporters as a mechanism for increased K+ uptake could
be mediated by the low-affinity K+ uptake system (such as AKT). At high Na levels (80 mM
NaCl or above), Na+ crosses the plasma membrane causing a significant membrane
depolarization and increases K+ leakage through depolarization-activated outward-rectifying
channels (Shabala & Cuin, 2008). In sharp contrast to 80 mM NaCl treatment, K+ efflux in 20
mM NaCl treatment was found to be very short-lived and K+ uptake became dominant from
elusive ‘osmosensing mechanism’ (Chen et al., 2005). At moderate salinity (20 mM NaCl in
barley), Na+ hyperpolarized the plasma membrane and increased K+ uptake via inward-
rectifying hyperpolarized-activated K+ channels (Chen et al., 2005; Shabala & Cuin, 2008).
Therefore, Na-induced K+ flux was clearly dose dependent, and could possibly explain
increased K+ uptake at low and moderate Na levels in the present study.
In the present short-term experiments in wheat, there was increased Rb+ uptake (as the tracer
for K+ uptake) only at 2 mM Na but not at higher Na levels. The difference in external Na+
concentrations between barley (20 mM Na) and wheat (2 mM Na) where K+ uptake was
increased could be due to the difference between barley and wheat in Na+ uptake and use. For
example, wheat has 10 to 15 fold higher K+/Na+ ratio than barley, indicating that high soil Na
would have greater effect on wheat than in barley (Ma et al., 2011). Also, in comparison to
current results, Na+ stimulated Rb+ uptake via a K+/Na+ symport at low external Na+
concentration of 1 mM Na, however, but not at higher concentration of about 16 mM Na
(Rubio et al., 1995).
The increase in Rb+ or K+ uptake due to Na-stimulated root elongation in wheat is an another
possibility as root elongation can determine nutrient uptake by providing access to additional
nutrient supply (Barber & Silberbush, 1984). However, in this study there was no effect of Na
at deficient K supply in root growth parameters in both cultivars, and therefore it is unlikely
that increased K+ uptake was a direct effect of increased root elongation, but more likely due
to Na+-energised K+ uptake through mechanisms explained above.
122
The results of this study indicate that K-use efficiency of the cultivars had an impact on K+
uptake. We found that Na stimulated greater Rb+ uptake (as a tracer of K+ uptake) in the K-
efficient cultivar Wyalkatchem but not in the K-inefficient Gutha with low external Na
addition (2 mM Na+) for 48 hours. The main mechanism identified by Damon and Rengel
(2007) for K efficiency in wheat cultivars like Wyalkatchem was greater utilization efficiency
of shoot K. However, K-utilization efficiency in terms of growth or yield was not measured
due to short duration of experiment. This experiment was designed to measure short-term
response of K+ uptake with low external Na+. Long-term response in K+ uptake to low
external Na treatments will be measured in the following experiment (Chapter 7).
6.5 Conclusion
In this study, shoot or root growth parameters and leaf gas exchange of wheat cultivars were
not affected by K and Na treatments for 48 hours. Wheat K+ uptake varied with K-use
efficiency of cultivars with significantly increased Rb+ uptake (as a tracer of K+ uptake) in the
K-efficient Wyalkatchem but not in K-inefficient Gutha at deficient K supply (0.05 mM K)
and low external Na+ concentration (2 mM Na). The effect of Na+ on high-affinity and low-
affinity K+ transporters probably contributed to increased K+ uptake under low external Na+
conditions. A long-term solution culture experiment with similar treatments to study the
effect of low to moderate Na+ concentrations on K+ uptake and plant growth is warranted.
123
CHAPTER 7
SOLUTION CULTURE EXPERIMENT: LONG-TERM RESPONSES
Evaluation of potassium (K+) uptake of wheat cultivars under low external sodium
(Na+) supply using rubidium (Rb+) tracer in a solution culture experiment: long-term
responses
7.1 Introduction
Sodium and potassium are structurally and chemically very similar elements and because of
this similarity, the presence of Na+ and its uptake by plants reduces the amount of K+ required
by many plants to meet basic osmotic functions (Benito et al., 2014). It is believed that under
limited K+ supply, Na+ can replace K+ in the vacuole as an alternative inorganic osmoticum,
and the released K+ is available for more K-specific processes (Kronzucker & Britto, 2011).
The presence of tissue Na+ is associated with reduced K+ content in leaves and reduced K+
requirement by plants (Benito et al., 2014). In contrast to this general understanding, my
earlier experiments showed that addition of low to moderate Na (25 and 50 mg Na/kg)
increased plant K uptake when soil K supply was deficient (Chapter 4) especially in K-
efficient wheat cultivars (Chapter 3). The Na-induced K uptake (Rb as a tracer) was evident
in a short-term solution culture experiment with a K-efficient but not in K-inefficient cultivar
after addition of K, Na treatments and Rb tracer for 48 hours (Chapter 6).
There are a number of K+ uptake mechanisms that operate in plants to ensure adequate K is
available for growth and metabolism. Depending on external K concentrations, at least two
transport systems are involved in K+ uptake: a low-affinity system that operates at high K
concentrations and a high-affinity system at lower concentrations (Nieves-Cordones et al.,
2014; Rubio et al., 1995). It has been suggested that one of the high-affinity K+ uptake
mechanisms, Na+- coupled K+ symport HKT1, may play an important role in K+ uptake when
external Na+ concentrations are low (Box & Schachtman, 2000). Solution culture
experiments were designed to investigate whether low external Na+ concentrations play a role
in energising K+ acquisition using Rb+ as a tracer for K+ uptake.
In previous soil culture experiments, there was increase in wheat K+ uptake, leaf
photosynthesis and stimulation of growth when low to moderate Na levels were added to K-
deficient soil (Chapter 3 and 4). Root growth at deficient K was better stimulated than shoot,
and K-efficient cultivars showed a stronger response (Chapter 3). In the solution culture
experiments, the effect of external Na concentration on root growth parameters was assessed
124
in detail by scanning roots as increase in root elongation could contribute to increased K
uptake by root. However, no root growth responses to Na were evident after the 2-day
treatment period (Chapter 6).
The main objective of the solution culture experiments was to examine the effect of Na on K
uptake, thereby avoiding any effect of Na on K availability that may occur in the soil
medium. The previous short-term uptake studies using Rb+ as a tracer suggested that Rb+
uptake in cv. Wyalkatchem was increased with 2 mM Na addition in low K solution (0.05
mM K) for 48 hours (Chapter 6). In this solution culture experiment, the long-term effects of
low external Na+ on K+ uptake and growth of K-efficient and K-inefficient cultivars were
examined.
7.2 Materials and methods
7.2.1 Plant culture
Wheat (Triticum aestivum L.) cultivars Wyalkatchem (K-efficient) and Gutha (K- inefficient)
were grown in a naturally-lit glasshouse at Murdoch University, Perth, Western Australia
(32°04′S, 115°50′E) during late winter to early spring in a nutrient solution culture. Seeds
were germinated and transplanted to 4 L pots in the same procedure as described in Chapter
6. The seedlings were held in plastic lids of the pots supported by polystyrene foam, and the
nutrient solution was continuously bubbled with compressed air. The nutrient solution was
changed every 3 days throughout the experiment. The experiment had a factorial combination
of two wheat cultivars, two K levels, three Na levels and four replicates (see below).
7.2.2 Basal nutrient solution
A modified Hoagland’s solution was used in this experiment with the same composition of
nutrient solution described in previous chapter (Chapter 6).
7.2.3 Potassium and sodium treatments
Two K levels of 0.05 mM (low) and 2 mM K (adequate) and three Na levels of 0, 2 and 10
mM Na, the same as those in Experiment 2 of Chapter 6, were used in this experiment. The
plants were initially grown in 0.05 mM K plus basal nutrient solution without Na for two
weeks. A pre-treatment harvest was made by sampling two plants per pot 17 days after
transplanting to analyse initial ion concentrations. From day 17, half of the pots were
supplemented with extra K to raise the concentrations to 2 mM and the remaining pots were
continuously supplied with 0.05 mM, together with application of the three Na treatments.
125
Two weeks later a second harvest prior to Rb treatment was made with two plants per pot to
evaluate plant growth and ion concentrations. After the second harvest, RbCl was added as a
tracer (0.5 mM Rb) to the K and Na treatments to measure the uptake of Rb, K and Na. All
plants were harvested 48 hours after Rb addition. This experiment was long enough to
observe wheat growth response as plants received the K and Na treatments for 18 days before
final harvest, compared with the harvest was made 48 hours after the K and Na treatments
and Rb+ addition in the previous experiments (Chapter 6).
7.2.4 Measurements
Three harvests were taken: initial harvest of 2 plants/ pot before the K and Na treatments (17
days after transplanting), pre-Rb harvest of 2 plants/ pot after two weeks of K and Na
treatments (32 days after transplanting) and final harvest at 48 hours after Rb addition (35
days after transplanting) of 4 remaining plants/ pot. Plant phenology (leaf numbers, tiller
numbers) was recorded throughout the experiment. Leaf net photosynthesis, stomatal
conductance and transpiration rate were measured before and after treatment application
using LCpro+ advanced photosynthesis system (ADC Bioscientific, UK). The measurements
were made in fully expanded young leaves at ambient relative humidity of 50 %, leaf
temperature of 25˚C, reference CO2 of 380 µmol/mol, and photosynthetically active radiation
of 1500 µmol/m2·s.
At each harvest, shoot fresh and dry weights, and root dry weight were determined. At final
harvest, roots from one plant per pot were harvested for determining root length, surface area,
average diameter, root volume using a root scanner controlled by WinRhizo software
(WinRhizo Pro 2007, Regent Instruments Quebec, Canada). The shoots of four plants were
separated into old leaves (basal 3-5 leaves on main tillers including the leaves that showed K
deficiency/Na toxicity symptoms), young leaves (top 2- 3 leaves on each tiller) and the rest of
shoot for elemental analysis. Roots were collected after washing in DI water. The shoot and
root samples were dried in an oven at 60˚C for 48 hours and their dry weights were recorded.
The samples were then milled, digested as described in Chapter 3 and concentrations of K,
Na and Rb were measured using inductively-coupled plasma-atomic emission spectroscopy.
7.2.5 Statistical analysis
Statistical analyses were conducted using the statistical program SPSS 18.0. Three-way
analysis of variance was conducted to assess the effects of soil K and Na supply and their
126
interactions with cultivars. Tukey’s HSD was computed at P ≤ 0.05 for pair-wise comparison
of treatment means.
7.3 Results
7.3.1 Plant growth
Seventeen days after transplanting, the pre-treatment shoot dry weight and root dry weights
were 0.19 and 0.06 g/plant, respectively in cv. Wyalkatchem, and 0.33 and 0.09 g/plant,
respectively in cv. Gutha.
7.3.1.1 Pre- rubidium harvest
Plants with adequate K supply (2 mM) for two weeks had significantly higher shoot dry
weight than those with continuous low K (0.05 mM) in both Wyalkatchem and Gutha (Fig.
7.1). The addition of Na (2 and 10 mM) had no significant effect in shoot dry weight
compared with nil Na treatment at both K levels. The three way interactions between K, Na
and cultivars were not significant for shoot dry weight (P ≤0.05) (Table 7.1). Root dry weight
of adequate K plants was significantly higher than low K plants at all Na levels. The addition
of 2 mM Na to low K solution increased root dry weight significantly in Wyalkatchem (Fig.
7.1). However, at adequate K supply addition of 2 or 10 mM Na reduced root dry weight in
both Wyalkatchem and Gutha. The interaction between K and Na was significant (Table 7.1),
as was the three-way interaction with the cultivars (P ≤0.05).
127
Sho
ot d
ry w
eigh
t (g
/plt)
0.0
0.5
1.0
1.5
2.0
2.5
0.05 mM 2 mM
Roo
t dr
y w
eigh
t (g
/plt)
0.0
0.1
0.2
0.3
0.4
K concentration (mM)
0.05 mM 2 mM
0 Na 2 Na 10 Na
Wy alkatchem Gutha
Fig. 7.1 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K
levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (harvested 32 days
after transplanting, pre-rubidium addition) (±SE, n=4).
128
Table 7.1 Statistical summary of plant growth in cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K
levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium
harvest), and after Rb treatment for 48 hours (post-rubidium or final harvest).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters K Na Cultivar K×Na K×cv Na×cv K×Na×cv
Pre- Rubidium harvest
Shoot dry wt *** n.s *** n.s *** n.s n.s
Root dry wt *** ** *** *** *** ** **
Post- Rubidium harvest
Shoot dry wt *** * *** *** *** * ***
Root dry wt *** n.s *** *** *** n.s *
Photosynthesis *** *** *** *** n.s n.s *
Stomatal conductance
*** *** *** *** *** n.s n.s
Transpiration ** ** *** * n.s n.s n.s
Post-Rb harvest
Consistent with the pre-Rb harvest, shoot dry weights of adequate K plants at post-Rb harvest
were significantly higher than low K plants in both cultivars (Fig. 7.2). The addition of 2 mM
Na to low K nutrient solution significantly increased shoot dry weight of Gutha. However, at
adequate K, addition of 10 mM Na significantly reduced shoot dry weight (Fig. 7.2). The
interactions between K, Na and cultivars were significant for shoot dry weight (Table 7.1).
Root dry weight of adequate K plants was also higher than that of low K plants. Sodium
addition had no effect on root dry weight between treatments at post- Rb harvest, except in
Gutha at adequate K (2 mM K) there was significant reduction in root dry weight with 10
mM Na (Fig. 7.2). The three way interaction for root dry weight between K, Na, and cultivars
was significant (P ≤0.05; Table 7.1). Root growth was not affected with 2 mM and 10 mM
Na addition at low K supply, but 10 mM Na reduced root surface area, root volume, number
of tips and forks of Gutha at adequate K supply when compared with nil Na (Table 7.2).
129
Sho
ot d
ry w
eigh
t (g
/pla
nt)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.05 mM 2 mM
Roo
t dr
y w
eigh
t (g
/pla
nt)
0.0
0.1
0.2
0.3
0.4
K concentration (mM)
0.05 mM 2 mM
0 Na 2 Na 10 Na
Wy alkatchem Gutha
Fig. 7.2 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K
levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (pre- rubidium
harvest), and after Rb treatment for 48 hours (post-rubidium harvest, 35 days after
transplanting) (±SE, n=4).
130
Table 7.2 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3),
number of tips and forks in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient)
treated with 0.05 mM K for 2 weeks, followed by treatment with two K levels (0.05 and 2
mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested after Rb treatment for
48 hours (35 days after transplanting). Means (n=4) with different letters differ at P≤0.05.
Wyalkatchem Gutha
K (mM)
Na (mM)
Total length (cm)
Diameter (mm)
Surface area (cm2)
Total length (cm)
Diameter (mm)
Surface area (cm2)
0.05 0 3937cde 0.36abc 468d 2578e 0.41a 300e
0.05 2 4426cde 0.35bcd 508cd 3579de 0.38ab 431de
0.05 10 3791de 0.34bcd 418de 3616de 0.39ab 448d
2 0 7282a 0.32cd 744a 6535ab 0.35bcd 707a
2 2 6976a 0.30d 643abc 5561a-d 0.35bcd 614abc
2 10 5898abc 0.34bcd 689ab 4563b-e 0.36abc 548bcd
Wyalkatchem Gutha
K (mM)
Na (mM)
Root Volume (cm3) Tips Forks
Root volume (cm3) Tips Forks
0.05 0 4.07cde 6041d 16356de 3.23e 5199d 9167e
0.05 10 4.75bcd 7760cd 17815de 4.26cde 5110d 13357e
0.05 20 3.89de 5812d 13338e 4.22cde 6063d 13147e
2 0 6.07ab 17593a 47488a 6.33a 14832a 43124a
2 10 5.33a-d 16779a 41388ab 5.41abc 11460b 32353bc
2 20 5.18a-d 16825a 41294ab 4.69b-e 9217bc 25084cd
7.3.2 Leaf gas exchange
Leaf gas exchange parameters were measured 2 weeks after K and Na treatment and before
Rb addition. At low K, 2 mM Na addition significantly increased leaf net photosynthesis rate
of both cultivars compared with nil Na addition (Fig. 7.3). Gutha at low K with nil and 10
131
mM Na had the lowest photosynthesis rate among the treatments. The interactions between
K, Na and cultivars were significant for photosynthesis measurements (Table 7.1). Adequate
K treatment had significantly higher stomatal conductance (Gs) than low K, except Gutha at
10 mM Na. Similarly, addition of 2 mM Na to low K nutrient solution increased stomatal
conductance. Transpiration rate was the lowest in Gutha at low K without Na and the highest
in Wyalkatchem at high K with 2 mM Na. The interaction between K and Na was significant
for stomatal conductance and transpiration (P ≤0.05), but not with cultivars (Table 7.1).
Wy alkatchem
Leaf
ne
t ph
otos
ynth
esi
s (µ
mol
CO 2/m
2 .s)
0
5
10
15
20
25
30
Sto
mat
al c
ondu
ctan
ce (
mm
olH 2O
/m2 .s
)
0
200
400
600
800
0.05 mM 2 mM
Tran
spir
atio
n (m
mol
H2O
/m2 .s
)
0
2
4
6
8
Gutha
K concentration (mM)
0.05 mM 2 mM
0 Na 2 Na 10 Na
Fig. 7.3 Leaf net photosynthesis, stomatal conductance, and transpiration in cultivars
Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks,
followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10
mM) for 2 weeks (±SE, n=4).
132
7.3.3 K, Na and Rb concentrations
Pre-treatment shoot K and Na concentrations (on a dry weight basis) were 19.7 mg K/g and
0.16 mg Na/g in Wyalkatchem, and 19.0 mg K/g and 0.18 mg Na/g in Gutha, respectively.
Root K and Na concentrations were 12.5 mg K/g and 0.77 mg Na/kg in Wyalkatchem, and
10.7 mg K/kg and 1.29 mg Na/kg in Gutha, respectively. Rubidium concentration was below
the detection level in shoots and roots of both cultivars (< 0.02 mg Rb/g).
Pre- Rb harvest
Seventeen days after K and Na treatments, shoots of both cultivars had nearly four- fold
higher K concentration at adequate K supply (53.3 mg K/kg) than at low K (14.9 mg K/kg)
(Table 7.3). At low K, addition of 2 mM Na did not increase shoot K concentration
significantly in either cultivar. Shoot Na concentrations were related to solution Na
concentrations, and increased with increasing Na concentrations in both cultivars from 0.15
mg Na/kg at nil Na to 1.54 mg Na/kg at 10 mM Na (Table 7.3). The three way interaction
between K, Na and cultivars was significant for shoot K concentrations but not for shoot Na
concentrations (P ≥0.05).
Shoot and root K concentration showed similar response to the K and Na treatments. Root K
concentration was 38 mg K/kg at adequate K supply compared with 6.8 mg K/kg at low K
supply. The addition of Na had no effect in root K concentrations at low K supply, whereas
root K concentration decreased with 20 mM Na in both cultivars at adequate K supply. Wheat
roots had lower K concentration than shoots in both cultivars, particularly at low K levels. In
contrast, roots had considerably higher Na concentration than shoots. Root Na concentration
increased with increase in solution Na concentrations. Similar to shoot, the interaction
between K and Na was significant for root K concentration but not for root Na concentration.
Also, the three way interaction between K, Na and cultivar was not significant (Table 7.4).
133
Table 7.3 Shoot and root K and Na concentrations in cultivars Wyalkatchem (K-efficient)
and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with
two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb
harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Shoot ion concentration (mg/g, dry wt.) (n=4)
K Na K Na
0.05 0 15.5de 0.15g 12.6e 0.22fg
0.05 2 17.3d 0.77e 15.3de 0.89de
0.05 10 15.2de 1.22cd 13.6e 1.99a
2.0 0 53.4ab 0.12g 56.2a 0.11g
2.0 2 56.1a 0.77e 52.1bc 0.57ef
2.0 10 52.0bc 1.33bc 49.9c 1.62ab
Root ion concentration (mg/g, dry wt.) (n=4)
K Na K Na
0.05 0 6.99d 0.52d 6.68d 0.48d
0.05 2 7.20d 3.01c 6.46d 3.86bc
0.05 10 6.98d 6.15a 6.63d 6.86a
2.0 0 36.2b 0.39d 44.1a 0.43d
2.0 2 37.5b 3.72bc 43.0a 4.50b
2.0 10 32.2c 5.89a 34.8bc 7.06a
134
Table 7.4 Statistical summary of shoot and root K, Na and Rb concentrations in cultivars
Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks
and then treated with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for
2 weeks (Pre-rubidium harvest), and harvested after Rb treatment for 48 hours (Post-
rubidium harvest). *P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters K Na Cultivar K×Na K×cv Na×cv K×Na×cv
Pre- rubidium harvest
Shoot K conc *** *** *** *** n.s ** ***
Shoot Na conc ** *** *** n.s ** *** n.s
Root K conc *** *** *** *** *** ** **
Root Na conc n.s *** *** n.s n.s * n.s
Post- rubidium harvest
Young leaves
K concentration *** *** *** *** n.s ** ***
Na concentration * *** * * n.s ** n.s
Rb concentration *** *** ** *** ** n.s n.s
Old leaves
K concentration *** *** n.s *** *** *** ***
Na concentration n.s *** n.s n.s n.s * n.s
Rb concentration *** *** *** *** *** n.s n.s
Rest of shoot
K concentration *** *** n.s n.s n.s n.s n.s
Na concentration ** *** ** n.s n.s n.s n.s
Rb concentration *** ** *** n.s *** n.s n.s
Root
K concentration *** *** n.s *** n.s n.s n.s
Na concentration *** *** *** * n.s * n.s
Rb concentration *** *** n.s *** n.s n.s n.s
135
Post-Rb harvest
Forty-eight hours after Rb addition, tissue K and Na concentrations remained similar to those
at pre-Rb harvest, i.e. young leaf K concentrations were higher at adequate K supply than low
K but did not vary with Na addition, except that 10 mM Na reduced young leaf K
concentration in Gutha with adequate K supply (Table 7.5). Young leaf Na concentrations
increased with addition of 2 and 10 mM Na. With Rb addition, young leaf Rb concentrations
in low K treatment were significantly higher than in adequate K treatment. At low K supply,
Rb concentrations in young leaves were significantly increased in both cultivars with addition
of 2 mM Na but decreased with 10 mM Na in Gutha, compared with nil Na (Table 7.5). At
adequate K, there was no change due to Na addition. The interaction between K and Na was
significant (P ≤0.05) for young leaf K, Na and Rb concentrations, while the three way
interaction among K, Na and cultivars was significant only for young leaf K concentrations
(Table 7.4).
Old leaf K concentration in low K treatment was lower than young leaf K concentrations.
Sodium concentration of old leaves was dependent on solution Na concentrations. Old leaf
Rb concentration increased significantly with 2 mM Na addition at low K treatment in
Wyalkatchem but not in Gutha. However, there was significant decrease in Rb concentration
with 10 mM Na addition at low K treatment in both cultivars. The interaction between K and
Na was significant for old leaf K and Rb concentrations while interaction between K, Na and
cultivar was significant only for K concentration (Table 7.4).
The rest of shoots also had similar treatment response as the leaves in terms of K and Na
concentrations. Adequate K treatment had considerably higher shoot K concentration than
low K treatment (Table 7.5). Low K treatment had significantly higher Rb concentration than
adequate K treatment, and the addition of 2 mM Na increased the Rb concentration
significantly in Wyalkatchem, but not in Gutha. The interaction between K, Na and cultivar
was not significant for rest of shoot K, Na and Rb concentrations (Table 7.4).
Roots had lower K concentrations compared with leaves and the rest of shoots. Adequate K
treatment resulted in nearly six-fold higher root K concentration than low K treatment, but Na
addition had no effect on root K concentration. Sodium addition increased root Na
concentration at both K levels. At low K, roots had considerably less Rb than the rest of
shoots, and Rb concentration varied significantly between low and adequate K treatments.
136
The addition of 2 mM Na to low K nutrient solution increased root Rb concentration in both
Wyalkatchem and Gutha.
Table 7.5 Young leaf, old leaf, and the rest of shoot and root K, Na, and Rb concentrations in
cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2
weeks, followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2
and 10 mM) for 2 weeks, and harvested 48 hours after Rb addition (35 days after
transplanting). Means (n=4) with different letters differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Young leaf ion concentration (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.05 0 14.0cd 0.33d 20.4bc 12.3d 0.33d 21.3b
0.05 2 15.7c 1.67c 23.9a 13.8cd 1.66c 23.2a
0.05 10 13.8cd 2.70ab 19.5c 12.3d 2.95a 19.5c
2.0 0 45.5a 0.36d 3.39d 47.5a 0.36d 2.56de
2.0 2 47.9a 1.41c 3.27de 47.4a 1.48c 2.37de
2.0 10 45.3a 2.43b 2.54de 41.5b 2.85a 2.02e
Old leaf ion concentration (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.05 0 10.6de 0.65d 18.4b 7.88e 0.66d 15.2c
0.05 2 11.6d 2.63c 19.6a 8.77de 3.63abc 15.2c
0.05 10 10.3de 4.34a 15.2c 7.84e 4.52a 10.9d
2.0 0 45.0bc 0.65d 2.71ef 51.3a 0.60d 2.14ef
2.0 2 45.9b 2.61c 2.85e 44.6bc 3.04bc 2.03ef
2.0 10 42.6c 4.10ab 2.35ef 42.5c 3.95ab 1.68f
137
Rest of shoot ion concentration (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.05 0 15.1cde 0.37e 29.9b 12.8de 0.37e 26.4cd
0.05 2 17.8c 1.39cd 32.9a 16.5cd 1.23d 26.2cd
0.05 10 13.9cde 2.39ab 27.3bc 12.1e 2.76a 23.8d
2.0 0 59.9a 0.23e 5.05e 60.3a 0.22e 5.53e
2.0 2 59.5a 0.77de 4.65e 59.6a 1.27d 3.89e
2.0 10 54.2b 1.96bc 3.39e 54.0b 2.45ab 3.08e
Root ion concentration (mg/g, dry wt.) (n=4)
K Na Rb K Na Rb
0.05 0 5.25c 0.90d 16.4bc 4.46c 0.84d 16.3bc
0.05 2 4.67c 4.18c 18.9a 4.07c 4.59c 18.0a
0.05 10 4.04c 6.36ab 15.8bc 3.82c 6.93a 15.0c
2.0 0 33.2a 0.72d 7.21de 33.4a 0.69a 7.52d
2.0 2 31.6a 3.93c 6.21de 32.4a 4.38c 6.52d
2.0 10 23.7a 5.72b 4.93ef 28.0ab 6.18b 3.41f
7.3.4 K, Na and Rb contents
At pre-Rb harvest shoot K content was significantly higher in adequate K treatment than low
K treatment (Table 7.6). There was little change in shoot K uptake due to Na addition or
between the cultivars Wyalkatchem and Gutha. Shoot Na content increased with increasing
Na levels (Table 7.6). Roots had lower K content but higher Na content than shoots (Table
7.6). The interaction between K and Na was significant for shoot and root K and Na content,
while three way interactions with cultivar were significant for shoot and root K content
(Table 7.7).
138
Table 7.6 Shoot and root K and Na contents in cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, and harvested after treatment with
two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks (Pre-Rb
harvest; 32 days after transplanting). Means (n=4) with different letters differ at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Shoot ion content (mg/plant) (n=4)
K Na K Na
0.05 0 15.5de 0.15g 12.6e 0.22fg
0.05 2 17.3d 0.77e 15.3de 0.89de
0.05 10 15.2de 1.22cd 13.6e 1.99a
2.0 0 53.4ab 0.12g 56.2a 0.11g
2.0 2 56.1a 0.77e 52.1bc 0.57ef
2.0 10 52.0bc 1.33bc 49.9c 1.62ab
Root ion content (mg/plant) (n=4)
K Na K Na
0.05 0 6.99d 0.52d 6.68d 0.48d
0.05 2 7.20d 3.00c 6.46d 3.86bc
0.05 10 6.98d 6.15a 6.63d 6.86a
2.0 0 36.2b 0.39d 44.1a 0.43d
2.0 2 37.5b 3.72bc 43.0a 4.50b
2.0 10 32.2c 5.89a 34.8bc 7.06a
139
Table 7.7 Statistical summary of shoot and root K, Na and Rb contents in cultivars
Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks,
followed by treatment with two K levels (0.05 and 2 mM) and three Na levels (0, 2 and 10
mM) for 2 weeks (Pre-Rb harvest) and harvested 48 hours after Rb treatment (post-rubidium
or final harvest).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters K Na Cultivar K×Na K×cv Na×cv K×Na×cv
Pre- rubidium harvest
Shoot K content *** *** *** *** *** *** ***
Shoot Na content *** *** *** *** n.s *** n.s
Root K content *** *** *** *** *** ** **
Root Na content *** *** * *** ** n.s n.s
Post- rubidium harvest
Shoot
K content *** *** *** *** *** *** ***
Na content *** *** *** * * *** n.s
Rb content *** *** *** *** *** n.s ***
Root
K content *** *** n.s *** ** n.s n.s
Na content n.s *** n.s n.s n.s n.s n.s
Rb content *** *** *** *** *** n.s ***
Post-Rb shoot K content at adequate K supply was higher than at low K supply in both
Wyalkatchem and Gutha. At low K supply, shoot K content was significantly higher with 2
mM Na addition than with 10 mM Na in both cultivars and with nil Na in Gutha. Shoot Na
content increased significantly with 2 and 10 mM Na addition (Table 7.8). Shoot Rb content
was lower at adequate K treatments than low K treatments. Shoot Rb increased significantly
with 2 mM Na addition to low K treatment. Root K and Rb contents were much lower than
the shoot contents (Table 7.8). The addition of 2 mM Na increased root Rb content of
140
Wyalkatchem and Gutha at low K. The interaction between K and Na was significant for
shoot K, Na and Rb contents, while three way interactions between K, Na and cultivar were
significant for shoot K and Rb contents. The interaction between K, Na and cultivars was also
significant for root Rb content.
Table 7.8 Shoot and root K, Na, and Rb contents in cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with 0.05 mM K for 2 weeks, followed by treatment with two K
levels (0.05 and 2 mM) and three Na levels (0, 2 and 10 mM) for 2 weeks, and harvested 48
hours after Rb addition (35 days after transplanting). Means (n=4) with different letters differ
at P≤0.05.
K (mM) Na (mM) Wyalkatchem Gutha
Shoot ion content (mg/plant) (n=4)
K Na Rb K Na Rb
0.05 0 17.2gh 0.49f 31.3c 18.6gh 0.61f 36.5b
0.05 2 19.8g 1.96e 34.7b 25.2f 2.86d 40.8a
0.05 10 15.5h 3.2cd 27.3d 19.8g 5.13b 36.2b
2.0 0 90.1d 0.51f 7.24fg 152a 0.78f 12.3e
2.0 2 92.2d 1.84e 6.9fg 134b 3.61cd 8.20f
2.0 10 83.5e 3.71c 5.05g 113c 6.07a 6.10fg
Root ion content (mg/plant) (n=4)
K Na Rb K Na Rb
0.05 0 1.95d 0.33c 6.12b 1.41d 0.26c 5.13d
0.05 2 1.84d 1.64b 7.46a 1.34d 1.52b 5.92bc
0.05 10 1.57d 2.48a 6.15b 1.35d 2.47a 5.35cd
2.0 0 14.6ab 0.32c 3.18ef 15.1a 0.32c 3.41e
2.0 2 14.0ab 1.73b 2.76fg 13.5b 1.82b 2.72fg
2.0 10 10.3c 2.49a 2.15g 11.2c 2.48a 1.35h
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7.4 Discussion
This experiment showed no significant increase in shoot dry weight with Na addition for two
weeks to low K treatment in both cultivars at pre-Rb and post-Rb harvest. This is in contrast
to the previous split-root experiment (Chapter 4) where there was significant increase in
shoot dry weight in Wyalkatchem with Na addition of 50 mg Na/kg to one or both the
compartments at deficient soil K supply for 5 to 6 weeks. It is consistent however, with the
lack of shoot dry weight response to low Na in K-deficient wheat in Chapter 3. The root dry
weight of Wyalkatchem showed an increase with 2 mM Na addition to low K (0.05 mM K)
for 2 weeks. In previous pot experiment (Chapter 3), root dry weight increased only in K-
efficient cultivars (including cv. Wyalkatchem) with soil K deficiency and Na addition, and
also in split-root experiment at low K with 50 mg Na/kg Na addition, where the increase in
root dry weight was comparable to that with adequate K supply. The stimulation in shoot and
root dry weight due to Na in previous studies at 25 and 50 mg Na/kg was equivalent to 7.25
and 14.5 mM Na in soil solution at 15 % field capacity which was slightly higher than the
concentrations of 2 and 10 mM Na in the present study. Unlike the present study, there were
moderate to severe K deficiency symptoms observed in previous studies where positive
response to Na addition was observed in shoot and root growth. In an earlier solution culture
experiment with much lower Na concentrations, wheat biomass increased in one of the
experiments when 500 µM Na was added to nutrient solution with deficient external K (20
µM K) when there were low levels of light (Box & Schachtman, 2000). In the present study,
there was an increase in root dry weight of Wyalkatchem but no change in shoot dry weight.
Potassium has a vital role in regulating photosynthesis and stomatal aperture, and when plants
are K deficient, the rate of photosynthesis is depressed (Cakmak, 2005). In K deficient plants,
it was believed that Na+ substitutes K+ in maintaining ionic balance, osmotic pressure and
stomatal conductance (Marschner, 1995; Subbarao et al., 2003). Also, under K-deficient
conditions, low levels of Na can be beneficial to physiological processes, and this is mainly
attributed to the function of K+ as an osmoticum in vacuoles which can be replaced by Na+
(Gattward et al., 2012; Gierth & Mäser, 2007).
In this long-term solution culture experiment there was a significant increase in leaf net
photosynthesis rate and stomatal conductance with 2 mM Na addition to low K treatment
solution in both cultivars. The increase in leaf net photosynthesis rate as a result of 2 mM Na
was almost equal to the presence of adequate K in nutrient solution (2 mM K). Similarly, in a
study by Gattward et al. (2012) on cocoa a significant increase in photosynthesis, nearly
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double than that of control (nil Na) was noticed with 40 % replacement of K+ by Na+ in the
soil medium. With increased photosynthesis in shoots of K-deficient wheat plants supplied
with low Na, more assimilates would be available for the need of root growth and nutrient
uptake. This was also the case in Chapter 3.
At the level of 2 mM K supply, there was enough K+ uptake to maintain adequate leaf
concentration and maximum growth. The K+ uptake mechanism which operates effectively at
2 mM K+ may, however, not operate efficiently to supply K+ required for plant growth at 0.05
mM K, as external K+ was too low to supply K requirements for growth. A recent evaluation
of mechanisms of K+ uptake at various external concentrations showed that non-selective
channels are the main pathway for K+ uptake at high concentrations (> 10 mM K). The
inward rectifying channel AKT1 dominates K+ uptake at intermediate concentrations (around
1 mM K), while at lower concentrations (0.1 mM K), AKT1 along with the high-affinity K+
uptake system (HAK) are the dominant K uptake systems. At extremely low concentrations
(< 0.01 mM), the only system capable of K+ acquisition is HAK5 or HAK1 (Nieves-
Cordones et al., 2014).
In this experiment, pre-Rb harvest showed no change in shoot or root K concentrations and K
uptake of both cultivars in the presence of low external Na+ at low K. Similarly the K
concentrations measured after Rb+ tracer addition for 48 hours showed no significant increase
in shoot parts of both Wyalkatchem and Gutha with Na addition to low K treatment solution.
However, 2 mM Na addition to 0.05 mM K treatment solution significantly increased Rb
concentrations (tracer for K+ uptake in this study) in young leaves, old leaves, rest of shoot,
i.e., in all parts of shoots and roots of Wyalkatchem, and in young leaves and roots of Gutha.
Shoot and root Rb+ uptake showed a significant increase in both cultivars with 2 mM Na
addition to low K treatments. Therefore, the findings showed evidence of Na+-coupled Rb+
influx in wheat roots and shoots with much stronger influx in K-efficient Wyalkatchem than
K-inefficient Gutha with external Na+ concentration of 2 mM Na but not at 10 mM Na.
In this experiment when Rb+ uptake was stimulated by low concentrations of Na+ addition,
K+ uptake increased but was not significant with Tukey’s HSD comparison. This may be due
to differences in Rb+ and K+ uptake behaviour. Negative feedback from K in plants may
decrease K+ uptake in long term studies but not Rb+ uptake since Rb do not have an essential
physiological role in plants. Moreover, the large amount of K in shoots already makes it
difficult to detect a small increase during the treatment period which can only be detected
using tracer such as Rb or radio-active K source.
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An earlier study in wheat using Rb+ as a tracer by Box and Schachtman (2000) showed that
low concentrations of Na+ do not increase K+ uptake to a large extent suggesting that Na+-
energised K+ is not a major mechanism for high-affinity K+ acquisition (Box & Schachtman,
2000). In this experiment, the increased K+ uptake with 2 mM Na addition to deficient supply
of K in nutrient solution observed could be explained by effect of Na+ on K transporters and
uptake mechanism. One possible mechanism is that in wheat roots, the high-affinity K+
uptake transporter HKT1 was shown to function as a K+-Na+ cotransporter with activation of
high-affinity K+ uptake by micromolar Na concentrations (Rubio et al., 1995). However,
more recent evidence emerged that HKT operates predominantly as a Na+ channel by
selecting Na+ over K+ (Benito et al., 2014). Another possible mechanism is mediated by low-
affinity K+ uptake involving Na+ hyper-polarization of the plasma membrane and increased
K+ uptake via inward-rectifying activated K+ transporters (Chen et al., 2005; Shabala & Cuin,
2008). The latter mechanism could also account for increased Rb+ uptake by wheat.
An increase in root Na concentration in K-deficient plants was shown to stimulate root
growth in cotton (Ali et al., 2009) and in previous soil culture experiments of wheat
(Chapters 3 and 4). Whether an increase in root elongation could contribute to increased plant
K uptake was examined by scanning of roots to assess length, surface area etc., as such an
effect could contribute to nutrient uptake by providing access to additional nutrient supply
(Barber & Silberbush, 1984). The results showed no significant increase in root dry weight,
or other parameters like root length, volume and surface area when Na+ was added to low K
treatments suggesting that increased root length and or surface area was not the reason for Na
stimulated K+ and Rb+ uptake..
The results from K+ uptake studies suggest that Na+-coupled Rb+ influx in wheat varied
between short-term and long-term experiments. In the short-term experiment, when Na
treatments and Rb were added for 48 hours, there was an increase in shoot and root Rb+
concentrations, and shoot Rb+ uptake in Wyalkatchem only, while in the long-term
experiment where plants were treated with Na treatments for 16 days before Rb+ addition
there was increased Rb+ influx due to Na+ in both cultivars. Hence the K-efficient cultivar
had quicker response to low to moderate Na supply relative to the K-inefficient cultivar as the
former increased Rb+ uptake with short-term exposure of Na treatment but the latter required
long-term Na exposure for showing Na+-energised Rb+ (K+) uptake. The solution culture
results showed that Na stimulated greater K uptake by K-efficient cultivars, consistent to the
finding from my pot experiment (Chapter 3).
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In previous study to rank wheat genotypes according to K-use efficiency there was no
examination of the role of Na (Damon & Rengel, 2007). In the present study, the relative K-
use efficiency of wheat cultivars was consistent across a range of Na levels from no added Na
up to toxic levels. Moreover in this study, K-efficient cultivar showed better response with
low to moderate Na supply relative to K-inefficient cultivar. In contrast to the suggestion that
increased capacity to substitute Na+ for K+ may be a mechanism underlying K-use efficiency
in wheat (Damon & Rengel, 2007), our results suggest that Na stimulated greater Rb+ uptake
(tracer for K+ uptake) by K-efficient cultivars. This raises the question of whether Na+
activation of K+ transporters should be re-examined in wheat cultivars with a range of K-use
efficiencies.
7.5 Conclusion
The addition of low Na (2 mM) to low K (0.05 mM) solution for 14 days increased root dry
weight of Wyalkatchem and increased leaf net photosynthesis and stomatal conductance in
both Wyalkatchem and Gutha. There was an 11 % increase in shoot Rb+ uptake in both
cultivars, and 22 % and 13 % increase in root Rb+ uptake of Wyalkatchem and Gutha,
respectively with addition of low Na. This study showed an increase in K+ uptake (as
measured with Rb+) suggesting the likely role of Na+ in energising K+ uptake by an effect of
Na+ on low-affinity K+ uptake involving Na+ hyper-polarization of the plasma membrane via
inward-rectifying activated K+ transporters or high-affinity transporter HKT1 which can
function as a K+-Na+ cotransporter.
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CHAPTER 8
GENERAL DISCUSSION AND CONCLUSIONS
8.1 Introduction
Under K deficiency, Na can substitute for K in maintaining ionic balance (Subbarao et al.,
2003), regulating osmotic pressure (Marschner, 1995), exerting vacuolar functions (Mäser et
al., 2002), and improving water balance via stomatal conductance (Gattward et al., 2012).
However, the degree of substitution varies among species and cultivars and so far there is a
paucity of information on effects of Na on K nutrition of wheat especially in soil-based
conditions (Ma et al., 2011). A good understanding of Na effects on K nutrition would
optimise soil K use and improve K fertilizer management and grain yield of wheat grown
under sodic and saline soils. Previously it has been suggested that cultivar differences in K-
use efficiency in wheat may be related to variation in Na substitution of K (Damon & Rengel,
2007) but there was no direct evidence available on whether the K-efficient genotypes would
be more or less responsive to soil Na supply than the K-inefficient genotypes. The main
objective of this thesis was to investigate the interaction between K and Na in wheat cultivars
at levels of NaCl that caused salinity and those that did not. A series of experiments were
designed to study the effect of NaCl on wheat K uptake, leaf gas exchange, cation ratios, and
plant growth in a range of K (deficient to adequate) and Na concentrations (low to toxic). The
experiments were conducted in both soil and solution culture systems and tested cultivars
differing in K-use efficiency and used a range of experimental approaches including split-
root, water deficit and tracer flux studies.
8.2 Growth stimulation by Na
Although Na is not an essential element for glycophytes such as wheat, the addition of Na
stimulated growth under limited K availability in three experiments of this study including
soils in standard pots, in split-root pots and in solution culture. Tiller number and shoot dry
weight were significantly increased in the split-root experiment when 50 mg Na/kg was
added to one or both compartments of 40 mg K/kg treatments which otherwise induced
severe K deficiency (Chapter 4). Low to moderate Na supply also alleviated K deficiency
symptoms in two pot experiments (Chapters 3 and 4). However, in the initial pot experiment
with cultivars differing in K-use efficiency, shoot dry weights were not significantly affected
by low to moderate Na (25 to 50 mg Na/kg equivalent to 7.25 and 14.5 mM Na in soil
solution at 15 % field capacity) in K deficient plants (Chapter 3). Similarly, shoot weight did
146
not show significant stimulation with low or moderate Na additions to low K treatments in
the column experiment (Chapter 5) or solution culture experiments (Chapter 6 and 7).
Root growth was more responsive to Na at low K supply than shoot in experiments where
growth stimulation was observed. The root growth in K-efficient cultivars increased
significantly with low to moderate Na addition (25 to 50 mg Na/kg) under low soil K
(Chapter 3). Similarly, in the long-term solution culture experiment root growth stimulation
was seen at pre-Rb harvest only in K-efficient Wyalkatchem treated with 2 mM Na and 0.05
mM K (Chapter 7). In pot experiment and long-term solution culture experiment (Chapters 3
and 7), Na-induced growth stimulation was clearly evident in roots but not in shoots. The root
growth stimulation was much stronger in split-root experiment in Wyalkatchem with addition
of 50 mg Na/kg to one or both of the compartments at deficient soil K (40 mg K/kg). Indeed
the magnitude of root growth stimulation with 50 mg Na/kg was comparable to that with
adequate K supply (Chapter 4).
Even though the same levels of low K treatments (40 mg K/Kg) were used in all three soil-
based experiments, the plant to soil weight ratio varied considerably and this altered the
degree of K deficiency. Potassium deficiency was very severe in the split-root experiment
with a plant (number) to soil weight (kg) ratio of 1: 2.0 (Chapter 4), moderately severe in the
pot experiment with a ratio of 1: 2.07 (Chapter 3), and in the column experiment with plant to
soil weight ratio of 1: 5.0 (Chapter 5) wheat did not show any K deficiency symptoms or Na-
induced growth stimulation at low K supply. These experiments differed in the depth and
volume of soil that can be exploited by roots, and the pool of K available for K uptake in the
column experiment was 2.5 times greater with an extra 160 mg K/plant available than in the
pot and split-root experiments. As the available soil K can be locally depleted by root K
uptake in the pot, continued K uptake is dependent on root extension to access K available
elsewhere in the pot or deeper in the soil profile (Damon & Rengel, 2007). Therefore, in the
pot and split-root experiments as available K was depleted, added Na replaced some roles of
K by maintaining better plant growth than nil Na treatment. Hence in retrospect the research
questions posed for the column experiment (Chapter 5) would have been more effectively
tested if the ratio of plant number to soil weight was similar to that in the pot and split root
experiments (Chapters 3 and 4).
Wheat growth was stimulated when 0.5 mM Na was added to 0.02 mM K solution in an
earlier study, however, at much lower concentration of K and Na compared to this study, and
moreover, there was no differentiation between shoot and root growth in the study (Box &
147
Schachtman, 2000). The present research in wheat showed contrasting effects of low to
moderate Na to those with barley where addition of 100 mg Na/kg stimulated significant
shoot growth increases but not root growth of barley (Ma et al., 2011). In salt-tolerant barley,
addition of 100 mg Na/kg stimulated significant shoot growth but not root growth, whereas
in wheat 100 mg Na/kg reduced the growth of K deficient plants (30 mg K/kg) (Ma et al.,
2011). Similar findings were obtained from the pot experiment in this thesis where 100 mg
Na/kg for 8 weeks had negative effects on plant growth (Chapter 3). The stimulative effect of
Na was at much lower Na levels in wheat (25- 50 mg Na/kg soil) than in barley (100 mg
Na/kg soil) and in wheat there was stronger response in roots than shoots.
8.3 Na effects on K deficient wheat
Typical K deficiency symptoms started to appear at low soil K (40 mg K/kg) with yellowing
of older leaves and brown spots in shoots at 6 weeks after germinated seeds were transferred
in the pot experiment in all wheat cultivars irrespective of K-use efficiency (Chapter 3). The
treatments of 25 and 50 mg Na/kg eliminated the deficiency symptoms and produced greener,
more erect leaves (Refer to earlier photos). Severe K deficiency symptoms developed and
persisted in the treatment of nil Na and low K supply. Under split-root condition, addition of
50 mg Na/kg in just one of the two root compartments was able to alleviate K deficiency
symptoms and produced healthy plants with increased tillering (Chapter 4). Previous study
also reported that rice plants grown without NaCl under K deficiency showed yellow
discoloration with droopy leaves and marginal necrosis, while in NaCl-treated plants the
leaves remained erect and greener (Yoshida & Castaneda, 1969).
Sodium supply had a beneficial role in leaf net photosynthesis, stomatal conductance and
transpiration at limited K supply in this study. At low K (40 mg K/kg), addition of low to
moderate Na (25 to 50 mg Na/kg) increased leaf photosynthesis and stomatal conductance to
measured values similar to those under adequate K and nil Na conditions (Chapter 3) and
there was three-fold increase in leaf gas exchange measurements in split-root experiment, at
low K, with addition of 50 mg Na/kg to just one of the compartments when compared with
low K without Na (Chapter 4). Similar increase in leaf gas exchange was seen in long-term
solution culture experiment with addition of 2 mM Na+ to 0.05 mM K solution (Chapter 7).
However, the lack of shoot growth response in the pot experiment even though gas exchange
increased with Na supply to low K plants is puzzling. It could be related to the late
appearance of K deficiency as symptoms first appeared after 6 weeks growth only 2 weeks
148
before harvest in the pot experiment (Chapter 3). Hence it is possible that the shoot response
lagged behind that of the root and given time would have been measurable. This is consistent
with low root-shoot ratio being a pronounced response to low K (Ma et al., 2011). Hence the
stimulation of gas exchange by low to moderate Na in low K plants may increase
carbohydrate supply first to roots and later to shoots. Similarly, in long-term solution culture
experiment, an increase in root growth of Wyalkatchem and photosynthesis of both cultivars
were measured with 2 mM Na addition to low K plants, even though there was no significant
shoot response (Chapter 7). However, in split-root experiment K deficiency symptoms were
severe and early (3-4 weeks after transplanting) compared to other experiments and the
beneficial effects of Na was evident in both shoots and roots (Chapter 4).
The positive effect of Na on photosynthetic and respiratory CO2 exchange has been long
reported in sugar beet under K deficiency, suggesting that Na+ may have substituted for K+ in
stomatal opening either as an alternative cation to K or by conserving K supply (Terry &
Ulrich, 1973). However, the evidence gathered in the present study suggests that the low Na
supply that stimulated photosynthesis and stomatal conductance did not increase leaf or shoot
Na concentrations sufficiently to account for Na substitution of K. For example in pot
experiment in the low K plants, shoot K concentration was 13.9 mg K/g, dry weight
(equivalent to 89.5 mM K in tissue water) at 25 mg Na/kg of soil, whereas, depending on
cultivars there was only 0.18-0.33 mg Na/g, dry weight (mean tissue water Na concentration
of 2.8 mM) which was too low to replace K+ functions. Instead, the addition of Na increased
shoot K concentration/uptake and it is most likely that the photosynthesis and stomatal
conductance responses were due to increased leaf K concentration.
Sodium has the potential to replace K+ in certain non-specific metabolic functions in certain
plant species (Wakeel et al., 2011). High-affinity Na+ uptake in several plants take place
when K+ has been exhausted (Subbarao et al., 2003) suggesting that Na+ plays a
physiologically programmed role at insufficient K+ supply (Wakeel, 2013). In contrast to
previous suggestions, our study suggest that beneficial effect of Na+ in wheat can be
attributed to Na+-induced K+ uptake not to Na+ uptake.
8.4 Stimulation of K uptake by Na
The increase in K+ uptake due to addition of Na+ at deficient K supply was evident in wheat
in both soil-based and solution culture experiments. There was significant increase in shoot K
content of K-efficient cultivars, Wyalkatchem and Cranbrook with 25 and 50 mg Na/kg
149
addition to low K soil (40 mg K/kg) (Chapter 3). Also, the addition of 50 mg Na/kg to one or
both of the compartments in split-root system with low K supply increased plant K content
significantly (Chapter 4). In tracer flux experiments, a short-term exposure to Rb+ (48 hours)
in low K solution (0.05 mM) containing 2 mM Na+ increased shoot Rb+ content of
Wyalkatchem (K-efficient cultivar) significantly (Chapter 6) but not Gutha (K-inefficient
cultivar). Long-term Na+ addition (14 days) showed Na+-induced K+ uptake (measured as
Rb+ uptake) in both cultivars. There was 11 % increase in shoot Rb+ uptake in both cultivars,
and 22 % and 13 % increase in root Rb+ uptake of Wyalkatchem and Gutha, respectively with
the treatment of 2 mM Na and 0.05 mM K (Chapter 7).
In this research, low to moderate Na induced beneficial effects in wheat at low K likely by
increasing K+ uptake. However, in an earlier research by Box and Schachtman (2000), there
was no evidence of enhanced K+ content in wheat due to Na supply, even though there was
an increase in wheat growth due to external Na+, indicating the positive effect of Na at low K
can be largely attributed to partial substitution of Na+ in wheat K functions and a direct effect
of Na+ on growth. In their experiment, tissue Na+ concentration increased significantly in the
presence of external Na+ when there was low solution K (20 µM K+), corresponding to
decrease in shoot K+ concentration. An increase in wheat K+ uptake (measured as Rb+
uptake) due to external low Na addition of 1 mM was seen in the uptake experiment by
Rubio et al. (1995) mediated by HKT1 transporter of wheat roots. Rubidium uptake was
increased significantly from 0.05 nmol/mg/min at nil Na to 0.40 nmol/mg/min at 1 mM Na in
solution containing 15 µM Rb+ as a tracer for K+ uptake (Rubio et al., 1995). Similarly in a
tomato growth experiment, there was significant increase in whole plant K content but not
root K+ content with 1 or 5 mM Na+ addition to low K (0.5 mM K+) treatments (Walker et al.,
2000). However, the increase in plant K uptake was not seen in their K+ uptake experiment of
tomato (Walker et al., 2000).
On the other hand, an increase in plant Na+ or shoot Na+ uptake and shoot Na+ concentrations
with addition of external Na at deficient K supply were attributed to Na+ substitution of K+
functions in few studies. There was an increase of up to 291 % in leaf Na+ content in cacao
tree with increasing replacement of K+ by Na+ at 2.5 mM soil K (Gattward et al., 2012), shoot
Na+ content of rice increased more than 10 fold with Na+ addition to K-deficient plants
(Yoshida & Castaneda, 1969), also there was significant increase in Na+ content of
eucalyptus (Almeida et al., 2010) and tomato (Tahal et al., 2000) where there was beneficial
effects seen with Na addition.
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8.5 Possible mechanisms of Na-induced K uptake
Plant roots have low and high-affinity K+ uptake mechanisms to absorb K+ from the
extracellular medium (Britto & Kronzucker, 2008; Szczerba et al., 2009). A possible
mechanism for increased K uptake is Na activation of K+ symporters in roots. Under K
deficiency, there is increased expression of the high-affinity K+ transporter (HKT) (Anschütz
et al., 2014). The HKT transporter mediates high-affinity K + uptake and high or low-affinity
Na+ uptake depending on external Na+ and K+ concentrations (Benito et al., 2014). At low
external Na+ and K+ concentrations, some transporters function as Na+-K+ symporters,
however, at high external Na+ concentrations, some of these transporters become Na+
uniporters, no longer transporting K+ (Benito et al., 2014; Rubio et al., 1995). Transporters of
the HKT-type discriminate less between K+ and Na+ or even select for Na+ over K+ (Benito et
al., 2014). However, Box and Schachtman (2000) reported the Na+ activation of K+
symporters increased K+ uptake only under low light conditions in wheat and concluded that
it was functionally a minor process for K+ uptake by wheat. Nevertheless, according to the
present study, there was increased K+ uptake due to Na+ in soil (Chapters 3 and 4), and
solution culture experiments (Chapter 7), suggesting there may be effects of Na+ on
transporters not identified by Box and Schachtman (2000).
An alternative mechanism for increased K+ uptake could be by a low-affinity K+ uptake
system (such as AKT). At high Na levels (80 mM NaCl or above in barley), Na+ causes a
significant membrane depolarization and increases K+ leakage, whereas, in sharp contrast to
80 mM NaCl treatment, K+ efflux in 20 mM NaCl treatment was found to be very short-lived
and K+ uptake became dominant from the elusive ‘osmosensing mechanism’ (Chen et al.,
2005). At moderate salinity (20 mM NaCl in barley), Na+ hyperpolarized the plasma
membrane and increased K+ uptake via inward-rectifying hyperpolarized-activated K+
channels (Chen et al., 2005; Shabala & Cuin, 2008). Therefore, Na-induced K+ flux was
clearly dose dependent, and could possibly explain increased K+ uptake at moderate Na
levels. In this study, estimated soil solution concentrations ranged from 0 to 60 mM Na (in
soil culture experiments, Chapter 3, 4 and 5) and the actual solution concentrations ranged
from 0 to 20 mM Na (solution culture experiments, Chapter 6 and 7). Since wheat is less salt-
tolerant than barley (Ma et al., 2011), it is possible for levels of 60 mM Na high enough to
cause membrane polarisation in wheat. However, the concentrations of Na+ that cause
membrane hyperpolarization and depolarization in wheat needs to be verified with
experiments.
151
Other than Na+ effect on K+ transport across membranes that facilitate K+ uptake there may
be other possibilities that could contribute to increased K+ uptake. The Na-induced growth
stimulation and K+ uptake could be due to an increase in extractable soil K availability by soil
Na supply. In the incubation experiment, soil exchangeable and Colwell K showed no
significant increase with addition of Na (Chapter 3). However, in soil-based experiments for
a pot with 6 kg of soil, the increase in Colwell K was equivalent to around 18 mg in the 50
mg Na/kg treatment and provided 6 mg of extra K+ to each plant in the 3-plant pots, which
would account for part but not the entire increase of K content in Na-added plants. Clearly in
the solution culture experiment where there was also a Na stimulated uptake of K (Rb), the
effect on soil available K can be ruled out. Hence Na effects on soil available K are a possible
mechanism but not the only one or the most likely mechanism in these soils.
The Na+-induced increase in root growth due to Na+ could increase K+ uptake in wheat. The
increase in root Na concentrations may stimulate root elongation of K-deficient plants by
turgor effects on cell expansion in soil-based systems where root elongation has a major role
in determining nutrient uptake by providing access to additional nutrient supply (Barber &
Silberbush, 1984). An increase in root dry weight with low to moderate Na addition at low K
was seen in pot experiment particularly for K-efficient cultivars but there was no direct
evidence of increased root K uptake (Chapter 3). In split-root experiment the increase in root
dry weight of cv. Wyalkatchem with 50 mg Na/kg to one or both the compartments was
accompanied with significant increase in K uptake (Chapter 4). In long-term solution culture
experiment (Chapter 7), root dry weight of cv. Wyalkatchem increased only at Pre-Rb
harvest. Detailed root scanning results did not show any change in root length, root volume or
area with low external Na addition to low K solution in both short-term and long-term
experiments, despite an increase in root K+ uptake (as measured using Rb+ tracer) (Chapters 6
and 7). It is unlikely that Na supply under K deficiency would directly increase root dry
weight because root growth is impaired by limited assimilate translocation to roots in low K
and hence an increase in photosynthesis would have to be triggered first before root growth
would respond (Lemoine et al., 2013). In this study, low to moderate Na increased leaf
photosynthesis, which would lead to greater assimilate supply to roots and thus the
stimulation of root growth as appeared to occur in pot and split-root experiments and the
long-term solution culture experiment.
A possible increase in shoot K content could be due to increased K partitioning to shoots in
wheat. An increase in root Na concentrations at low to moderate Na may release vacuolar K+
152
that is made available for cytoplasmic functions in the root cells or for translocation to the
shoot (Walker et al., 2000). In tomato roots, decreased K+ content and increased Na+ content
with 1 or 5 mM NaCl suggest release of K+ to maintain K+-dependent biochemical processes
in the cytoplasm or be available for translocation to shoots (Walker et al., 2000). In the
present experiments, Na+ content in roots of wheat greatly exceeded that in shoots, and
concentrations of Na+ in roots exceeded those of K+. The substitution of K by Na in roots
seems feasible and may release more K to the shoots which could account for increased shoot
K content of wheat with low to moderate Na addition to low K plants (Chapter 3). Shoot K
content increased with Na addition in K-efficient cultivars of wheat grown in pot-experiment
(Chapter 3) and short-term solution culture experiment (Chapter 6) without significant
increase in root K content.
8.6 Toxicity effects of Na
In this study, toxic effects of Na were evident at high soil Na concentrations (100 and 200 mg
Na/kg) and solution concentrations of 20 mM Na (Chapter 6) both at low and high soil K.
However, in this study less emphasis has been placed on Na toxicity effects on wheat as there
is a large body of research available on this topic already (Kronzucker et al., 2013) but much
less on effects of low to moderate Na levels. High Na levels reduced wheat growth (Chapters
3 to 5), and high Na levels of 200 mg Na/kg at both low (40 mg K/kg) and adequate K (100
mg K/kg) supply drastically reduced the photosynthesis and stomatal conductance of wheat
cultivars. It is suggested that high levels of NaCl can cause stomatal closure which limits CO2
diffusion to the chloroplast or inducing a stress-related decline in PSII photochemistry with
consequent PSII photoinhibition and/or photodamage (Degl'Innocenti et al., 2009).
Under NaCl stress conditions, reduction in K+ uptake occurs due to inhibition by Na+ ions of
K+ influx into the cell and stimulation of K+ efflux (Britto et al., 2010). The Na+-coupled K+
uptake mediated by both high-affinity and low-affinity transporters are affected by high Na
levels. According to Rubio et al. (1995), K+ uptake in wheat was stimulated at low external
Na+ (1 mM Na+) mediated by high-affinity K+ uptake transporter HKT1, but at
physiologically detrimental concentrations of Na+, K+ uptake mediated by HKT1 was
blocked and low-affinity Na+ uptake occurred (approximately 16 mM Na+), which correlated
to Na+ toxicity in plants. Moreover, at high Na levels (80 mM NaCl or above), Na+ crosses
the plasma membrane causing a significant membrane depolarization and increases K+
leakage through depolarization-activated outward-rectifying channels (Shabala & Cuin,
2008).
153
8.7 Na effects on cultivars differing in K-use efficiency
The increased capacity to substitute Na+ for K+ was suggested as one possible mechanism
underlying K utilization efficiency (Rengel & Damon, 2008). However, in their previous
studies to rank wheat genotypes according to K-use efficiency (Damon & Rengel, 2007) there
was no examination of the role of Na in cultivar rankings. In this research, the ranking of
cultivars for K-use efficiency was consistent with those for the same cultivars in earlier report
in wheat by Damon and Rengel (2007). Moreover, the relative K-use efficiency of wheat
cultivars was consistent across a range of Na levels from no added Na up to toxic levels.
According to this study, K-efficient cultivars (Wyalkatchem and Cranbrook) had greater
response to low to moderate Na supply than K-inefficient cultivars (Gutha and Gamenya),
and the Na benefit was more restricted to K deficient plants. In contrast to the suggestion that
increased capacity to substitute Na+ for K+ may be a mechanism underlying K-use efficiency
in wheat, our results show that Na stimulated greater K uptake by K-efficient cultivars than
K-inefficient cultivars in experiments (Chapter 3 and 7). Sodium stimulation of
photosynthesis, stomatal conductance and root dry weight were greater in the K-efficient
cultivars (Chapter 3 and 7). These responses were consistent with greater utilization
efficiency of shoot K in the K-efficient cultivars which was the main mechanism identified
earlier by Damon and Rengel (2007) for K-use efficiency in wheat.
8.8 Implications of low to moderate Na for plant K nutrition
The present research on Na+ stimulation of growth by low-K wheat plants may be of
importance in fertiliser management strategies. There may be economic, nutritive and
environmental perspectives associated with K substitution by Na (Wakeel et al., 2011).
The application of expensive K+ fertilisers is hardly affordable by poor farmers especially in
developing countries, and partial substitution of K+ by Na+ in plant nutrition can decrease
cost of production. Particularly, soils dominant in clay minerals (vermiculite and smectite)
need a lot of K+ fertilisers, and Na+ can be a partial replacement for K+ fertilisers. Indeed,
smaller amounts of Na are required when compared to K as Na is not fixed by clay particles
(Wakeel et al., 2011). Research has shown that application of Na application at low rates
reduced K fertiliser requirement in sugar beet (Wakeel et al., 2010), and cotton was found to
grow under partial K substitution by Na (Ali et al., 2009; Ali et al., 2006). However, the
blending of Na in K fertilisers to decrease the K requirement and fertiliser cost, does not
appear to have been adopted in commercial agriculture.
154
Potassium deficiency in wheat can depress root development more than shoot (Ma et al.,
2011). In this study, wheat root growth was better stimulated than shoot due to Na addition in
three instances (Chapter 3, 4 and 6). These results suggest that wheat grown under K-
deficiency and in presence of low to moderate Na may have greater benefit in stress than
well-watered conditions as Na can increase root biomass under drought. This could have an
implication under rainfed conditions where soil water is limited and where low K will further
hinder root elongation. However, under drought stress, root length or root: shoot ratio is
increased which help access to available water (Comas et al., 2013) but the effect on root
development on low K soils probably depends on which occurs first, drought or K-
deficiency.
The increase in wheat K uptake, photosynthesis, stomatal conductance and growth due to low
to moderate Na addition was evident in three experiments (Chapter 3, 4 and 7) when plants
suffered K deficiency symptoms. Moreover, the effect varied with K-use efficiency of
cultivars with K-efficient cultivars being more responsive to Na than K-inefficient cultivars.
In regards to practical implications under field conditions, based on this study, when K-
efficient wheat cultivars are grown under low to moderately saline conditions, K fertiliser
application can be reduced without risking yield declines, however, the substitution of K by
Na was not strong enough to recommend Na-based fertilisers in place of K in wheat.
Nevertheless, some encouraging results of alleviating K deficiency symptoms, stimulation in
growth and leaf gas exchange measurements by addition of moderate Na provide the
motivation for conducting further studies to improve our understanding and perspectives for
potassium fertilizer application in moderately saline and sodic soils. Further research
recommendations are discussed below.
8.9 Conclusions and recommendations
8.9.1 Conclusions
The main conclusions from this study can be summarised as follows:
• Wheat showed positive responses to low to moderate Na supply under K deficiency
with increased biomass production. Roots were more responsive to Na+ than shoot
when growth stimulation was observed.
• Wheat cultivars differing in K-use efficiency showed varied responses in growth
stimulation to added Na. There was a significant increase in root dry weight of K-
155
efficient cultivars in a standard pot soil culture experiment and long-term solution
culture experiment but not in K-inefficient cultivars.
• The growth response to NaCl varied with K and Na levels. High Na concentrations
(100 and 200 mg Na/kg in soil-based experiments) suppressed shoot and root dry
weight regardless of soil K levels.
• Genotypic differences in K-use efficiency influenced Na uptake and salt tolerance
with K-efficient cultivars being more salt tolerant than K-inefficient cultivars.
• When supplied with low to moderate Na under K deficiency, wheat cultivars showed
an increase in leaf net photosynthesis, transpiration and stomatal conductance in both
soil-based and long-term solution culture experiments with values comparable to
adequate K without Na.
• Split-root experiment showed that Na stimulation of growth in low-K plants occurred
regardless of the supply of K and Na in the same or different parts of the root system.
• Potassium uptake of wheat cultivars increased with low to moderate external Na+
supplied under K deficiency in this study in both soil-based and solution culture
experiments with Rb+ as a tracer. It was found that Na-induced increase in K+ uptake
was dose dependent.
• K-use efficiency of wheat cultivars influenced K+ uptake in presence of low to
moderate Na under K deficiency. There was a significant increase in shoot K content
of only K-efficient cultivars in a soil-based pot experiment and also in a short-term
solution culture experiment with Na addition for 48 hours using Rb+ as a tracer for
K+. However, in long-term solution culture experiment with Na addition for nearly 20
days there was significant Na-induced K+ uptake (measured as Rb+ uptake) in both K-
efficient and K-inefficient cultivars.
• The main mechanism for Na+-energized K+ uptake under limited K availability with
low external Na+ supply is attributed to the effect of Na+ on K+ transporters, both on
high-affinity and low-affinity K+ uptake transporters. However, under salinity stress
conditions, reduction in K+ uptake occurs due to inhibition of K+ influx into the cell
by Na+ ions and stimulation of K+ efflux by excess Na+.
• An increase in extractable soil K availability due to added Na may also increase root
elongation and contribute in part to the increase in K content of low K plants in soil-
based systems.
156
• This research facilitates novel understanding of the Na effects on K nutrition of wheat
cultivars differing in K-use efficiency, and will improve decision making and
management of K fertiliser in salt-affected, sodic and K-deficient soils.
8.9.2 Further research recommendations
The following recommendations are made based on the conclusions of this study to foster
new research:
• The present research based on glasshouse experiments needs to be evaluated under
field conditions with varying soil and agro-climatic conditions to define critical soil
levels of Na that stimulate wheat growth.
• More detailed research on Na+-activated low and high-affinity K+ uptake mechanisms
in wheat genotypes would improve our understanding on K uptake under moderately
saline conditions.
• It would be useful to enhance our understanding of K and Na interactions in relation
to varied levels of other major cations like Ca and Mg.
• Research on molecular expression underlying the effect of Na supply on low K wheat
genotypes. Molecular biology approach using mapped germplasm of wheat will help
to correlate the physiological responses of wheat to Na and K with the underlying
gene expression.
157
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166
APPENDIX 1
Chapter 3
1.1 K and Na concentrations in leaves, spikes and stem
Potassium concentration in young leaves, old leaves and stems of all four cultivars was
significantly higher with adequate K supply than K deficit conditions when soil Na levels
ranged from nil to moderate. In general, old leaves of K-efficient cultivars Wyalkatchem and
Cranbrook had considerably higher K concentrations at adequate K supply (100 mg K/kg). At
low K, high soil Na treatment (200 mg Na/kg) concentrated high K in leaves and stem mainly
due to concentration- effect. The interaction between soil K and Na levels was significant
(P≤0.05, Table A1.2) for leaf K concentrations; however, three way interactions between K,
Na and cultivars were not significant for K concentrations in leaves and stem. Ears/spikes had
almost similar K concentrations irrespective of K and Na treatments (Table A1.1).
Sodium concentration in leaves and stem were closely associated with soil Na levels in all
cultivars (Table A1.3). Old leaves and stem concentrated more Na than young leaves and
spikes, and concentration in young leaves was four times less than in old leaves and stem.
Sodium concentration in spikes was least influenced by soil Na irrespective of soil K and
genotypes, and there were only negligible concentrations of Na measured in spikes (Table
A1.3).
167
Table A1.1 Mean (n=3) K concentrations in young leaves, old leaves, stem and spikes (mg/g,
dry weight) of four wheat cultivars treated with two K levels (40 and 100 mg K/kg) and five
Na levels (0, 25, 50, 100 and 200 mg Na/kg) for 8 weeks. See Table A1.2 for significance of
treatment effects and interactions.
K concentrations
Na levels
Wyalkatchem Cranbrook Gutha Gamenya
K 40 K 100
K 40 K 100
K 40 K 100 K 40 K 100
Young leaves
mg/g
dry wt.
Nil Na 7.88 25.2 9.37 21.7 9.24 25.6 9.08 25.0
25 9.65 25.1 12.2 21.6 14.0 24.9 10.6 22.8
50 13.9 24.0 9.79 21.7 13.8 25.1 13.2 24.1
100 12.5 23.1 14.2 22.7 12.4 27.6 13.8 24.8
200 19.4 26.1 19.5 25.1 17.0 26.4 21.1 26.7
Old leaves
mg/g
dry wt.
Nil Na 6.64 29.8 10.8 29.7 8.7 25.7 8.15 25.9
25 11.1 28.8 19.1 28.8 14.5 23.0 11.6 25.6
50 11.0 28.5 18.4 29.4 14.6 25.5 11.6 27.9
100 8.88 30.1 13.4 31.8 13.6 26.5 10.8 27.7
200 17.9 32.1 19.4 32.6 19.4 31.9 17.1 28.2
Stem
mg/g
dry wt.
Nil Na 12.2 26.1 12.2 24.6 9.54 25.5 11.3 24.1
25 16.4 24.4 14.2 24.1 13.8 23.1 13.3 23.8
50 16.1 26.6 13.4 24.1 14.4 22.4 14.4 24.9
100 16.7 28.5 18.7 26.2 16.3 29.6 16.1 27.1
200 20.1 28.1 21.5 27.5 18.2 26.5 16.8 31.5
Spikes
mg/g
dry wt.
Nil Na 12.9 15.9 13.2 15.7 14.9 14.1 13.5 16.6
25 12.9 14.7 14.8 15.3 13.8 15.8 14.9 16.4
50 14.0 15.6 12.9 16.1 14.8 16.3 15.7 16.7
100 14.6 16.8 15.1 14.9 15.6 16.4 16.3 17.0
200 16.4 18.2 14.9 16.9 17.0 17.5 15.7 18.9
168
Table A1.2 Statistical summary of cation concentrations and contents in four wheat cultivars
(Wyalkatchem, Cranbrook, Gutha and Gamenya) treated with two levels of soil K (40 and
100 mg kg-1) and five levels of Na (0, 25, 50, 100 and 200 mg kg-1) for 8 weeks. *P≤0.05;
** P≤0.01; ***P≤0.001; n.s., not significant
Parameters Soil K Soil Na cultivar K×Na K×cv Na×cv K×Na×cv
K conc. young *** *** n.s *** n.s n.s n.s
K conc. old *** *** *** *** *** n.s n.s
K conc. stem *** *** n.s n.s n.s n.s n.s
K conc. spikes *** *** * n.s n.s n.s n.s
Na conc. young *** *** ** *** n.s * n.s
Na conc. old ** *** n.s n.s n.s n.s n.s
Na conc. stem ** *** n.s * * n.s n.s
Na conc. spikes n.s *** n.s n.s n.s n.s n.s
Ca conc. young *** *** *** ** *** n.s n.s
Ca conc. old *** *** n.s * ** * n.s
Mg conc. young *** n.s *** n.s *** n.s n.s
Mg conc. old *** *** * n.s *** n.s n.s
169
Table A1.3 Na concentrations in young leaves, old leaves, stem and spikes of four wheat
cultivars treated with two K levels (40 and 100 mg K/kg) and five Na levels (0, 25, 50, 100
and 200 mg Na/kg) for 8 weeks. Values are mean of three replicates. See Table A1.2 for
significance of treatment effects and their interactions.
Na concentrations
Na levels
Wyalkatchem Cranbrook Gutha Gamenya
K 40 K 100 K 40 K 100 K 40 K 100 K 40 K 100
Young leaves
mg/g
dry wt.
Nil Na 0.015 0.029 0.017 0.01 0.035 0.02 0.013 0.02
25 0.04 0.03 0.05 0.025 0.04 0.04 0.025 0.03
50 0.07 0.08 0.12 0.07 0.10 0.13 0.09 0.05
100 0.27 0.18 0.22 0.15 0.27 0.27 0.36 0.17
200 0.58 0.22 0.47 0.22 0.86 0.43 0.65 0.29
Old leaves
mg/g
dry wt.
Nil Na 0.07 0.08 0.08 0.05 0.065 0.06 0.08 0.06
25 0.22 0.17 0.39 0.12 0.29 0.27 0.30 0.34
50 0.57 0.56 0.89 0.51 0.81 0.77 0.80 0.75
100 1.36 1.20 1.55 1.38 1.86 1.16 2.07 1.07
200 3.90 2.98 4.00 4.25 4.97 3.89 4.14 3.08
Stem
mg/g
dry wt.
Nil Na 0.098 0.19 0.12 0.10 0.14 0.16 0.13 0.14
25 0.24 0.31 0.38 0.25 0.35 0.44 0.39 0.43
50 1.01 0.66 1.12 0.53 1.19 0.76 1.65 0.64
100 1.76 2.25 2.97 1.58 2.06 3.5 3.72 2.40
200 4.17 3.72 5.23 3.25 5.25 3.94 4.61 3.70
Spikes
mg/g
dry wt.
Nil Na 0.12 0.12 0.14 0.10 0.12 0.10 0.11 0.10
25 0.11 0.12 0.18 0.10 0.12 0.11 0.18 0.13
50 0.17 0.11 0.22 0.11 0.11 0.12 0.18 0.11
100 0.27 0.20 0.28 0.12 0.17 0.20 0.20 0.16
200 0.32 0.36 0.24 0.27 0.23 0.36 0.35 0.23
170
1.2 Ca and Mg concentrations in young and old leaves
Leaf Ca concentrations were lower in K adequate plants when compared with K deficient
plants (Fig. A1.1) at all Na levels which might be due to ‘dilution-effect’ as Ca content was
almost similar between the two K levels. The difference in Ca concentration between two soil
K levels was greater in Wyalkatchem and Cranbrook when compared with Gutha and
Gamenya. Leaf Ca concentrations in old leaves when compared with young leaves were
higher in genotypes Gutha and Gamenya, but was about the same in Wyalkatchem and
Cranbrook. Calcium concentrations in young leaves across various Na levels showed little
difference at adequate K supply. High Na treatments reduced Ca concentrations in both
young and old leaves at low soil K. Similarly, leaf Mg concentration was higher in the low K
soil than in the adequate K soil ranging from 0.8 to 2.1 mg Mg/g, dry weight (Fig. A1.2). The
interaction between K and Na levels among the genotypes was not significant for leaf Ca and
Mg concentrations (Table A1.2).
171
Gutha
0 50 100 150 2000
2
4
6
8 Gamenya
Soil Na levels (mg kg-1)
0 50 100 150 200
Wyalkatchem
Ca
conc
ent
ratio
n of
old
leav
es
(mg/
g, d
ry w
eig
ht)
0
2
4
6
8Cranbrook
Gutha
0
2
4
6
8 Gamenya
WyalkatchemC
a co
nce
ntra
tion
of y
oung
leav
es
(mg/
g, d
ry w
eig
ht)
0
2
4
6
8
10Cranbrook
Fig. A1.1 Ca concentration (mg/g, dry weight) in young (upper sub-figures) and old leaves
(lower sub-figures) (± S.E., n=3) of four wheat cultivars, treated with 40 mg K/kg (closed
circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg
Na/kg) for 8 weeks. See Table A1.2 for analysis of variance results.
172
Gutha
0 50 100 150 2000.0
0.5
1.0
1.5
2.0Gamenya
Soil Na levels (mg kg-1)
0 50 100 150 200
Wyalkatchem
Mg
conc
ent
ratio
n of
old
leav
es
(mg/
g, d
ry w
eig
ht)
0.0
0.5
1.0
1.5
2.0Cranbrook
Gutha
0.0
0.5
1.0
1.5
2.0 Gamenya
WyalkatchemM
g co
nce
ntra
tion
of y
oung
leav
es
(mg/
g dr
y w
eig
ht)
0.0
0.5
1.0
1.5
2.0
2.5Cranbrook
Fig. A1.2 Mg concentration (mg/g, dry weight) in young (upper sub-figures) and old leaves
(lower sub-figures) (± S.E., n=3) of four wheat cultivars, treated with 40 mg K/kg (closed
circle) and 100 mg K/kg (open circle), and five soil Na levels (0, 25, 50, 100 and 200 mg
Na/kg) for 8 weeks. See Table A1.2 for analysis of variance results.
173
APPENDIX 2
Chapter 5
Root: Shoot ratios 5 weeks after sowing
Root: Shoot- harvest at 5 WAS
Soil K
40/40(W) 120/40(W)
root
: sho
ot ra
tio
0.0
0.1
0.2
0.3
0.4
0.5
0 Na 50 Na 200 Na
Fig. A2.1 Root: shoot ratios of wheat at 11 weeks after sowing (±SE, n=3). For treatment
descriptions refer to Table 5.1.
174
APPENDIX 3
Fig. A3.1 Experimental setup used in solution culture experiments
175
APPENDIX 4
Chapter 6
A4.1 Plant growth (Experiment 1)
Forty eight hours after K, Na treatments and Rb addition there was no significant treatment
effect on shoot or root biomass in both cvv Wyalkatchem and Gutha (Fig. A4.1). Gutha
produced nearly thrice the shoot weight and twice the root weight of Wyalkatchem (Table
A4.1). Root length measurements with the WinRhizo scanner showed no difference in
various parameters among the treatments (Table A4.2). Gutha had significantly higher root
length, projected root area, surface area, root volume and number of forks than Wyalkatchem.
0.2 mM 2 mM
Roo
t dry
wei
ght(
g/pl
ant)
0.00
0.02
0.04
0.06
0.08
K concentration (mM)
0.2 mM 2 mM
0 Na 10 Na 20 Na
Sho
ot d
ry w
eigh
t(g/
plan
t)
0.00
0.05
0.10
0.15
0.20
0.25
Wyalkatchem Gutha
Fig. A4.1 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2
mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for 48 hours (harvested 17
days after transplanting) (Experiment 1) (±SE, n=4).
176
Table A4.1 Statistical summary of plant growth in cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2
mM K), three Na levels (0, 10 and 20 mM Na) and Rb tracer (0.5 mM) for a further 48 hours
(Experiment 1).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Solution K
Solution Na
Cultivar K×Na K×cv Na×cv K×Na×cv
Shoot dry wt n.s n.s *** n.s n.s n.s n.s
Root dry wt * n.s *** n.s n.s n.s n.s
177
Table A4.2 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3),
number of tips and forks in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient)
(n=2 plants/replicate) treated with low K (0.2 mM K) for 2 weeks and two K levels (0.2 and 2
mM), three Na levels (0, 10 and 20 mM) and Rb tracer (0.5 mM) for a further 48 hours
(harvested 17 days after transplanting) (Experiment 1). Means with different letters differ at
P≤0.05.
Wyalkatchem Gutha
K (mM)
Na (mM)
Total length (cm)
Diameter (mm)
Surface area (cm2)
Total length (cm) Diameter
(mm)
Surface area (cm2)
0.2 0 810b 0.39a 97.5b 1703a 0.37a 199a
0.2 10 908b 0.39a 113b 1861a 0.37a 214a
0.2 20 854b 0.37a 100b 1838a 0.37a 211a
2 0 993b 0.37a 116b 1843a 0.36a 210a
2 10 864b 0.38a 104b 1785a 0.37a 206a
2 20 881b 0.40a 110b 1884a 0.36a 215a
Wyalkatchem Gutha
K (mM)
Na (mM)
Root Volume (cm3) Tips Forks
Root Volume(cm3) Tips Forks
0.2 0 0.94b 1834b 1697b 1.86a 3962ab 4717a
0.2 10 1.11b 2882b 1960b 1.96a 4401ab 4388a
0.2 20 0.94b 3796ab 1766b 1.90a 3222b 4335a
2 0 1.08b 2311b 2057b 1.90a 3566b 4393a
2 10 1.01b 1952b 1604b 1.91a 4592ab 4302a
2 20 1.10b 2452b 1759b 1.96a 6801a 5300a
178
A4.2 Pre-treatment leaf gas exchange measurements (Experiment 1)
Table A4.3 Leaf net photosynthesis, stomatal conductance, and transpiration of wheat
cultivars Wyalkatchem and Gutha before treatment (±SE, n=24) at 14 days after transplanting
(Experiment 1).
Leaf gas exchange measurements Wyalkatchem Gutha
Photosynthesis (µmolCO2m-2 s-1) 14.4±0.17 15.6±0.71
Stomatal conductance (mmolH2Om-2s-1) 382±6.43 373±9.40
Transpiration (mmolH2O m-2 s-1) 6.39±0.16 5.95±0.29
A4.3 Plant growth (Experiment 2)
The growth response to K and Na treatments for 48 hours was mostly consistent with
Experiment 1. There was no significant difference among the treatments (Table A4.4),
despite higher biomass in Gutha than Wyalkatchem (Fig. A4.2).The growth difference
between the two cultivars was smaller when compared to Experiment 1. Total root length of
cv. Gutha treated with high K (2 mM) for 2 days were significantly higher than the
continuous low K treatment at all three Na levels (Table A4.5).
179
Table A4.4 Statistical summary of plant growth and leaf gas exchange in cultivars
Wyalkatchem (K-efficient) and Gutha (K-inefficient) treated with low K (0.05 mM K) for 2
weeks and two K levels (0.05 and 2 mM K), two Na levels (0, 2 and 10 mM Na) and Rb (0.5
mM) (n=4) added for 48 hours (Experiment 2).
*P≤0.05; **P≤0.01; ***P≤0.001; n.s., not significant
Parameters Solution K
Solution Na
cultivar K×Na K×cv Na×cv K×Na×cv
Shoot dry wt n.s n.s *** n.s n.s n.s n.s
Root dry wt n.s n.s *** n.s n.s * n.s
Photosynthesis n.s * n.s n.s ** n.s n.s
Stomatal conductance n.s n.s n.s n.s n.s n.s n.s
Transpiration n.s n.s n.s n.s n.s n.s n.s
180
K concentration (mM)
0.05 mM 2 mM
Roo
t dr
y w
eigh
t (g
/pla
nt)
0.00
0.05
0.10
0.15
0.20
0.05 mM 2 mM
0 Na 2 Na 10 Na
Sho
ot d
ry w
eigh
t (g
/pla
nt)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Wy alkatchem Gutha
Fig. A4.2 Shoot dry weight, and root dry weight of cultivars Wyalkatchem (K-efficient) and
Gutha (K-inefficient) treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05
and 2 mM), three Na levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for 48 hours (harvested
19 days after transplanting) (Experiment 2) (±SE, n=4).
181
Table A4.5 Root total length (cm), surface area (cm2), diameter (cm), root volume (cm3),
number of tips and forks in cultivars Wyalkatchem (K-efficient) and Gutha (K-inefficient)
treated with low K (0.05 mM K) for 2 weeks and two K levels (0.05 and 2 mM), three Na
levels (0, 2 and 10 mM) and Rb tracer (0.5 mM) for a further 48 hours (harvested 19 days
after transplanting) (Experiment 2). Means (n=4) with different letters differ at P≤0.05.
Wyalkatchem Gutha
K (mM)
Na (mM)
Total length (cm) Diameter (mm)
Surface area (cm2)
Total length (cm) Diameter
(mm)
Surface area (cm2)
0.05 0 1106d 0.41ab 146de 1462bc 0.41ab 183bcd
0.05 2 1173cd 0.40ab 147de 1520b 0.40ab 190abc
0.05 10 1083d 0.42a 143e 1443bc 0.41ab 184a-d
2 0 1198bcd 0.39ab 154cde 1906a 0.38ab 219ab
2 2 1377bcd 0.37ab 163cde 1880a 0.37ab 218ab
2 10 1256bcd 0.39ab 158cde 1945a 0.36b 223a
Wyalkatchem Gutha
K (mM)
Na (mM)
Root volume(cm3) Tips Forks
Root volume (cm3) Tips Forks
0.05 0 1.57cd 2558a 3799b 1.87a-d 2796a 3940b
0.05 2 1.55cd 2815a 3832b 1.93a-d 2930a 4166b
0.05 10 1.53d 2653a 3724b 1.93a-d 2463a 4211b
2 0 1.63bcd 2936a 3970b 2.13a 3212a 5446a
2 2 1.79a-d 3172a 4229b 2.03abc 3244a 5459a
2 10 1.65a-d 3078a 4189b 2.12ab 3268a 5780a