Physiological and photosynthetic characters of Polygonum species in response to aluminum stress

Qiang Liu*, Yian Xiao, Genhe He, Wenhai Hu, Wanwan Long College of Life Sciences, Jinggangshan University, Ji’an 343009, China

E-mail: qliu2006@163.com Abstract—Two kinds of Polygonum species

(Polygonum hydropiper and Polygonum lapathifolium) grown vigorously on acid soils were compared to investigate the physiological and photosynthetic responses under five Al3+ treatments (0, 25, 50, 100 and 400 μmol L-1) in a hydroponic experiment. The results showed that under 400 μmol L-1 Al3+, the malondialdehyde (MDA) content and transpiration rate (Tr) were significantly increased, and the proline content, activities of peroxides (POD) and catalase (CAT), net photosynthetic rate (Pn), water utilization efficiency (WUE), quantum yield of electron transport at PS II (φPS II) were decreased significantly in the two plant leaves, with photochemical quenching coefficient (qp) and non-photochemical quenching coefficient (qN) decreased only in P. hydropiper, compared to the control. However, under 25 and 50 μmol L-1 Al3+, the MDA content decreased significantly, the proline content, activities of POD and CAT increased significantly, and the photosynthetic/chlorophyll fluorescence parameters did not inhibited, especially in P. lapathifolium. These results suggested that lower MDA content, more content of proline, higher activities of POD and CAT, and keeping proper function of photochemical system contributed to the tolerance to Al stress under low or middle Al concentrations, and P. lapathifolium was more tolerant than P. hydropiper.

Keywords-aluminum stress; chlorophyll fluorescence; photosynthetic; physiological; Polygonum species

I. INTRODUCTION Aluminum (Al) is the most abundant metal in the earth’s

crust, and obtained as harmless oxides and aluminosilicates. Nevertheless, under acidic conditions (pH < 5), as is now the case in 40% of the arable lands worldwide, Al is solubilized into toxic forms (usually Al3+), which threaten plants and environments [1]. Therefore finding ways to alleviate the adverse effect of Al on plant growth has become an important goal.

There is a wide variation in the resistance to Al toxicity between species and between cultivars within species. Such variation provides breeders with a strategy for improving the ability of agricultural systems to cope with Al toxicity. Although mechanisms involved in Al toxicity and resistance in plants have not been clearly elucidated, there were increasing evidence suggesting that Al toxicity induced the formation of reactive oxygen species and lipid peroxidation

in plasma membrane, thereby destroyed metabolism in plant cells [2]. At the same time, plants under Al stress may mobilize a variety of antioxidant enzymes such as peroxides (POD) and catalase (CAT), and some other antioxidant compounds such as proline and ascorbate to scavenge reactive oxygen species. There is a close relationship between antioxidant activity and tolerance to Al stress in plants [3]. Therefore, Al tolerance mechanisms may be revealed by investigating changes of lipid peroxidation, antioxidant enzymes, and other antioxidant compounds in response to Al stress.

It is also well known that micromolar concentrations of Al can inhibit root elongation and consequently influence water and nutrients uptake resulting in poor plant growth [1]. And water or nutrients deficiency can greatly affect photosynthesis of plants [4]. It was reported that the photosynthesis rate of red pine seedlings was reduced by increasing the Al concentration in the soil solution [5]. Ridolfi and Garrec [6] indicated that excess Al caused stomatal closure and reduced photosynthesis of beech seedlings. However, more research on leaves, especially the chlorophyll fluorescence characteristics, is needed, as the mechanism by which Al affects photosynthesis is not well understood.

Wild plants show strong adaptability and competitiveness in nature, many of which must have some physiological and ecological mechanisms because they distribute ubiquitously and grow vigorously on acid soils. In recent decades, researches of Al toxicity and resistance mechanisms have mainly focused on crops, while few studies considered wild plants. Without the courses of breeding for other agronmic features, wild plants may conserve more resistance genes and provide good materials to better understand the Al resistance mechanisms or search for novel Al resistance mechsnisms. In the present study, physiological and photosynthetic responses of wild plants (Polygonum hydropiper and Polygonum lapathifolium belonging to the same species) under Al stress were investigated to better reveal Al resistance mechanism and to prevent Al toxicity.

II. MATERIALS AND METHODS

A. Plant Materials and Culture Two cultivars of Polygonum species, Polygonum

hydropiper and Polygonum lapathifolium, were selected because of their wide distribution and well growth on acid red soils of Jinggangshan university, Jiangxi province. The plants

This work was supported by fund from Natural Science Foundation of China (No. 30760108) and Scientific Program of Education Department of Jiangxi Province (No.GJJ08419, GJJ09340) *Corresponding author: Tel: +86-796-8100493 E-mail: qliu2006@163.com

978-1-4244-4713-8/10/$25.00 ©2010 IEEE

were transplanted into the 1.1-L plastic pots (4 seedlings per pot) containing 1-L aerated nutrient solution. One fifth strength Hoagland solution was used to maintain plant nutrition. B. Experiment Design

After one week acclimation in nutrient solution, five treatments were carried out with various amounts of AlCl3

.6H2O added into nutrient solution to make the Al3+ concentration to 0, 25, 50, 100 and 400 μmol L-1. Leaves were sampled on the 14th day after Al treatment for measuring contents of malondialdehyde (MDA), proline, activities of peroxides (POD) and catalase (CAT), photosynthetic and chlorophyll fluorescence parameters. C. Measurement Methods

The MDA content was determined using thiobarbituric acid (TBA) method. The proline content and activities of POD and CAT were measured using methods based on Institute of Plant Physiology of Shanghai [7].

The net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance rate (Gs) and intercellular CO2 concentration (Ci) were measured on the middle part of fully developed third leaves using a portable photosynthesis apparatus (Ciras-1, PP Systems, UK) with a leaf clip control system. Water utilization efficiency (WUE) was defined as the percentage of Pn/Tr. The measurement was made 1 h after the onset of the light period. The photosynthetic photon flux density, temperature and CO2 concentration were 1600 μmol m-2 s-1, 25°C and 350 μl L-1, respectively. Meanwhile, chlorophyll fluorescence parameters, such as maximal photochemical efficiency (Fv/Fm), quantum yield of electron transport at PS II (φPS II), photochemical quenching coefficient (qp) and non-photochemical quenching coefficient (qN), were recorded with a pulse amplitude modulation fluorometer (FMS-2, Hansatech, UK) according to Hu et al. [8].

III. RESULTS AND DISCUSSIONS A. Effect of Al on MDA Contents of Polygonum hydropiper and Polygonum lapathifolium

Although Al itself is not a transition metal and cannot catalyze redox reactions, the Al-enhanced peroxidation of lipids has been reported in various systems. MDA is routinely used as an indicator of lipid peroxidation [2]. In the present study, the MDA contents of P. hydropiper and P. lapathifolium were determined under different Al concentrations. P. hydropiper and P. lapathifolium showed a significant increase under 400 μmol L-1 Al concentration (P < 0.05). The MDA contents of P. hydropiper and P. lapathifolium increased by 66.2% and 39.3%, respectively, which indicated that Al at this concentration caused stress to plants. As the Al concentration increased from 0 to 50 μmol L-1, the MDA contents in P. hydropiper and P. lapathifolium decreased significantly (P < 0.05). While under 100 μmol L-1 Al3+, the MDA contents increased by 24.5% in P. hydropiper (P < 0.05) and 13.5% in P. lapathifolium (Fig.1). These results indicated that Al at low or middle concentration was

beneficial to plants in resisting lipid peroxidation, and P. lapathifolium was more tolerant than P. hydropiper under different Al concentrations.

0 25 50 100 4000

10

20

30

40

Aluminum concentration (μmol L-1)

MD

A c

onte

nt (n

mol

g-1

FW)

Polygonum hydropiperPolygonum lapathifolium

a

b

c

d d

a

abb

c c

Fig.1. Effect of Al on MDA contents of P. hydropiper and P. lapathifolium (Note: The differences between the treatments without the same letters were significant (P < 0.05), the same as follows)

B. Effect of Al on Proline Contents of Polygonum hydropiper and Polygonum lapathifolium

Under stress such as water deficiency, plants can accumulate a great deal of proline to decrease cell osmotic potential, stabilize macromolecules and maintain normal functions of cellular membrane [9]. Therefore, proline contents under different Al concentrations were investigated, and the results are presented in Fig.2. The proline content increased gradually as the Al concentration increased from 0 to 100 μmol L-1, with the proline content increased by 25.5% in P. hydropiper and 60.6% in P. lapathifolium under 100 μmol L-1 Al3+ compared with control. However, under 400 μmol L-1 Al3+ treatment, the proline content reduced to the lowest level. Compared with control, the proline content reduced by 18.2% in P. hydropiper and 13.6% in P. lapathifolium (Fig.2). These results suggested that Al, at middle or low concentrations, promoted the synthesis of proline in plant leaves, whereas Al at high concentration led to decrease of the proline content. This is in agreement with Al study in tea plant [10], suggesting that proline synthesis contributed to Al tolerance in plant to some extent.

0 25 50 100 4000

40

80

120

160

Aluminum concentration (μmol L-1)

Prol

ine

cont

ent (

μg g

-1FW

)

Polygonum hydropiperPolygonum lapathifolium

aaa

b

c

c

a

bb

cd

Fig.2. Effect of Al on proline contents of P. hydropiper and P. lapathifolium

C. Effect of Al on Activities of POD and CAT in Polygonum hydropiper and Polygonum lapathifolium

Plants generate large amount of reactive free radicals (ROS) such as O2

-, H2O2-, etc., leading to lipid peroxidation and

damage of cell membrane in response to Al toxicity. POD and CAT are antioxidant enzymes that are important in plant protective system. These antioxidant enzymes can scavenge reactive free radicals mentioned above effectively, avoid lipid peroxidation and protect plants against damage under stress [11]. The POD and CAT activities increased gradually as Al concentration increased from 0 to 50 μmol L-1, with activities of POD and CAT at 50 μmol L-1 Al3+ significantly higher than those under other treatments, P < 0.01). While at 400 μmol L-1

Al3+, activities of POD and CAT decreased sharply to the lowest level (Fig.3). These results were in consistent with an Al study on Pharbitis nil or Aeschynomene indica [12], indicating that the increment of POD and CAT activities at middle or low Al concentration can alleviate Al toxicity by decreasing ROS in plant system.

0 100 200 300 4000.0

0.5

1.0

1.5

2.0

2.5

3.0

Aluminum concentration (μmol L-1)

POD

act

ivity

( ΔA

470 g

-1FW

min

-1)

Polygonum hydropiper Polygonum lapathifolium

0 100 200 300 4000

4

8

12

16

20

24

28

CAT

activ

ity (m

g g-1

FW m

in-1

)

Fig.3. Effect of Al on activities of POD and CAT in P. hydropiper and P. lapathifolium

D. Effect of Al on Photosynthetic Parameters in Polygonum hydropiper and Polygonum lapathifolium

As it can be seen from Table I, the net photosynthetic rate

(Pn) in the two plant leaves decreased evidently under 400 μmol L-1 Al3+ treatment (P < 0.05). Treatment with different Al concentrations had no significant effect on intercellular CO2 concentration (Ci) and stomatal conductance rate (Gs) in P. lapathifolium, which convinced us that high concentrations of Al-induced inhibition of Pn was due to a non-stomatal restriction. From the results of transpiration rate (Tr) and water utilization efficiency (WUE) in response to Al stress, we found out that 400 μmol L-1 Al3+ significantly enhanced Tr or lowered WUE in the two plant leaves, especially in P. hydropiper. Thus, based on our results we suggested that the Al caused inhibition of Pn was more likely coupled to a reduction in WUE, resulting in inhibition of plant growth at 400 μmol L-1 Al concentration (data not shown).

E. Effect of Al on Chlorophyll Fluorescence Parameters in Polygonum hydropiper and Polygonum lapathifolium

Chlorophyll fluorescence parameters are frequently used to detect stress-induced effects on light utilization efficiency of the photosynthetic machinery [13]. As shown in Table II, different concentrations of Al treatment had no significant effect on maximal photochemical efficiency (Fv/Fm), whereas quantum yield of electron transport at PS II (φPS II) was depressed at 400 μmol L-1 Al3+ in the two plant leaves (Table II). These results suggested that low or middle concentration of Al (0-100 μmol L-1) did not lead to photoinhibition in PS II, while some kind of injury to PS II was produced under high Al concentration. Photochemical quenching coefficient (qp), representing exoteric degree of PS II to some extent, was decreased significantly at 400 μmol L-1 Al3+ in P. hydropiper, suggesting that Al caused partial closure of PS II. Furthermore, 50-400 μmol L-1 Al3+ treatment greatly reduced the non- photochemical quenching coefficient (qN) only in P. hydropiper, indicating that P. lapathifolium had more capability in eliminating excess light energy than P. hydropiper, thus protecting plants from photodamage.

TABLE I EFFECT OF AL ON PHOTOSYNTHETIC PARAMETERS IN P. HYDROPIPER AND P. LAPATHIFOLIUM

Polygonum species Al concentration (μmol L-1)

Pn (μmol m-2 s-1)

Tr (mmol m-2 s-1)

Gs (mmol m-2 s-1)

Ci (μL L-1)

WUE (μmol mmol-1)

Polygonum hydropiper

0 16.5±1.01 a 5.10±0.47 b 303.4±17.9 c 247.4±26.3 a 3.24±0.24 a 25 16.9±1.25 a 5.36±0.59 b 356.7±28.4 b 252.6±31.2 a 3.25±0.32 a 50 17.8±0.85 a 5.89±0.44 ab 383.4±34.6 a 258.4±13.8 a 3.02±0.24 ab

100 16.4±1.34 a 5.82±0.71 ab 314.2±14.9 c 245.9±11.5 a 2.82±0.21 b 400 13.3±1.11 b 6.35±0.68 a 316.5±23.3 c 252.1±23.1 a 2.09±0.15 c

Polygonum lapathifolium

0 20.8±2.21 a 7.01±0.44 b 318.9±22.1 a 239.1±12.7 a 2.97±0.17 a 25 21.1±1.37 a 6.94±0.31 b 321.6±19.4 a 243.4±21.4 a 3.04±0.24 a 50 20.4±1.91 a 7.03±0.45 b 313.6±16.7 a 256.3±14.4 a 2.90±0.19 a

100 19.8±1.67 a 6.92±0.68 b 302.9±16.2 a 245.3±27.3 a 2.86±0.23 a 400 17.3±1.12 b 7.45±0.84 a 341.7±27.4 a 246.2±23.9 a 2.32±0.12 b

TABLE II EFFECT OF AL ON CHLOROPHYLL FLUORESCENCE PARAMETERS IN P. HYDROPIPER AND P. LAPATHIFOLIUM

Polygonum species Al concentration (μmol L-1) Fv/Fm φPS II qp qN

Polygonum hydropiper

0 0.851±0.037 a 0.639±0.023 a 0.961±0.034 a 0.414±0.029 a 25 0.849±0.046 a 0.634±0.054 a 0.963±0.116 a 0.398±0.042 a 50 0.853±0.062 a 0.631±0.041 a 0.978±0.077 a 0.357±0.047 b

100 0.848±0.018 a 0.554±0.053 ab 0.874±0.075 ab 0.341±0.039 b 400 0.828±0.015 a 0.461±0.018 b 0.756±0.036 b 0.289±0.021 c

Polygonum lapathifolium

0 0.837±0.021 a 0.768±0.034 a 0.986±0.048 a 0.311±0.012 a 25 0.833±0.012 a 0.736±0.081 a 0.929±0.041 a 0.321±0.017 a 50 0.831±0.026 a 0.757±0.016 a 0.943±0.035 a 0.322±0.008 a

100 0.829±0.009 a 0.754±0.025 a 0.996±0.126 a 0.331±0.041 a 400 0.833±0.011 a 0.658±0.031 b 0.943±0.012 a 0.337±0.023 a

IV. CONCLUSION

Two kinds of Polygonum species (Polygonum hydropiper and Polygonum lapathifolium) well adapted to acid soils were collected to investigate the physiological and photosynthetic mechanisms involved in their high aluminum (Al) resistance. Hydroponic experiments showed that Al at low or middle concentration (25 and 50 μmol L-1 Al3+ in this study) decreased MDA content, and increased proline content and activities of POD and CAT in the two plant leaves. Besides, the photosynthetic apparatus was in good condition as photosynthetic and chlorophyll fluorescence characters were not inhibited under these Al concentrations. However, under 400 μmol L-1 Al3+, the physiological characters (MDA, proline, POD and CAT) were changed opposite mentioned above, and photosynthetic/chlorophyll fluorescence parameters such as Pn and φPS II were inhibited significantly, especially in P. hydropiper. These results suggested that lower MDA content, more content of proline, higher activities of POD and CAT, and keeping good condition of photosynthetic apparatus improved the tolerance to Al stress under low or middle Al concentrations, and P. lapathifolium was more tolerant than P. hydropiper.

ACKNOWLEDGMENT The authors gratefully acknowledge the financial support

from Natural Science Foundation of China (No. 30760108) and Scientific Program of Education Department of Jiangxi Province (No.GJJ08419, GJJ09340) for this work.

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