PHYSIOLOGICAL AND MOLECULARprr.hec.gov.pk/jspui/bitstream/123456789/1233/1/2412S.pdfI must not...

PHYSIOLOGICAL AND MOLECULAR

CHARACTERIZATION OF TWO GENETICALLY

DIVERSE SPRING WHEAT (Triticum aestivum L.)

CULTIVARS FOR SALT TOLERANCE

By

Muhammad Arslan Ashraf M. Phil (QAU)

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

BOTANY

D E P A R T M E N T O F B O T A N Y

FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE

FAISALABAD

PAKISTAN

2012

To

The Controller of Examinations,

University of Agriculture,

Faisalabad.

“We, the Supervisory Committee, certify that the content and form of

thesis submitted by Muhammad Arslan Ashraf, Reg. No. 2002-ag-1125 ,

have been found satisfactory and recommend that it be processed for

evaluation by the external Examiner (s) for the award of the degree”

Supervisory Committee 1. Chairman ---------------------------------- Dr. Muhammad Ashraf 2. Member ----------------------------------- Dr. Muhammad Shahbaz 3. Member ----------------------------------- Dr. Amer Jamil

DECLARATION “I hereby declare that the content of the thesis, entitled “Physiological and molecular

characterization of two genetically diverse spring wheat (Triticum aestivum L.) cultivars

for salt tolerance” are product of my own research and no part has been copied from any

published source (except the references, standard mathematical or genetic

models/equation/formulae etc.). I further declare that this work has not been submitted for

award of any other diploma/degree. The university may take action if the information

provided is found inaccurate at any stage”.

Muhammad Arslan Ashraf Regd. No. 2002-ag-1125

Dedicated

to

my Parents

ACKNOLEDMENT

All my praises and appreciations are for Almighty Allah for bestowing upon me the

wisdom and potential for successful completion of this manuscript. I offer my humblest

salutations upon the Holy Prophet (Peace be Upon Him), who is forever a source of

guidance and knowledge for all mankind.

It is sense of immense pleasure to express my heartiest gratitude to my esteemed

supervisor and elite scientist Prof. Dr. Muhammad Ashraf, Dean Faculty of Sciences,

University of Agriculture, Faisalabad. Without his guidance, proper direction and support,

it would have never been possible for me to complete my Ph.D work, especially the Ph.D

manuscript.

I am also greatly indebted to my supervisory committee, Dr. Muhammad

Shahbaz, Assistant Professor University of Agriculture, Faisalabad and Dr. Amer Jamil,

Associate Professor, University of Agriculture, Faisalabad, for their humble guidance,

wise counseling and encouragement. I am thankful to Dr. Abdul Wahid, Chairman of

Department of Botany, University of Agriculture, Faisalabad.

I must not forget Dr. Sajid Aqeel and all my lab fellows, especially Mr. Qasim

Ali for his kind help. I am also thankful to Mr. Nadeem Asghar (L.A.) for his consistent

help during lab work. I wish to express my sincere thanks to Dr. Habib-ur-Rehman

Athar, Assistant Professor, BZU, Multan for his guidance and kind attitude. Finally I

have no words to thank my parents who always wish to see me successful and prosperous.

I gratefully acknowledge the financial support by Higher Education Commission

for my Ph.D studies. It would have been impossible for me to complete my studies

without this support by HEC. I am also thankful to all the members of HEC dealing with

my indigenous scholarship. They always sent me funds well in time and have been very

polite to me.

Muhammad Arslan Ashraf

CONTENTS

No. Title Page DEDICATION

ACKNOWLEDGEMENT

ABSTRACT

I INTRODUCTION 1

II REVIEW OF LITRATURE 11

III MATERIALS AND METHODS 44

IV RESULTS 59

V DISCUSSION 108

GENERAL DISCUSSION 134

SUMMARY 147

LITERATURE CITED 150

LIST OF TABLES Table No. TITLE Page No.

4.1 Analysis of variance (mean squares) of data for growth attributes measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

61

4.2 Analysis of variance (mean squares) of data for photosynthetic pigments measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponics culture.

66

4.3 Analysis of variance (mean squares) of data for yield attributes of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

68

4.4 Analysis of variance (mean squares) of data for water relation parameters measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

71

4.5 Analysis of variance (mean squares) of data for gas exchange attributes measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

74

4.6 Analysis of variance (mean squares) of data for gas exchange attributes measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

74

4.7

Analysis of variance (mean squares) of data for different chlorophyll fluorescence attributes measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

80

4.8


80

4.9


81

4.10 Analysis of variance (mean squares) of data for enzymatic antioxidants measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

87

4.11

Analysis of variance (mean squares) of data for total soluble protein, ascorbic acid, H2O2, MDA and phenolics measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

91

4.12

Analysis of variance (mean squares) of data for leaf alpha tocopherols, proline and glycine betaine contents measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

95

4.13 Analysis of variance (mean squares) of data for ions measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

100

4.14 Analysis of variance (mean squares) of data for ions measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

100

4.15

Analysis of variance (mean squares) of data for shoot and root K+/Na+ and Ca2+/Na+ ratios measured at different growth stages of two wheat (Triticum aestivum L.) cultivars when grown in salinized hydroponic culture.

101

5.1 Correlation among different physiological and biochemical attributes of two wheat cultivars recorded at different growth stages 133

LIST OF FIGURES Fig. No. TITLE Page No.

3.1 Meterological data showing minimum and maximum of temperature (°C), relative humidity (%), and rainfall for the entire crop growth period during 2008-2009.

45

3.2 Meterological data showing minimum and maximum of temperature (°C), relative humidity (%), and rainfall for the entire crop growth period during 2009-2010.

45

4.1a Result of RAPD amplification of genotypes MH-97 (V1) and S-24 (V2) with primers (AB, OP) “M” is 1 kb DNA ladder. 59

4.1 Shoot fresh and dry weights of two wheat (Triticum aestivum L.) cultivars at different growth stages when grown in a salinized hydroponic culture (n=4±S.E.)

62

4.2 Root fresh and dry weights of two wheat (Triticum aestivum L.) cultivars at different growth stages when grown in a salinized hydroponic culture (n=4±S.E.)

63

4.3 Shoot length of two wheat (Triticum aestivum L.) cultivars at different growth stages when grown in a salinized hydroponic culture (n=4±S.E.) 64

4.4 Photosynthetic pigments of two wheat (Triticum aestivum L.) cultivars at different growth stages when grown in a salinized hydroponic culture (n=4±S.E.)

67

4.5 Various yield attributes of two wheat (Triticum aestivum L.) cultivars at different growth stages when grown in a salinized hydroponic culture (n=4±S.E.).

69

4.6 Water relation parameters of two wheat (Triticum aestivum L.) cultivars at different growth stages in a salinized hydroponic culture (n=4±S.E.) 72

4.7 Gas exchange attributes (A, E, gs) of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.).

75

4.8 Gas exchange attributes (A/E, Ci, Ci/Ca) of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.).

76

4.9 Intinsic water use efficiency (A/gs) of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.).

77

4.10 Chlorophyll fluorescence attributes of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E).

82


83


84


85

4.14 Superoxide dismutase (SOD) and peroxidase (POD) activities of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.)

88

4.15 Catalase (CAT) and ascorbate peroxidase (APX) activities of two wheat (Triticum aestivum L.) cultivars measured at the different growth stages in a salinized hydroponic culture (n=4±S.E.)

89

4.16 Ascorbic acid and hydrogen peroxide of two wheat (Triticum aestivum L.) cultivars at different growth stages when grown in a salinized hydroponic culture (n=4±S.E.)

92

4.17 Leaf phenolics and leaf MDA contents of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.)

93

4.18 Leaf proline and total soluble protein of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinzed hydroponic culture (n=4±S.E.)

96

4.19 Leaf alpha tocopherols and glycine betaine contents of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.).

97

4.20 Shoot and root Na+ of two wheat (Triticum aestivum L.) cultivars measured

at different growth stages in a salinized hydroponic culture (n=4±S.E.) 102

4.21 Shoot and root K+ of two wheat (Triticum aestivum L.) cultivars measured


4.22 Shoot and root Ca2+ of two wheat cultivars (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.)

104

4.23 Shoot and root K+/Na+ of two wheat cultivars (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.)

105

4.24 Shoot and root Ca2+/Na+ of two wheat (Triticum aestivum L.) cultivars measured at different growth stages in a salinized hydroponic culture (n=4±S.E.)

106

4.25 Shoot and root Cl- of two wheat (Triticum aestivum L.) cultivars measured


Abstract Hydroponic experiments were conducted to appraise variation in the salt tolerance potential of two wheat cultivars (salt tolerant, S-24 and moderately salt sensitive MH-97) at different growth stages. Salinity stress caused a marked reduction in plant biomass and grain yield of both wheat cultivars. However, cv. S-24 was superior to cv. MH-97 in maintaining higher plant biomass and grain yield under saline stress. Furthermore, salinity caused a significant variation in different physiological attributes measured at different growth stages. For example, salt stress caused a marked reduction in net photosynthetic and transpiration rate in both wheat cultivars but to a varying extent at different growth stages. Higher photosynthetic and transpiration rates were recorded at the boot stage than at other growth stages in both wheat cultivars. The response of other gas exchange attributes was also variable at different growth stages. Salt sensitive wheat cultivar MH-97 was more prone to salt-induced adverse effects on gas exchange attributes as compared to cv. S-24. Salt stress caused considerable reduction in different water relation attributes of wheat plants. A significant reduction in leaf water, osmotic and turgor potentials was recorded in both wheat cultivars at different growth stages. Maximal reduction in leaf water potential was recorded at the reproductive stage in both wheat cultivars. In contrast, maximal turgor potential was observed at the boot stage. Salt-induced adverse effects of salinity on different water relation attributes were more prominent in cv. MH-97 as compared to those in cv. S-24. The integrity of PS II was greatly perturbed in both wheat cultivars at different growth stages and this salt-induced damage to PS II was more in cv. MH-97. A significant alteration in different biochemical attributes was also observed in both wheat cultivars at different growth stages. For example, salt stress caused a substantial decrease in chlorophyll pigments, ascorbic acid, phenolics and tocopherols. In contrast, it increased the endogenous levels of ROS (H2O2), MDA, total soluble proteins, proline, glycine betaine and activities of enzymatic antioxidants (SOD, POD, CAT, APX). These biochemical attributes exhibited significant salt-induced variation at different growth stages in both wheat cultivars. For example, maximum accumulation of glycine betaine and proline was recorded at the early growth stages (vegetative and boot). However, cv. S-24 showed higher accumulation of these two organic osmolytes and this could be the reason for maintenance of higher turgor than that of cv. MH-97 under stress conditions. The activities of various enzymatic antioxidants increased markedly in both wheat cultivars, particularly at the vegetative stage. However, cv. S-24 exhibited consistent increase in the activities of various enzymatic antioxidants, whereas, this phenomena occurred erratically in cv. MH-97 at different growth stages. Salt stress significantly increased the endogenous levels of toxic ions (Na+and Cl-) and decreased essential cations (K+ and Ca2+) in both wheat cultivars at different growth stages. Furthermore, K+/Na+ and Ca2+/Na+ ratios decreased markedly due to salt stress in both wheat cultivars at different growth stages and this salt-induced reduction was more prominent in cv. MH-97. Moreover, higher K+/Na+ and Ca2+/Na+ ratios were recorded at early growth stages in both wheat cultivars. It can be inferred from the results that wheat plants are more prone to adverse effects of salinity stress at early growth stages than that at the reproductive stage.

Chapter 1 INTRODUCTION

There has been a rapid increase in human population during the past 50 years. In

1950, the total human population was 2.5 billion and in 2005 it dramatically increased to

6.5 billion. According to an estimate, the world human population will reach to 9.5

billion if it increases with this rapid pace (http:// www. prb. org/ Educators/ Teachers

Guides/ Human Population/Population Growth.aspx). Increasing human population

demands more food. However, due to limited arable land resources, the availability of

food to every person becomes badly affected especially in the developing countries.

Furthermore, the crop productivity is reduced by different abiotic stresses such as salt

stress, water stress, temperature stress, radiation stress etc. (Ashraf, 2009). Of different

abiotic stresses, salt stress is one of the major causes of decline in crop productivity. For

example, 7% of the world’s land and about 5% of the cultivated land are adversely

affected by salinity stress (Flowers et al., 1997; Macrum ,2006). In Pakistan, of 20.36

Mha area, 6.67 Mha are salt affected (Ali et al., 2004).

This problem of salinization becomes worse due to poor drainage and use of bad

quality irrigation water (Chinnusamy et al., 2005). The soil with electrical conductivity of

4 dS m-1 is categorized as saline soil (Chinnusamy et al., 2005). Plants are generally

grouped into two categories on the basis of their potential to tolerate salt stress, i.e.,

halophytes and glycophytes. Halophytes are those plants which can tolerate higher levels

of salinity in the growth medium. In contrast, glycophytes (most grain crops) cannot

tolerate higher concentration of salinity (Ashraf, 2004) and their productivity is greatly

hampered when electrical conductivity (ECe) of the growth medium is more than 4 dS m-

1 (Chinnusamy et al., 2005).

Salinity stress imposes several deleterious effects on plant growth in the form of

osmotic stress, ion toxicity, nutrient deficiency, hormonal imbalance, ROS-induced

oxidative stress (Ashraf and Harris, 2004; Ashraf, 2009). Plants grown on soils with

higher salinity levels often face the problem of low soil water potential which results in

reduced plant water uptake (Ashraf, 2004). This in turn decreases turgor of crop plants.

Reduced turgor of plants is one of the major causes of growth cessation of plants under

1

salt stress (Ashraf, 2004; Munns, 2009). Plants grown on saline soils take up higher

levels of toxic ions like Na+ and Cl- that perturb a variety of cellular processes (Parida

and Das, 2005; Nawaz et al., 2010). Likewise, salinity stress causes imbalance in the

endogenous levels of plant hormones (Parida and Das, 2005; Nawaz et al., 2010). Plant

hormones are actively involved in plant signal transduction during different abiotic

stresses (Kaya et al., 2009). For example, it is generally known that salt stress down-

regulates the production of gibberellins and cytokinins, whereas the production of

abscisic acid is up-regulated (Pedranzani et al. 2003; Kaya et al., 2009). Furthermore,

plants under saline conditions exhibit alteration in the levels of ABA and higher levels of

leaf ABA cause stomatal closure (Zheng et al., 2001).

Salt stressed plants also accumulate higher levels of reactive oxygen species

(ROS) in different organs such as chloroplast, mitochonderia and microbodies in the cell

(Ashraf, 2009; Ashraf and Akram, 2009). These ROS are highly reactive in nature

inducing damage to various cellular metabolites e.g., pigments, proteins, lipids, DNA etc

(Parida and Das, 2005; Ashraf, 2009; Ashraf and Akram, 2009). However, plants have

well developed defense mechanism in the form of different enzymatic and non-enzymatic

antioxidants (Ashraf, 2009; Ashraf and Akram, 2009). The major enzymatic antioxidants

include ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT),

peroxidase (POD), superoxide dismutase (SOD), etc., whereas, tocopherols, phenolics,

carotenoids, ascorbic acid are included in the non-enzymatic category. It has been

reported that plants grown under salt stress exhibit up-regulation of various enzymatic

antioxidants (Sairam et al., 2002 ; Sariam et al., 2005; Mandhania et al., 2006; Ashraf,

2009; Ashraf and Akram, 2009), whereas, main non-enzymatic antioxidants such as

ascorbic acid, tocopherols, carotenoids, generally, decrease due to their reaction with

different oxidants under salt stress (Sairam et al., 2002; Sairam et al., 2005; Munne´-

Bosch, 2005). For example, α-tocopherols decrease under salt stress because of their

chemical scavenging of singlet oxygen leading to the production of such by-products that

cannot be recycled back to α-tocopherols (Munne´-Bosch, 2005).

Various biochemical and physiological mechanisms are adversely affected by salt

stress resulting in reduced biomass. Such inhibitory effects of salt stress could be

observed on all parts of the plant at different growth stages, i.e., germination, seedling,

2

vegetative, boot and reproductive stages (Nawaz et al., 2010). However, the degree of

salt sensitivity at different developmental growth stages depends on the plant species

(Nawaz et al., 2010). For example, variation in salt sensitivity at different growth stages

has been reported in a variety of plants, e.g., rice (Akbar and Yabuno, 1977), wheat

(Ashraf and Khanum, 1997; Ashraf and Parveen, 2002), barley (Norlyn, 1980). This

variation in salt sensitivity at different growth stages primarily depends on the ability of

plants to exclude Na+ or the potential of plants to compartmentalize Na+ into the vacuole

(Ashraf, 2004; Munns, 2005; Nawaz et al., 2010). Salt tolerant plants generally exhibit

lower cytosolic Na+ than that in salt sensitive ones (Carden et al., 2003; Ashraf, 2004;

Nawaz et al., 2010). The higher endogenous levels of toxic ions like Na+ and Cl- induce a

variety of inhibitory effects on plants at cellular level in terms of hampered enzymatic

reactions and other processes like CO2 assimilation and protein synthesis (Ashraf, 2004;

Munns, 2005) which results in leaf death (Nawaz et al., 2010). Therefore, in glycophytes

(most crop plants), exclusion of Na+ from the cell is considered as an essential

component of salt tolerance (Ashraf, 2004; Nawaz et al., 2010). Moreover, plants grown

on saline soils often suffer from nutrient deficiency, because Na+ competes with the

uptake of other essential cations like Ca2+ and K+. The adequate levels of these cations

are required by plants to maintain the integrity of cell membranes and cell wall. Also for

various enzymes potassium is used as a cofactor (Ashraf et al., 2011).

Salt stress induces certain alterations in the important physiological processes of

plants (Ashraf, 2004). For example, elevated salt concentrations lower the water potential

of soil, reducing influx of water into the plant (Ashraf, 2004). Drought- or salt-induced

osmotic stress reduces turgor potential that leads to growth suppression (Hsiao, 1973;

Greenway and Munns, 1980; Ashraf, 2004). However, plants grown on soils with high

salt levels accumulate higher levels of inorganic ions (Na+, K+) and organic osmolytes

(glycine betaine, proline etc.) which lowers the osmotic potential resulting in turgor

maintenance under stress conditions (Ashraf, 2004; Parida and Das, 2005; Nawaz et al.,

2010), a phenomenon known as osmotic adjustment. Generally, salt tolerant cultivars

exhibit better osmotic adjustment than do the salt sensitive cultivars (Ashraf, 2004; Singh

et al., 2010). Salt stress affects plant water relations at different growth stages (Sing et

al., 2010). For example, salinity stress caused considerable reduction in osmotic potential

3

of two wheat cultivars (salt tolerant, KRL-19 and salt-sensitive, WH-542) at the seedling

stage (Mandhania, et al., 2010). Likewise, salinity stress reduced water and osmotic

potentials of 63-day old plants of two tomato cultivars (Daniela F1 and Moneymaker),

but in contrast, an increase in turgor potential was recorded in tomato plants under salt

stress (Romero-Aranda, 2001). Recently, salt-induced reduction in leaf water potential of

wheat plants has also been observed at the seedling stage (Abdelmalek and Khaled,

2011). It is clear from these reports that plant water relations vary at different growth

stages. However, plant water relations have not been investigated in wheat at distinct

growth stages from seedling till maturity in one study. In most of the cases, salt-induced

variation in plant water relations has been studied at one particular growth stage. Since,

plant water relations could be considered as important selection criteria for salt tolerance,

this intensifies the need to correlate plant salt tolerance with salt-induced variation in

water relation attributes at different growth stages.

Like degradation of a variety of biomolecules, salt stress also causes degradation

of chlorophyll contents (Parida and Das, 2005; Shahbaz et al., 2011). This decrease in

chlorophyll contents is attributed to the decrease in accumulation of 5-aminolevulinic

acid (ALA) which is the precursor of protochlorophyllide. This protochlorophyllide is

further converted into chlorophyll when exposed to light (Santos, 2004). In addition, 5-

aminolevulinic acid is formed from glutamic acid which is reported to decrease in salt

stressed plants (Beale and Castelfranco, 1974; Santos and Caldeira, 1999; Santos et al.,

2001; Santos, 2004). The first step in salt-induced degradation of chlorophyll is the

elimination of phytol due to the enhanced activity of chlorophyllase enzyme (Fang et al.,

1998). The salt-induced degradation of chlorophyll has been reported earlier in a number

of plants, e.g., sunflower (Santos, 2004), wheat (Khatkar and Kuhad, 2000), tomato

(Doganlar, 2010), Arabidopsis (Huang et al., 2005), maize (Molazem et al., 2010), canola

(Nazarbeygi et al., 2011). The degradation of chlorophyll contents at different growth

stages of wheat has been reported earlier by Khatkar and Kuhad (2000). Since salt-

induced degradation of chlorophyll has a direct impact on net photosynthetic rate

(Ashraf, 2004), so studying the degradation of chlorophyll at distinct developmental

growth stages seems meaningful to investigate the variation in salinity tolerance with

plant age.

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http://www.springerlink.com/content/?Author=D.+Khatkarhttp://www.springerlink.com/content/?Author=M.S.+Kuhadhttp://www.springerlink.com/content/?Author=D.+Khatkarhttp://www.springerlink.com/content/?Author=M.S.+Kuhad

Salinity stress is also known to decrease different gas exchange attributes such as

photosynthesis, transpiration rate, stomatal conductance, etc. (Ashraf, 2004). Salt-induced

stomatal closure results in decreased photosynthesis (Drew et al. 1990; Downton 1977;

Ashraf, 2004). Osmotic stress caused by higher salt concentrations results in the up-

regulation of ABA production which accumulates in leaves leading to reduced

intercellular CO2 concentration, stomatal conductance, activity of rubisco, etc.

Thaylakoid membranes are damaged due to elevated salt concentration in the

photosynthetic tissue. Salt-induced ionic imbalance reduces the K+ concentration in

chloroplasts, thereby adversely affecting the photosystem II (Ashraf, 2004). Salinity

tolerance of plants can be attributed to higher photosynthetic rate and stomatal regulation

(Salama et al., 1994; Ashraf, 2004). Generally, higher photosynthetic rates result in

improved biomass production and yield under salt stress (Ashraf, 2004). Recently, Sun et

al. (2011) have observed increased photosynthesis and stomatal conductance in Periploca

sepium seedling at mild salt stress. However, these attributes decreased significantly at

higher salt concentrations. Likewise, salt stress reduced net photosynthesis and stomatal

conductance of maize seedlings (Hichem et al., 2009). The reduction in net

photosynthesis of Arabidopsis seedlings has also earlier been reported (Zhang and Xing,

2008). In wheat, salt-induced reduction in photosynthesis has also been recorded at the

vegetative and grain filling stage (Ashraf and Parveen, 2002) and at the boot stage

(Abdeshahian et al., 2010). From these reports it could be stated that gas exchange

attributes exhibit significant salt-induced variation with plant age. Therefore,

investigation of gas exchange attributes at different growth stages could contribute

towards screening of crops for salt tolerance at different developmental growth stages.

Of the two primary photosystems, photosystem II is more prone to adverse effects

of salinity as compared to PS I (Apostolova et al., 2006; Mehta et al., 2010). Salt-induced

damage to electron transport may decrease the photosynthetic rates (Borsani et al., 2001).

It is evident from the previous studies that salt tolerance is a stage-specific mechanism

because salt tolerance at one developmental growth stage is not certainly correlated with

the salinity tolerance at the other growth stages (Mehta et al., 2010; Borsani et al., 2001).

Salinity stress degrades certain proteins (a membrane linker protein, chlorophyll protein)

which are required for attachment of phycobilisomes to thylakoids (Garnier et al., 1994).

5

http://www.refdoc.fr/?traduire=en&FormRechercher=submit&FormRechercher_Txt_Recherche_name_attr=auteursNom:%20%28HICHEM%29

Higher salt concentrations induce considerable damage to thylakoid membranes resulting

in changed membrane protein profile, which is responsible for decrease in oxygen

evolving activity of PS II and increase in the activity of PS I. Altered membrane protein

profile could result in minimal energy transfer from the light harvesting antenna complex

to photosystem II (Mehta et al., 2010). Plants grown on saline soils usually down-

regulate the reaction centers of photosystem II, thereby improving the conversion

efficiency of excitation energy (Lu and Vonshak, 2002). Therefore, chlorophyll

fluorescence is an effective method to detect and quantify the salt-induced damage to

photosynthetic machinery (Mehta et al., 2010). The salinity-induced damage to PS II has

been studied earlier using chlorophyll fluorescence technique in wheat at the seedling

stage (Mehta et al., 2010). Moradi and Ismail (2007) studied the adverse effects of

salinity on photosystem II using chlorophyll fluorescence method in rice at the vegetative

and reproductive stages. They reported decrease in ETR and increase in qN with

increasing salinity regimes. The increase in qN is an indication of photoinhibition

(Moradi and Ismail, 2007). Houimli et al. (2008) reported non-significant effects of salt

stress on Fv/Fm indicating the resistance of photosynthetic machinery to salt stress

(Belkhodja et al. 1994, 1999). It can be inferred from the literature presented here that

salinity variably affects the photosystem II at different developmental growth stages.

Therefore, insights into salt-induced damage to PS II using chlorophyll fluorescence

could yield meaningful results which can be used to screen crops for salt tolerance at

different growth stages.

Plants grown on saline soils exhibit marked changes in various biochemical

processes (Nawaz et al., 2010). For example, salt stress has been reported to decrease

ascorbic acid, chlorophyll content, relative water content, membrane stability index, and

increase the endogenous levels of H2O2, leaf MDA content (a measure of lipid

peroxidation) and activities of some potential enzymatic antioxidants listed earlier in

plants, e.g., in wheat at the seedling stage (Sairam and Srivastava, 2002) as well as at the

vegetative stage (Sairam et al., 2005; Mandhania et al., 2006). In cereals, the early

growth stages are least tolerant to salt stress as compared to the later flowering and grain

filling stages (El-Hendawy et al., 2005), but the performance of different genotypes is

variable at different growth stages (Maas and Poss, 1989). It has been reported widely in

6

the literature that production of antioxidants is up-regulated in a number of crops in

response to abiotic stresses including salinity stress to counteract the salt-induced

elevated levels of ROS in cells. For example, salt-induced increase in the activities of

various antioxidants enzymes was observed in barley (Khosravinejad et al., 2008), wheat

(Sairam et al., 2002; 2005), sesame (Koca et al., 2007), strawberry (Turhan et al., 2008),

and tomato (Gapin´ska et al., 2008). Thus, the plants containing high levels of

antioxidants can effectively scavenge/detoxify ROS thereby contributing to increased salt

tolerance (Wise and Naylor, 1987; Keles and Öncel, 2002; Parida and Das, 2005). Since

salt-induced alterations in different biochemical attributes and activities of a number of

enzymatic antioxidants as well as concentrations of non-enzymatic antioxidants of wheat

have been examined only at one growth stage, so it was felt necessary to study the

changes in different biochemical attributes at different growth stages, because there are a

number of reports in the literature which indicate that salt tolerance potential of most

crops including wheat changes with plant age (Ahmad et al., 2005; El-Hendawy et al.,

2005; Genc et al., 2007).

Membrane stability is considered as one of the important selection criteria to

discriminate between salt sensitive and salt tolerant cultivars (Jain et al., 2001; Sairam

and Srivastava, 2002; Demiral and Türkan, 2005). The integrity of membranes is greatly

hampered due to ROS-induced lipid peroxidation and MDA content (the product of lipid

peroxidation), a good measure of membrane stability (Meloni et al., 2003; Azevedo Neto

et al., 2006). Generally, salt sensitive plants are more prone to membrane lipid

peroxidation due to ROS as compared to salt tolerant plants (Sairam and Srivastava,

2002). For example, salt tolerant cultivars exhibit lower levels of MDA than that of salt

sensitive cultivars (Sairam et al., 2002; Demiral and Türkan, 2005). Therefore, measure

of membrane lipid peroxidation in terms of MDA contents could be used as a potential

determinant of stress tolerance (Luna et al., 2000; Demiral and Turkan, 2005).

A strong evidence exists in the literature that salinity stress induces enhanced

accumulation of organic osmolytes (most studied ones are proline and glycine betaine) in

the cytosol where they effectively participate in osmoregulation (Hasegawa, 2000; Ashraf

and Foolad, 2007). Proline is known to stabilize a number of sub-cellular structures such

as proteins and membranes, and scavenge free radicals. In addition, proline is considered

7

as a source of nitrogen for plants during stress recovery (Ashraf and Foolad, 2007). It up-

regulates the expression of salt responsive genes (Hmida-Sayari et al., 2005; Hoque et

al., 2008; Banu et al., 2009). It has been widely reported in the literature that salt tolerant

plants generally accumulate more proline than that of salt sensitive plants (Kumar et al.,

2003; Ashraf and Foolad, 2007; Szabados and Savoure, 2010). Enhanced free proline

accumulation due to salt stress has been recorded in a number of crops, e.g., wheat

(Mattioni et al., 1997; Sairam et al., 2002), Brassica juncea (Madan et al., 1995;

Gangopadhyay et al., 1997), soybean (Moftah and Michel, 1987), rice (Lin and Kao,

1996; Demiral and Türkan, 2005), onion (Mansour, 1998), and pea (Ahmad and Jhon,

2005; Najafi et al., 2007). This shows the importance of proline to be used as a potential

indicator of salt tolerance in most crop plants (Ashraf and Foolad, 2007). Glycine betaine

is one of the quaternary ammonium compounds actively involved in conferring salt

tolerance to plants (Ashraf and Foolad, 2007). For example, accumulation of glycine

betaine is positively correlated with osmotic adjustment and it also protects different

cellular metabolites. Furthermore, glycine betaine also protects photosystem II because it

is known to stabilize the association of extrinsic PS II protein complex under saline

regimes (Murata et al., 1992). Most of the cereals except rice exhibit higher endogenous

levels of glycine betaine when exposed to salinity regimes (Ashraf and Foolad, 2007).

For example, higher salinity tolerance of wheat is positively correlated with the

accumulation of GB in the leaves (Colmer et al., 1995). However, there exists a

significant variation in the accumulation of these organic osmolytes at different growth

stages (Sariam et al., 2002). As these osmolytes are known to play a potential role in

osmotic adjustment under salt stress, so studying the variation in their production at

different growth and developmental stages would be helpful in pinpointing the most

susceptible and tolerant stages of a plant under salt stress.

Wheat is the main staple food in many regions of the world (Stone and Savin,

2000; FAO Stat 2009). In Pakistan, the area under wheat cultivation is 8.216 million

hectares (Govt. of Pakistan, 2005). However, its production is being badly affected due to

salinity (Mehta et al., 2010; Ashraf et al., 2010). Wheat is known as moderately salt

tolerant crop (Munns et al., 2006). In field, where salinity may reach 10 dS m-1 rice will

die, whereas, wheat could tolerate this salt concentration giving reduced yield (Munns et

8

al., 2006). Salinity stress imposes several inhibitory effects on wheat growth in the form

of altered physiological and biochemical processes as mentioned earlier (Munns et al.,

2006; Ashraf et al., 2010). These inhibitory effects of salinity are not the same at all

growth stages in plants (Nawaz et al., 2010). Before screening the crops for salt

tolerance, the most susceptible and most tolerant growth stages of plant should be known.

This information would be useful in breeding programs as well as for alleviating the

adverse effects of salinity on most sensitive growth stage of wheat by taking counter-

measures like excessive irrigation to leach down the elevated levels of salts. The

information on the genetic variation of cultivars is a pre-requisite to characterize plants

for variation in salt tolerance potential at different growth stages. Several PCR-based

techniques could be used to determine the genetic distances between cultivars. Of

different molecular techniques, RAPD (randomly amplified polymorphic DNA) analysis

is an easy and fast method to achieve this goal (Asif et al., 2005). In RAPD analysis,

random oligo-primers are used to randomly amplify the genomic DNA of cultivars and

then the comparison of random amplified segments gives the information of genetic

differences (Asif et al., 2005). This technique has been used earlier to determine the

genetic differences in wheat cultivars by Khan et al. (2010). Keeping in view the need to

study variation in salt tolerance mechanisms at different growth stages, the present

investigation was conducted to determine the salt sensitivity of economically important

wheat crop at different developmental stages.

Objectives

The main objectives of the present investigation were to:

1. since salt tolerance in most crops varies at different growth stages, so our primary

objective to carry out this investigation was to uncover differences in

physiological and biochemical processes at the vegetative, boot and reproductive

stages in two wheat cultivars differing in salt tolerance,

2. determine up to what extent polymorphism exists between the two wheat cultivars

using the RAPD marker analysis,

9

3. assess how far various physiological and biochemical characteristics could

discriminate the two cultivars differing in salt tolerance and how far some of them

could be used as potential physiological/biochemical selection criteria for salt

tolerance in wheat.

10

Chapter 2 REVIEW OF LITERATURE 2.1 Food insecurity Increase in human population is very rapid and it is estimated that this alarming increase

in human population may reach 9.3 billion by the year 2050 (httl:// www. unfpa. org/

swp/ 200/). This will put a considerable pressure on food production system as directly or

indirectly plants are considered to be the basis of human nutrition (Chrispeels and

Sadava, 2003). The problem of food insecurity is more prominent in developing countries

like Pakistan. Although over the past ten years there is a steady economic growth, the

availability of food to the vulnerable population remains a major issue in Pakistan

(http://www.fao.org/countries/55528/en/pak/). The recent increase in food prices coupled

with frequent natural disasters has resulted in food insecurity in Pakistan. According to an

estimate, the total population of Pakistan is 155.4 millions with 34% urban population. Of

this population, 35 million people are undernourished making 23% of total population

(http://www.fao.org/countries/55528/en/pak/). However, various abiotic stresses further

decrease the crop production to a great extent. Salinity, drought, nutrient imbalances and

extremes of temperature are among the major abiotic stresses that limit crop productivity

worldwide.

2.2 Salinity-a major threat to crop production

Of various abiotic stresses, salinity is one of the major abiotic stresses that

severely affect crop productivity and quality. According to an estimate, more than 800

million ha land throughout the world are salt-affected (FAO, 2008; http:// www. fao.org/

ag/ agl/ agll/ spush/). The use of poor quality water for irrigation and poor drainage

further worsen the problem of soil salinity. However, the problem of salinity is more

pronounced in most of the developing countries. As Pakistan is an agricultural country,

most of its economic growth is dependent on agricultural production. But this agricultural

production is being affected by salinity stress as 26% of irrigated agriculture land is salt

affected. In Pakistan, total cultivated land is about 20 Mha and about 6.3 Mha are salt

affected (Khan et al.,1998; Anwar et al., 2006) and this problem of salinization is

becoming bad to worse. The government of Pakistan has allocated US$ 785 million to

counteract waterlogging and salinity problems, but still these problems are not

11

http://%20www.%20fao.org/%20ag/%20agl/%20agll/%20spush/http://%20www.%20fao.org/%20ag/%20agl/%20agll/%20spush/

under control (Chaudhry 2002). Soil is considered as saline if the electrical conductivity

of the saturated paste of the soil is (ECe) is 4 dS m-1 or more (US Salinity Laboratory

Staff, 1954). Plants are categorized into glycophytes (salt sensitive) and halophytes (salt

tolerant), depending upon their ability to grow on substrates containing high salt

concentrations. Halophytes can grow in saline soils as these plants can maintain high

pressure potential by accumulation of ions (Flowers et al., 1977) whereas, glycophytes

are not able to adapt osmotically under salt stress. Most grain crops are glycophytes and

are susceptible to salinity even when the soil ECe is < 4 dS m-1.

2.3 Major effects of salinity on plant growth

The adverse effects of salt stress on plants could be in the form of: a) osmotic

stress b) specific ion toxicity, c) nutrient imbalance, d) hormonal imbalance, e)

production of reactive oxygen species (Ashraf, 2009).

2.3.1 Osmotic Stress

Water potential of the saline soil drops due to high concentration of salts in the

rooting zone. As a result of this, plant root water conductivity decreases which affects

cell membrane permeability leading to reduced influx of water to the plant (Munns, 2002;

2009). It is reported in the literature that major contribution in growth reduction of plants

at initial phase of salinity is due to osmotic stress (Munns and Tester, 2008). Plant growth

under salt stress is primarily correlated with turgor potential and decrease in turgor

potential is the major cause of stunted growth under saline conditions (Munns, 2002;

Ashraf, 2004). Salt-induced osmotic stress has greater inhibitory effects on plant growth

as compared to those of specific ionic effect. Castillo et al. (2005) reported that when rice

plants were subjected to osmotic stress produced by NaCl or PEG at the vegetative and

reproductive stages, a significant reduction in the plant biomass was observed.

Furthermore, osmotic stress delayed maturity of the plants and caused a marked reduction

in grain yield particularly when NaCl or PEG-induced osmotic stress was applied at the

vegetative stage of the plant than that at the reproductive stage. Salt-induced arrest in

plant growth is mainly dependent on the severity of stress. For example, mild osmotic

stress rapidly induces growth inhibition of stems, and leaves, whereas roots might

continue to elongate (Nonami and Boyer, 1990; Spollen et al., 1993; Bartels and Sunkar,

2005). The extent of growth inhibition under salt-induced osmotic stress depends on time

12

scale of response, the species in question, the particular tissue and how the stress was

applied (rapid or gradual).

2.3.2 Specific ion toxicity

Irrigation water contains certain toxic ions that are taken up by plants and their

accumulation results in specific ion toxicity. Mainly sodium sulphate and chloride are

included in these toxic constituents (Ashraf, 2004; Nawaz et al., 2010). Specific ion

toxicity reduces the crop productivity that eventually results in crop failures. Ion toxicity

does not affect all crops equally, however, most crops plants are sensitive (Nawaz et al.,

2010). Plants grown on the medium with elevated salt concentrations take up high

amount of salts and accumulate in old leaves. However, this accumulation of salt in the

transpiring leaves over long period of time results in elevated tissue Na+ and Cl-

concentrations leading to leaf death. Due to high salt load, cells could not

compartmentalize salts into vacuole which is probably the main cause of injury to leaves

under salt stress. Enzyme activity is also inhibited due to the rapid build up of salts in the

cytoplasm. Munns (2005) reported that cells may dehydrate due to the build up of salts in

cell walls. However, no evidence for this was found in maize cultivars differing in salt

tolerance (Mühling and Läuchli, 2002).

Plants can tolerate specific ion toxicity in two ways: 1) minimize the entry of salts

into plants; 2) minimize the concentration of salt in cytoplasm. The root cytosolic Na+

concentrations may fall in the range of 10-30 mM (Tester and Davenport, 2003).

However, cytosolic leaf Na+ concentration is considered to be much lower than 100 mM

(Wyn Jones and Gorham, 2002). Dissolved Na+ and Cl- ions in soil solution must be

excluded by the roots so as to avoid the gradual build up of toxic levels of these ions in

shoot (Munns, 2005). For example, two durum wheat genotypes with variable rates of

Na+ transport to leaf were exposed to NaCl stress in order to develop a correlation among

Na+ exclusion, leaf injury and high yield. The older leaves of high-Na+ lines lost

chlorophyll more rapidly and died earlier as compared to those of low-Na+ lines. Twenty

percent increase in yield was observed in low-Na+ line under mild salt stress. However,

reduction in yield was observed at high salinity levels. It means that traits other than Na+

exclusion are important at high NaCl salinity where the salt-induced osmotic stress

outweighs its salt specific effect on growth and yield (Husain et al., 2003). Na+

13

accumulation inside the plants had inhibitory effects on seed germination because its

accumulation affected the water relations and build up of Na+ in cell walls by displacing

Ca2+ could disrupt the cell wall synthesis resulting in reduced plant growth (Xue et al.,

2004). Similarly, Cl- concentration greater than 80 mM in total tissue water may alter

plant morphology, stomata might become less responsive to the climatic changes and leaf

thickness is reduced. Chloride readily moves with soil water as it is not adsorbed by soils.

Consequently, Cl- is taken up by the plant roots and toxic levels of Cl- are accumulated in

leaves causing leaf burn or drying of leaf tissue. Toxic levels of Cl- could cause an early

leaf drop (Marchner, 1995).

In view of the available information, it is evident that different toxic ions cause

injurious effects in plants but the extent of injury varies among species and also it

depends on salt concentration of the growth medium.

2.3.3 Nutritional imbalance

Most crop plants are glycophytes and evolved under the conditions of low soil

salinity. Therefore, the mechanism adopted by the crop plants to absorb, transport and

utilize the mineral nutrients does not work as efficiently in saline soils as in non-saline

soils. In saline soils, the high concentrations of Na+ and/or Cl- often exceeds those of

macro-and micro-nutrients and may depress the nutrient-ion activities resulting in the

plant being more susceptible to osmotic stress, specific ion injury and nutritional

disorders that could result in reduced yield or quality (Grattan and Grieve, 1999). This

excessive amount of soluble salt in the rooting medium decreases the water potential of

the soil (osmotic stress) which may lead to the disturbance of plant water relations,

uptake and utilization of mineral nutrients and also in the accumulation of certain toxic

ions. The plant metabolic processes including activities of various enzymes are affected

as a result of these changes (Munns, 2002; Lacerda et al., 2003). The interactions

between salts and mineral nutrients may cause considerable nutrient deficiencies and

imbalances (McCue and Hanson, 1990). For example, excessive accumulation of Na+ and

Cl- in the cell causes ionic imbalance, thereby causing impaired uptake of other nutrients

(e.g., K+, Ca2+ and Mn2+) (Karimi et al., 2005). Excess concentrations of Na+ and Cl- in

the rooting medium inhibits the uptake of K+ and leads to K+ deficiency which eventually

results in chlorosis and subsequently necrosis (Gopa and Dube, 2003).

14

Potassium plays a vital role in osmoregulation and protein synthesis, maintains

cell turgor and stimulates photosynthesis (Freitas et al., 2001; Ashraf, 2004; Ashraf et al.,

2011). The cell membrane integrity and functioning is maintained by both K+ and Ca2+

(Wenxue et al., 2003). Potassium is maintained in plant tissues to adequate level under

salt stress by selective K+ uptake and selective cellular Na+ and K+ compartmentation and

distribution in shoots (Munns et al., 2000; Carden et al., 2003; Nawaz et al., 2010).

Presence of high NaCl in the growth medium competes with the uptake of other nutrient

ions (Parida and Das, 2005). Endogenous Na+ and Cl- contents increase with increasing

NaCl concentration in the growth medium while the concentrations of K+, Ca2+ and Mg2+

decrease in a number of plants (Khan et al., 1999, 2000; 2001). For example, Tunçtürk et

al. (2011) reported salt-induced decrease in K+ and Ca2+ in genetically different canola

cultivars. Similarly, when two strawberry cultivars (Korona and Elsanta) differing in their

salt tolerance were exposed to 40 and 80 mM of NaCl salinity, the symptoms of toxic

Na+ and Cl- were detected on leaf that eventually resulted in leaf scorching. The cv.

Korona maintained significantly higher leaf K+ contents as compared to that of cv.

Elsanta, while Ca2+ uptake under the varying NaCl levels was not affected. The

concentration of leaf Mg2+, Mn2+ and Fe2+ decreased significantly in both cultivars due to

NaCl salinity. However, an increase in the uptake of N and P was observed in leaf and

root. Uptake of Zn2+ and Cu2+ under NaCl stress could not be detected in the strawberry

plants (Keutgen and Pawelzik, 2008). Salt-induced decrease in K+ and K+/Na+ ratio and

increase in Na+ contents have also been reported in different maize cultivars (Carpici et

al., 2010). Mandhania et al. (2006) while studying the adverse effects of salt stress on the

ion content of two wheat cultivars (salt tolerant cv. KRL-19 and salt sensitive cv. WH-

542) observed a decline in K+/Na+ ratio of both cultivars under salt stress, but cv. KRL

maintained higher K+/Na+ ratio than that of cv. WH-542. When two rice cultivars (cv.

Lunishree and cv. Begunbitchi) differing in salt tolerance were subjected to NaCl salinity,

a significant alteration in ionic compositions was observed (Khan and Panda, 2008). With

increasing NaCl levels, endogenous Na+ contents increased while a decrease in K+

content was observed in both rice cultivars. Salinity stress resulted in enhanced Na+, Ca2+

and Cl- contents but reduced K+/Na+ ratio in Vicia faba (Gadallah, 1999). In leguminous

15

plant, 45 times increase in Na+ concentration was observed at 200 mM of NaCl level as

compared to that of control plants (Kurban et al., 1999).

2.3.4 Hormonal imbalance

Plant hormones are the organic substances produced in one part of the plant and

translocated to other parts, where they stimulate physiological response even at very low

concentration. Plant hormones act as second messengers involved in the induction of

plant responses to abiotic stress (Pedranzani et al., 2003). An increase in the levels of

plant hormones such as ABA and cytokinins is triggered by high salt concentration of the

growth medium (Thomas et al., 1992; Aldesuquy, 1998; Vaidyanathan et al., 1999).

Abscisic acid causes alteration in the salt-stress-induced genes (de Bruxelles et al., 1996).

In rice, ABA-inducible genes are predicted to play a vital role in the mechanism of salt

tolerance (Gupta et al., 1998). Salt stress caused increase in ABA levels, ethylene and

ACC (aminocycloprpane-1-carboxylic acid) in Citrus sinensis (Gómez-Cadenas et al.,

1998). ABA is thought to be involved in alleviation of inhibitory effects of salt stress on

growth, photosynthesis and translocation of assimilates (Popova et al., 1995). ABA is

also involved in stomatal closure as it rapidly alters ion fluxes in guard cells under stress

conditions. Uptake of Ca2+ is associated with the rise in ABA contents under salt stress,

and thus it maintains membrane integrity hence enabling the plants to regulate uptake and

transport under high intensity of salt stress (Chen et al., 2001). It has been reported in the

literature that leaf abscission and ethylene release were reduced by ABA under salt stress

in citrus probably by decreasing the toxic Cl- ion accumulation in leaves (Gómez-

Cadenas et al., 2002). Noaman et al. (2002) reported that salt tolerance of facultative

halophyte Lophopyrum elongatum and the closely related but less salt tolerant wheat

(Triticum aestivum L.) was improved when plants were allowed to gradually acclimatize

the salt stress conditions in comparison with when the plants were shocked suddenly.

Abcsisic acid was thought to regulate this acclimation to salt stress; a pretreatemnet with

ABA acted as a substituent for acclimation and enhanced the tolerance to salt shock. This

acclimation due to ABA pretreatment is very rapid and coincides with enhanced

expression of early salt-induced genes in the roots. The tolerance of Lophopyrum

elogatum to sudden salt shock was found to be better than wheat and its genome confers

more salt tolerance to sudden salt shock in its amphploid than that in wheat. In L.

16

elongatum genome, chromosome 3E is involved in the tolerance of salt shock, whereas

homologous chromosomes 3A and 3D in wheat also control salt shock response. It could

be speculated that chromosome 3 in both species regulates salt shock response via

abcsisic acid (Noaman et al., 2002).

Jasmonates also play a vital role in salt tolerance. Experimental evidence indicates

that higher levels of jasmonates are found in salt tolerant tomato cultivars than those in

salt sensitive cultivars (Hilda et al., 2003). Generally, jasmonates are considered to

mediate signaling, such as senescence, flowering and defense responses. However, the

factors involved in jasmonate signal transduction pathway are not clear. Furthermore,

exogenous application of jasmonates resulted in accumulation of free proline and a

decrease in shoot Na+ and Cl- accumulation in pea (Fedina and Tsonev (1997).

Jasmonates are potentially involved in osmoregulation or osmoprotection through

enhanced accumulation of proline and decreased ion accumulation.

2.3.5 Production of reactive oxygen species

Molecular oxygen is vital for life on earth but its reduction by any means leads to

the formation of reactive oxygen species (ROS). ROS have been reported to disturb

different metabolic processes in plant (Asada, 1994; Ashraf, 2009). The reduction of

molecular oxygen (O2) results in the production of ROS such as hydrogen peroxide

(H2O2), superoxide (O·−2) and hydroxyl radical (·OH). When molecular oxygen reacts

with excited chlorophyll, singlet oxygen (1O2) is produced which is also considered as

one of the potential reactive oxygen species. As ROS are extremely reactive, they can

react with a number of metabolites and other molecules such as lipids, DNA, proteins,

pigments and other vital cellular molecules leading to a series of destructive processes

(Lamb and Dixon, 1997; Mittler, 2002).

Reactive oxygen species are also produced under normal conditions but their

concentration is very low (Polle, 2001). Enhanced production of reactive oxygen species

is induced under various environmental stresses (Desikan et al., 2001; Pastori and Foyer,

2002; Karpinski et al., 2003; Laloi et al., 2004; Ashraf, 2009). For example osmotic

stress results in stomatal closure thereby limiting CO2 availability for photosynthesis.

This results in elevated production of superoxide in chloroplast which can cause

photooxidation and photoinhibition damage. Under various environmental stresses, ROS

17

are produced in plants via different pathways, e.g., from the photosynthetic apparatus,

mitochondrial respiration and photorespiration (Ashraf, 2009). Salt stress also induces

the generation of ROS (Ali and Alqurainy, 2006). Therefore, reactive oxygen species are

considered to be the indicators of stresses and also act as second messengers that are

actively involved in stress-response signaling pathways.

ROS are free radicals (atoms or group of atoms) with at least one unpaired

electron. This configuration is highly unstable and the radicals tend to react with other

molecules to produce more free radicals in search of stable electronic configuration

(Ashraf, 2009). For example, electrons are leaked from respiratory chain and result in the

reduction of molecular oxygen to superoxide anion (Mittler, 2002). Furthermore,

hydrogen peroxide is produced from non-enzymatic or enzymatic dismutation of

superoxide. Photorespiration reactions and β-oxidation of fatty acids also result in H2O2

generation (Parida and Das, 2005; Ashraf, 2009; Nawaz et al., 2010). This is lethal for

plants, because it is considered to be one of the strong inhibitors of Calvin cycle (Shen et

al., 1997; Ashraf, 2009). Furthermore, in the presence of certain metal ions or metal

chelates, H2O2 and superoxide interact to produce highly reactive ·OH radical (Temple et

al., 2005).

Different subcellular compartments (peroxisomes, chloroplasts and

mitochonderia) are the sites of ROS production (Ashraf, 2009). Ubiquitous production of

ROS is observed during metabolism and all plants have the ability to cope with them.

However, significantly higher production of ROS results in a substantial cellular damage

(Sairam and Srivastava, 2002; Mittler, 2002).

Presence of molecular oxygen as final electron acceptor poses a threat of ROS

induced oxidative damage (Ali and Alqurainy, 2006). Superoxide cannot react with lipids

or proteins, but its protonated form may result in lipid peroxidation (Asada and

Takahashi, 1987). Superoxide can cause inhibition of ribonucleotide reductase and

peroxidases (Ashraf, 2009). Presence of superoxide results in accelerated production of

hydroxyl radicals and hydrogen peroxide (Hideg, 1997). Since H2O2 is the most

destructive ROS, it can cause more than 50% reduction in carbon dioxide assimilation in

plants at 10 μM concentration in the chloroplast (Kaiser, 1979). For example, when two

salt tolerant (Kharchia 65, KRL 19) and two salt sensitive (HD 2009, HD 2687) wheat

18

genotypes were exposed to different levels of salt stress, Kharchia 65 showed minimum

oxidative damage due to H2O2 as it had lower levels of hydrogen peroxide as compared

to the other wheat genotypes (Sairam et al., 2005). Similarly, Mittova et al., (2003)

reported an increase in hydrogen peroxide contents of tomato species under salt stress.

Salt-induced increase in hydrogen peroxide has also been reported in pea (Gomez et al.,

2004). Oxidative damage due to enhanced levels of hydrogen peroxide is also evident in

potato plants when grown in a growth medium containing high amounts of salts (Fidalgo

et al., 2004). Furthermore, production of reactive oxygen species is also variable at

different developmental stages (Ashraf, 2009).

2.4 Inhibitory effects of salinity on growth and physiological attributes

Salt stress affects biomass production and different physiological attributes of

most crop plants. These adverse effects of salt stress could be observed at different

developmental stages of plant growth. However, the response of plants to salt stress at

different growth stages is variable among plant species (Nawaz et al., 2010). In general,

the vegetative stage of plants is considered to be more responsive to the adverse effects of

salinity as compared to other growth stages (Cuartero et al., 2006; Bybordi, 2010;

Bandehagh et al., 2011).

As salt stress involves both ionic and osmotic stress (Hagemann and Erdmann,

1997; Hayashi and Murata, 1998), growth reduction is directly linked to the total

concentration of soluble salts or osmotic potential of soil water (Flowers et al., 1977;

Greenway and Munns, 1980). The inhibitory effect is seen at the whole-plant level as

decreased productivity or death of plants. Growth suppression occurs in all plants but

their tolerance abilities and growth reduction rates at lethal salt concentration vary widely

among different plant species. All the major processes such as photosynthesis, growth,

protein synthesis and energy and lipid metabolism are adversely affected by salt stress.

2.4.1 Salinity sensitivity in relation to developmental growth stage

It has long been known that sensitivity of crop plants to salt stress varies at

different growing stages (Bernstein and Hayward, 1958). For example, germination and

emergence stages or in other words early vegetative stages of crop plants have been

found sensitive to salt stress (Läuchli and Epstein, 1990; Maas and Grattan, 1999). It has

been reported that tolerance to salinity increases with the maturity of plants. Thus, salt

19

http://www.ncbi.nlm.nih.gov/pubmed?term=%22Bandehagh%20A%22%5BAuthor%5D

tolerance definition is not the same for different developmental growth stages. For

example, percent survival is considered as salt tolerance ability of plants at germination

and emergence stages, while at later growth stages salt tolerance ability is defined in

terms of relative growth reduction due to salt stress (Läuchli and Grattan, 2007).

Vegetative and reproductive development stages are severely affected by salt

stress, but the salinity-induced effects vary depending on whether the harvested organ is

leaf, root, shoot, stem, fiber, fruit or grain. Shoot growth is more affected by salinity than

root growth (Läuchli and Epstein, 1990). Salt stress can decrease the number of florets

per ear, increase the sterility and affect the maturity and flowering time in wheat (Maas

and Poss, 1989) and rice (Khatun et al., 1995). Therefore, management strategies must be

developed, so as to minimize the adverse effects of stress at critical stages. This could be

achieved by understanding the mechanisms of salinity effects at the plant vegetative and

reproductive developmental stages (Läuchli and Grattan, 2007).

Most crop plants are tolerant to salt stress during germination stage. However, salt

stress causes considerable delay in the germination despite the insignificant difference in

the percentage of germinated seeds (Maas and Poss, 1989). These observations lead to

the categorization of salt tolerance at different developmental stages. For example,

germination of Limonium perezii seeds (commonly grown as an ornamental plant) is

stimulated by 10 dS/m of salinity (Carter et al., 2005). In contrast, it has been widely

reported in the literature that elevated salt concentrations will reduce the germination

percentage (Badia and Meiri, 1994; Mauromicale and Licandro, 2002). Läuchli and

Epstein (1990) reported some degree of salt sensitivity in sugar beet (characterized as salt

tolerant) at germination stage (Läuchli and Epstein, 1990). Tajbakhsh et al. (2006) was

of the view that screening at germination stage is important for assessing salt tolerance of

crops because this early growth stage is very important for crop establishment.

It has been reported in a number of crops that plant growth is severely affected

due to salt stress at the seedling and early vegetative developmental growth stages in

comparison with germination stage, e.g., barley (Ayers et al., 1952), rice (Pearson and

Ayers, 1966), cotton (Abul-Naas and Omran, 1974), corn (Maas et al., 1983), sorghum

(Maas et al., 1986), wheat (Maas and Poss, 1989a), cowpea (Maas and Poss, 1989b),

spinach (Wilson et al., 2000), red orach (Wilson et al., 2000), tomato (del Amor et al.,

20

2001), melon (Botia et al., 2005) etc. When wheat and corn were grown under green-

house conditions, a significant reduction in shoot biomass and grain yield was recorded

(Maas et al., 1983; Maas and Poss, 1989a).

Zheng et al. (2010) while studying the effect of salinity stress on wheat at the

reproductive stage reported salt-induced reduction in leaf area index, leaf area duration

and dry biomass. Similarly, the reproductive stage of wheat plant is more susceptible to

adverse effects of salinity as compared to the vegetative growth stage. Reproductive stage

has also been hastened by salt stress which inhibits spike development and decreases

yield potential in wheat (Läuchli and Grattan, 2007). Rice was found to be more salt

susceptible at the vegetative and reproductive growth stages (Zeng et al., 2001; Moradi

and Ismail, 2007). Furthermore, 6.65 dS m-1salinity stress in rice could result in 50

percent yield reduction (Zheng and Shannon, 2000).

While summarizing all the reports of adverse effects of salinity at different

developmental stages, it can be concluded that salt tolerance varies among different plant

species at different growth stages. Therefore, study of tolerance mechanisms at different

growth stages will provide a tool to specify the most susceptible growth stage of a plant

and then countermeasures can be taken to prevent the salt-induced damage at that

particular growth stage.

2.4.2 Water relations

With increase in salinity, tissue osmotic and water potentials become more

negative, whereas both increase and decrease in turgor pressure are recorded with

increasing salt levels (Morales et al., 1998; Hernández et al., 1999; Khan et al., 1999;

Meloni et al., 2001; Khan et al., 2001; Romero-aranda et al., 2001; Taiz and Zeiger,

2006). The uptake of ions from the growth medium and/or accumulation of organic

osmotica play an important role in osmotic adjustment of plant under salt stress

(Hernandez and Almansa 2002; Taiz and Zeiger 2002; Chaparzadeh et al., 2003).

Generally, when turgor decreases below the yield threshold of the cell wall, growth is

ceased (Taiz and Zeiger 2002). The cells respond to reduced turgor by osmotic

adjustment. Leaf osmotic and water potentials decline depending on how stress has been

imposed and the osmotic potential of the rooting medium. A greater reduction in leaf

osmotic potential compared with the total water potential resulted in turgor maintenance

21

of plants under prolonged or progressive NaCl stress (Rajasekaran et al., 2001). Salt-

induced changes in turgor have been reported in a number of crops, e.g., reduction in this

attribute has been reported in barley (Thiel et al., 1988) and wheat (Ashraf and O’leary

1996), whereas increase in turgor potential under salt stress has been recorded in spinach

(Robinson et al., 1983), sugarbeet (Heuer and Plaut, 1989), Sorghum bicolor ( Yang et

al., 1990), citrus (Lloyd et al., 1987), and tomato (Romero-Aranda et al., 2001).

Leaf osmotic and water potentials as well as xylem tension were reported to

increase with increasing salinity in Rhizophora mucronata (Aziz and Khan, 2001).

Similarly, in jute water use efficiency, water retension, transpiration rate, water uptake,

leaf water potential and relative water content decrease under short-term sodium chloride

stress (Chaudhuri and Choudhuri, 1997). Stomatal conductance and water and osmotic

potentials became more negative in Urochondra setulosa, the halophytic perennial grass,

with increase in external salt levels, while turgor potential decreased in this grass with

increasing salinity (Gulzar et al., 2003). When Suaeda salsa was subjected to salt stress, a

significant reduction in evaporation rate and leaf water potential was recorded, whereas

leaf relative water content was not affected (Lu et al., 2002). It has been reported that

water relation parameters (water potential, osmotic potential and turgor potential) are

differently affected at different developmental stages of a plant under saline regimes

(Millar et al., 1968; Ashraf and Shahbaz, 2003; de Azevedo Neto et al., 2004; Singh et

al., 2010). For example, Siddiqi and Ashraf (2008) used water relation parameters as

selection criteria for assessing the inter-cultivar variation among ten safflower lines at the

vegetative stage under 150 mM level of NaCl. A significant reduction in shoot fresh

biomass and different water relation parameters (water potential, osmotic potential,

relative water content) except turgor pressure was observed in all safflower lines.

Similarly, Nawaz and Ashraf (2007) observed inhibitory effects of salt stress on plant

fresh and dry biomass as well as on water, turgor and osmotic potentials at the vegetative

and reproductive stages of maize. While studying the physiological behavior of two

Brassica cultivars at the vegetative stage under salinization, Singh et al. (2010) reported a

decline in water and osmotic potentials, whereas turgor potential increased under salt

stress indicating the substantial role of osmotic adjustment. Salt-induced reduction in

22

relative water content at the seedling stage of 12 Vigna genotypes has also been reported

(Win et al., 2011).

By summarizing all these reports, it can be inferred that water relations could be

used as potential selection criteria for screening crops at different developmental stages

under salt stress. However, water relation parameters are differently affected by salt stress

at different growth stages of plants.

2.4.3 Photosynthetic pigments

The total carotenoid and chlorophyll contents generally decrease in plants under

salinity stress. Chlorosis is developed in older leaves and prolonged period of salt stress

may result in the fall of these leaves (Hernandez et al., 1995, 1999; Gadallah, 1999,

Agastian et al., 2000; Ashraf 2004). However, Wang and Nil (2000) reported an increase

in chlorophyll contents in Amaranthus under the conditions of salinity. In contrast,

protochlorophyll, chlorophyll and carotenoids were markedly reduced in Grevilea due to

NaCl stress, whereas anthocyanin pigments increased significantly in this plant (Kennedy

and De Fillipis, 1999). In tomato, total cholorphyll contents, Chl.a and β- carotene

decreased under sodium chloride salinity (Khavarinejad and Mostofi, 1998). When nine

rice genotypes were subjected to salt stress, a significant reduction in photosynthetic

pigments was recorded (Alamgir and Ali, 1999). Zheng et al. (2008) reported a decline

in photosynthetic pigments of two wheat cultivars (DK961, salt-tolerant; JN17, salt-

sensitive) under salt stress, but this decrease was more prominent in salt sensitive

cultivar. Salt stress caused a marked reduction in photosynthetic pigments in blue

panicgrass (Panicum antidotale Retz.) (Ashraf, 2003). Sairam et al. (2005), while

studying the effect of long-term sodium chloride salinity on two tolerant wheat cultivars

(Kharchia 65, KRL 19) and two sensitive cultivars (HD 2009, HD 2687) reported a salt-

induced decline in chlorophyll contents. Kharchia 65 showed lowest decline in

photosynthetic pigments as compared to other wheat cultivars. When pea plants were

subjected to salt stress, a rapid decline in the chlorophyll contents was observed

(Hernandez et al., 1999). The concentration of chlorophyll pigments was also found to

be decline at different growth stages of plants under salinity stress (Sairam et al., 2002).

Khatkar and Kuhad (2000) studied the adverse effects of salinity on chlorophyll pigments

in two wheat cultivars (KRL 1-4 and HD 2009) at different growth stages. They observed

23

salt-induced decline in photosynthetic pigments with age of the plant. They were of the

view that this reduction in chlorophyll was perhaps due to the enhanced activity of

chlorphyllase or reduced de novo synthesis of chlorophyll. Dhanapackiam and Ilyas

(2010) observed a significant reduction in chlorophyll contents of Sesbania grandiflora at

the seedling stage. They reported that this salt-induced reduction in chlorophyll depends

upon the growth stage of plant and type of salt used, e.g., NaCl, Na2SO4. When Brassica

juncea L. (cv. Kraniti) was exposed to higher salinity levels, adverse effects of salinity on

chlorophyll contents were recorded during vegetative, boot and reproductive stages.

However, this salt-induced decrease in chlorophyll contents was more prominent at the

reproductive stage as compared to that at the vegetative or boot stage. Similarly,

chlorophyll contents were significantly reduced in salt tolerant wheat cultivar DK961 and

salt sensitive JN17 at the reproductive stage under salinity stress (Zheng et al., 2008).

When Momordica charantia plants were subjected to salt stress at three different growth

stages (pre-flowering, flowering and post-flowering), a significant reduction in

photosynthetic pigments was recorded at all three growth stages (Agarwal and Shaheen,

2007). Chookhampaeng (2011) studied the effects of salt stress on hydroponically grown

pepper (Capsicum annum L.) at the seedling stage. A marked reduction was observed in

the chlorophyll contents of pepper on exposure to high salt concentrations at the seedling

stage. Sairam et al. (2002) reported a salt-induced decline in chlorophyll contents of

wheat at different growth stages.

2.4.4 Soluble proteins

Salt stress is generally known to cause reduction in soluble protein contents of

most plants (Alamgir and Ali, 1999; Gadallah, 1999; Wang and Nil, 2000;

Muthukumarasamy et al., 2000; Parida et al., 2002). Agastian et al. (2000) have reported

an increase in soluble proteins at mild salt stress and decrease in this attribute was

observed at high salinities in mullberry. Similarly, when three different tomato cultivars

were subjected to salt stress, a significant decline in total soluble proteins was observed

(Doganlar, 2010). Amirjani (2010a) reported a decline in total soluble proteins of rice

under salt stress. In contrast, Afzal et al. (2006) reported increase in total soluble proteins

in wheat plants when grown under varying concentrations of salt stress. Lobato et al.

(2009) also found an increase in total soluble proteins in cowpea when different salt

24

concentrations were applied in the growth medium. When radish plants were germinated

under three concentrations of NaCl (50, 100 and 200 mM), a significant decrease in total

soluble proteins was recorded at 100 and 200 mM levels of NaCl except an increase at

mild salt stress (50 mM) . Total soluble proteins in cells vary at different growth stages

(Garg et al.,2006). For example, Sairam et al. (2002) while studying the adverse effects

of salt stress at different growth stages of wheat plants reported an increase in total

soluble proteins at varying salt levels and at all plant growth stages.

2.4.5 Photosynthesis

Salt stress reduces transpiration rate, stomatal conductance and photosynthetic

rate in plants (Tezara et al. 2002; Gibberd et al. 2002; Burman et al. 2003; Ashraf, 2004).

Chlorophyll contents increase at mild salt stress (Winicov and Button 1991; Locy et al.

1996) but degradation of chlorophyll takes place at high salinities (Salma et al., 1994;

Ashraf, 2004). Stomatal closure is the main cause of reduced photosynthesis in plants

under salt stress (Rajesh et al., 1998; Stepien and Klobus, 2006). The salt-induced

osmotic effects causes the accumulation of abscisic acid (ABA) resulting in reduced

stomatal conductance, chlorophyll content, intercellular carbon dioxide concentration and

activity of Rubisco. Abscisic acid accumulation also induces change in electron transport

and accumulation of sucrose (Ashraf, 2004). High salt concentration in photosynthetic

tissues results in stacking of adjacent membranes and shrinkage of thylakoids, reduction

in K+ contents of chloroplasts and degradation of photosystem II (PSII) (Sharma and

Hall, 1991; Murata et al., 2007). In addition, reduction in photosynthesis may be due to

feedback inhibition of high concentration of sugars in mesophyll cells that could be

observed on leaves when plants are exposed to salt stress, because impairment of normal

sugar utilization in the growing tissues results in high sugar concentrations in mesophyll

tissues (Munns et al., 1982; Ashraf, 2004). The maintenance of stomatal conductance

and net photosynthetic rate and high chlorophyll concentration confer salt tolerance in

plants (Lakshmi et al., 1996; Krishna Raj et al. 1993; Salama et al. 1994; Khan et al.,

2009). In contrast, a positive correlation between yield and photosynthesis under salt

stress has been reported in a number of crops, e.g., Vigna mungo (Chandra Babu et al.,

1985), Zea mays (Crosbie and Pearce 1982), Spinacia oleracea (Robinson et al., 1983),

Phaseolus vulgaris (Seemann and Critchley, 1985), Gossypium barbadense (Cornish et

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al., 1991), Gossypium hirsutum (Pettigrew and Meredith 1994), Asparagus officinalis

(Faville et al., 1999), Panicum hemitomon, Spartina patens, and Spartina alterniflora

(Hester et al., 2001), six Brassica diploid and amphiploid species (Ashraf 2001). There

has been little or no association between photosynthetic capacity and growth, e.g.,

Hordeum vulgare (Rawson et al., 1988), Hibiscus cannabinus (Curtis and Läuchli 1986),

Trifolium repens (Rogers and Noble 1992), Triticum aestivum (Hawkins and Lewis 1993;

Ashraf and O’leary 1996b), Olea europea (Loreto et al. 2003).

The net CO2 assimilation rate is affected to varying extents at different growth

stages of plant (Ashraf and Parveen, 2002; Walia et al., 2005; Moradi and Ismail, 2007).

For example, rice is considered to be the most salt sensitive crop and its sensitivity to salt

stress is variable at different growth stages. Rice is found to be more sensitive to salt

stress at early vegetative stage as compared to that at the reproductive stage (Lutts et al.,

1995; Walia et al., 2005; Moradi and Ismail, 2007). When Arabidopsis was subjected to

salt stress at the seedling stage, a significant reduction in net photosynthetic rate was

recorded (Li-Wei Ho, et al., 2010). While investigating the inhibitory effects of salt stress

on two spring wheat cultivars (salt tolerant cv. SARC-I and salt sensitive cv. Potohor),

Ashraf and Parveen (2002) reported decline in photosynthetic rate at the vegetative and

reproductive stages. However, the reduction in net CO2 assimilation was more in salt

sensitive cv. Potohar as compared to that in tolerant cv. SARC-I at both stages. When

four linseed (Linum usitatissimum L.) were exposed to salt stress at different growth

stages, a significant reduction in net photosynthetic rate was observed at all growth stages

(Khan et al., 2007).

From the above-given reports, it is amply clear that photosynthetic capacity varies

at different plant growth stages. Furthermore, measurement of photosynthetic capacity at

different growth stages under salt stress provides a good means of selecting most

susceptible growth stage of plant under salt stress.

2.4.6 Chlorophyll fluorescence

Chlorophyll fluorescence is considered as an efficient non-destructive and rapid

method to detect and quantify the damage to leaf and photosynthetic apparatus under

different abiotic stresses (Palta, 1992; Sestak and Stiffel, 1997; Percival, 2004; Baker and

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Rosenqvist, 2004; Kauser et al., 2006; Baker, 2008). It could also be used as one of the

potential selection criteria for salt tolerance in breeding programs to improve crop

production. This technique determines the change in chlorophyll ‘a’ fluorescence due to

altered activity of photosystem II (P II) caused by salt stress. However, Shabala et al.

(1998) reported no change in Fv and Fm of maize cultivars grown under salt stress. This

indicates that salt stress did not affect PS II. In contrast, Smillie and Nott (1982) found

salt-induced modification in fluorescence induction characteristics and were of the view

that chlorophyll fluorescence has the potential to be used a physiological selection

criterion for salt tolerance. In Brassica, salt stress adversely affected the electron

transport system of thylakoid membrane and the activities of PS II (Alia et al., 1993).

Netondo et al. (2004) reported a significant decrease in electron transport rate (ETR),

photochemical quenching coefficient (qP) and maximum quantum yield of photosystem

II in sorghum. In contrast, Amirjani (2010b) while studying the effect of salt stress on

wheat reported an increase in electron transport rate (ETR) and decrease in Fv/Fm.

Dionisio-Sese and Tobita (2000) studied the effects of salt stress on rice cultivars

differing in salt tolerance and found that Fv/Fm remained unaffected under salt stress

whereas in salt sensitive rice cultivars qN increased with increase in salt concentration of

the external medium. While studying the adverse effects of salt stress on four wheat

cultivars Abdeshahian et al. (2010) reported that salt stress caused decrease in quantum

yield of photosystem II of light adapted leaf (ΦPSII), photochemical quenching (qP),

quantum yield of dark adapted leaves (Fv/Fm) and increase in non-photochemical

quenching (NPq).

Variation in different parameters of chlorophyll fluorescence exists at different

growth stages of plants under salt stress (da Silva et al., 2011). For example, Moradi and

Ismail (2007), while studying the adverse effects of salinity on rice at vegetative and

reproductive stages revealed that electron transport rate decreased, whereas non-

photochemical quenching increased under salt stress. Similarly, Jatropha curcas was

exposed to salt stress and data for chlorophyll fluorescence was recorded at three

different harvest times

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