IN THE NAME OF ALLAH THE MOST BENEFICENT THE MOST MERCIFUL
DEMOGRAPHY AND SEASONAL ECOPHYSIOLOGY OF A LEAF SUCCULENT HALOPHYTE SALSOLA DRUMMONDII ULBRICH.
AYSHA RASHEED
Institute of Sustainable Halophyte Utilization (ISHU) University of Karachi Karachi-75270, Pakistan 2014 DEMOGRAPHY AND SEASONAL ECOPHYSIOLOGY OF A LEAF SUCCULENT HALOPHYTE SALSOLA DRUMMONDII ULBRICH.
THESIS SUBMITTED TO THE FACULTY OF SCIENCE UNIVERSITY OF KARACHI, IN THE FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
AYSHA RASHEED
Institute of Sustainable Halophyte Utilization (ISHU) University of Karachi Karachi-75270, Pakistan 2014
Acknowledgements
I am deeply indebted to my adviser, Prof. Dr. Bilquees Gul for her unwavering support, encouragement, stimulating insights, patience and guidance during the research work and thesis writing. I feel honored to be her first Ph.D. student. I am also thankful for the excellent example she has provided as a successful woman botanist and professor.
I feel great pleasure to express my deep sense of gratitude to Prof. Dr. M. Ajmal Khan, for his guidance and continuous support from the very beginning of this research. I am also grateful to him for patiently reviewing and editing the dissertation. His dynamic personality, immense knowledge and devotion to pursue high quality research make him a great mentor.
Special thank is due to Dr. Salman Gulzar for his generous help during the field work and valuable comments on my dissertation. He has been supportive since the days I began my research work. I am extremely grateful to Prof. Dr. Hans-Werner Koyro (Institute of Plant Ecology, Justus Liebig University, Giessen, Germany) for his sincere guidance, thought-provoking discussions and suggestions during this study. I am also thankful to Dr. Irfan Aziz, Dr. Raziuddin Ansari and Dr. M. Zaheer Ahmed for their encouragement throughout this work. I would like to acknowledge the “Higher Education Commission of Pakistan” for providing the Ph.D. merit scholarship (Indigenous Scholarship Program, Batch IV).
I am thankful to all my colleagues and friends for their moral support and help especially Mr. Zainul Abideen, Mr. Tabassum Hussain, Mr. Haibat Ali, Mr. M. Qasim, Ms. Sarwat Ghulam Rasool, Ms. Sadaf Asif and of course Ms. Mishal Birgees Khan for her sweet company and friendship. Thanks are also due to Mr. Irfanuddin and Mrs. Saira Salman for their assistance in office related issues and Mr. Sawa Khan (Lab attendant) for his help during field work. Words are lacking to express my profound gratitude to my beloved brother Dr. Abdul Hameed for his help, support, encouragement, and love. I would also like to thank my younger brother Abdul Haseeb, for his love and support. With a deep emotion of gratitude, I dedicate this thesis to the memories of my grandfather Abdul Rauf, who is not amongst us now but his love and prayers made me what I am today. Last but not the least I would extend my deepest gratitude to my father Abdul Rasheed and mother Kishwar Rasheed for their love, continuous prayers, untiring support and encouragement during this research and throughout my life. I always feel the strength of their love and prayers in all the achievements I have made and it will remain with me during my whole life.
Thank You Table of Contents
Page
Table of Contents I
List of Tables III
List of Figures V
List of Abbreviations X
Summary in English XIII
Summary in Urdu XV
Chapter 1 General introduction 1
References 15
Chapter 2 Effects of salinity, temperature, light and dormancy 27 regulating chemicals on seed germination of Salsola drummondii
Abstract 28
Introduction 29
Materials and methods 31
Results 34
Discussion 42
References 47
Chapter 3 Variation in temperature and light but not salinity 54 invoke antioxidant enzyme activities in germinating seeds of Salsola drummondii
Abstract 55
Introduction 56
I
Materials and methods 57
Results 60
Discussion 71
References 75
Chapter 4 Growth, oxidative damage and antioxidant enzyme 84 activities in NaCl-treated seedlings of Salsola drummondii
Abstract 85
Introduction 86
Materials and methods 87
Results 90
Discussion 98
References 102
Chapter 5 Physiological responses of Salsola drummondii population 109 to seasonal variations
Abstract 110
Introduction 111
Materials and methods 113
Results 117
Discussion 133
References 137
Chapter 6 General Conclusion 145
II
List of Table
Page
Table 2.1: Two-way analysis of variance (ANOVA) indicating significance of the 34 individual and collective effects of various experimental factors on the percentage of germinated (G), recovered (R), viable (V) and dead (D) seeds when recovered from salt. Where, numbers represent F-values. * = P < 0.05 and *** = P < 0.001.
Table 2.2: Two-way analysis of variance (ANOVA) indicating significance of the 35 individual and collective effects of various experimental factors on the percentage of germinated (G), recovered (R), viable (V) and dead (D) seeds when recovered from temperature. Where, numbers represent F- values. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 and ns = non- significant.
Table 2.3: Two-way analysis of variance (ANOVA) indicating significance of the 35 individual and collective effects of various experimental factors on the percentage of germinated (G), recovered (R), viable (V) and dead (D) seeds when recovered from dark. Where, numbers represent F-values. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 and ns = non-significant.
Table 3.1: Seed characteristics of Salsola drummondii. 60
Table 3.2: Analysis of variance showing the effect of different abiotic factors on 61 percent germination (G), relative water uptake (RWU), MDA content and antioxidant enzyme activities of Salsola drummondii seeds. Values represent F- values. Where, * = P < 0.05; ** = P < 0.01; *** = P < 0.001 and ns = non-significant.
Table 4.1: Analysis of variance showing the effect of different NaCl treatments at 91 20/30 oC in 12-H light on various growth parameters and antioxidant enzyme activities in seedlings of Salsola drummondii. Values represent F- values. Where, * = P < 0.05; ** = P < 0.01; *** = P < 0.001 and ns = non-significant.
III
Table 5.1: Description of sampling time. 114
Table 5.2: Two-way analysis of variance (ANOVA) of soil cation content due to seasons 119 (S), soil depth (D) and S x D interactions on the soil mineral content of the Salsola drummondii community. Numbers indicate F-values at * = P < 0.05, ** = P < 0.01, *** = P < 0.001 and ns = non-significant.
Table 5.3: Seasonal variations in the soil cations (Na+, K+, Ca++ and Mg++; mg Kg-1 DW) 120 contents drawn from different depths of the Salsola drummondii community.
Table 5.4: Summary of F-values from one way analysis of variance (ANOVA) in growth 124 and physiological parameters of Salsola drummondii during the study year.
Table 5.5: Seasonal variations in the photosynthetic pigments and gas exchange 131 parameters of Salsola drummondii.
IV
List of Figures
Page
Figure 1.1: Salsola drummondii in its habitat. 9
Figure 1.2: Google Earth image of the study site. 9
Figure 1.3: Economic importance of Salsola drummondii. 10
Figure 1.4: Phenology diagram of Salsola drummondii in salt desert of Balochistan, 11 Pakistan.
Figure 1.5: Conceptual flow diagram of seed bank dynamics for Salsola drummondii 12 growing in the salt deserts of Balochistan, Pakistan.
Figure 1.6: Parts of this PhD thesis in which the role of Salsola drummondii under 14 natural habitat conditions and its ecological requirements and ecophysiological responses were studied in the context of 1). Germination ecology, biochemistry and seedling growth and 2). Seasonal eco- physiology.
Figure 2.1: Effect of salt, light/dark and temperature treatments on the seed 36 germination of Salsola drummondii. A. 10/20 ◦C, B. 15/25 ◦C, C. 20/30 ◦C and D. 25/35 ◦C. Circles represent mean ± standard errors. F- Values were obtained from analysis of variance (ANOVA) by using L (light/dark treatments) and S (NaCl treatments). Where, * = P < 0.05; ** = P < 0.01 *** = P < 0.001 and ns = non-significant.
Figure 2.2: Effect of salt, light/dark and temperature treatments on the rate of seed 37 germination of Salsola drummondii. A. 10/20 ◦C, B. 15/25 ◦C, C. 20/30 ◦C and D. 25/35 ◦C. Each circle represents mean ± standard errors. Symbols having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test). F- values were obtained from Analysis of variance (ANOVA) by using S (NaCl treatments). Where, *** V
= P < 0.001.
Figure 2.3: Percentage of germinated ( ), recovered ( ), viable 39 ( ) and dead ( ) seeds of Salsola drummondii treated with various concentrations of NaCl under 12-H photoperiod.
Figure 2.4: Percentage of germinated ( ), recovered ( ), viable 40 ( ) and dead ( ) seeds of Salsola drummondii treated with different temperatures under 24-H dark.
Figure 2.5: Effects of GA3 (10 µM), GA4, (10 µM), GA4+7 (10 µM), thiourea(100 µM), 41 kinetin (3 mM) and fussicoccin (5 µM) on mean final germination of S. drummondii seeds in 12-H photoperiod (A), 24-H dark (C) and rate of germination (E) under control (0 mM NaCl) and saline condition (800 mM NaCl). Relative changes (folds) due to DRCs in mean final germination of S. drummondii seeds in 12-H photoperiod (B), 24-H dark (D) and rate of germination (F) are also given in comparison to respective non-saline and saline controls. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test). Asterisk (*) indicates significant (P < 0.05) difference between a DRC treatment and saline control (t-test).
Figure 3.1: Effect of A) salinity, B) temperature and C) light on the percent 62 germination in seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
Figure 3.2: Effect of salinity on the relative water uptake in seeds of Salsola 65 drummondii after 50 minutes of soaking. B) Arrow indicates embryo protrusion. Bars and circles represent mean ± standard error. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 3.3: Effects of A) salinity, B) temperature and C) light on the MDA content in 66 the germinating seeds of S. drummondii. Bars represent mean ± standard
VI
error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
Figure 3.4: Effects of A) salinity, B) temperature and C) light on the activity of SOD 67 in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
Figure 3.5: Effects of A) salinity, B) temperature and C) light on the activity of CAT 68 in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
Figure 3.6: Effects of A) salinity, B) temperature and C) light on the activity of APX 69 in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
Figure 3.7: Effects of A) salinity, B) temperature and C) light on the activity of GPX 70 in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
Figure 4.1: Biomass of the seedlings of Salsola drummondii growing under different 92 NaCl concentrations expressed as mg plant-1. Stack bars represent mean. Stack bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 4.2: Succulence of the seedlings of S. drummondii growing under different 93 -1 concentrations of NaCl expressed as mg H2O mg DW. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 4.3: Seedlings of S. drummondii growing under different salt stained for vitality 95 by Tetrazolium chloride staining. In (A) the picture was taken 20 days after
VII
germination. Scale bars represent 1 cm. (B) Bars represent area in cm of the stained regions for the seedlings measured with ImageJ in arbitrary units (mean ± standard error). Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 4.4: Seedlings of S. drummondii growing under different salt stained for 96 localization of loss of plasma membrane integrity with Evan blue staining. In (A) the picture was taken 20 days after germination. Scale bars represent 1 cm. (B) Bars represent area in cm of the stained regions for the seedlings measured with ImageJ in arbitrary units (mean ± standard error). Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 4.5: Seedlings of S. drummondii growing under different salt stained for 97
concentrations histochemical detection of H2O2 with DAB staining. In (A) the picture was taken 20 days after germination. Scale bars represent 1 cm. (B) Bars represent area in cm of the stained regions for the seedlings measured with ImageJ in arbitrary units (mean ± standard error). Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 4.6: Activities of different antioxidant enzymes in the seedlings of S. 99 drummondii under different NaCl treatments at 20/30 oC in 12-H light. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
Figure 5.1: Mean monthly precipitation (bars), maximum (red symbols) and minimum 118 (blue symbols) temperatures (oC) in the study site during 2009. Asterisks (green symbols) indicates the sampling months.
Figure 5.2: Seasonal variations in the soil electrical conductivity (ECe 1:5; Bars) and 121 moisture (%) content (circles) at 20, 60 and 180 cm depth from the soil surface.
Figure 5.3: Seasonal variations in leaf succulence, inorganic, and organic contents of 125
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Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
Figure 5.4: Seasonal variations in the leaf osmolality (mOsmol kg-1 plant water) of 126 Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
Figure 5.5: Seasonal variations in the xylem pressure potential (-MPa) of shoot in Salsola drummondii. Bars represent mean ± standard errors. Bars having 127 same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
Figure 5.6: Seasonal variations in the leaf proline (mg g-1 DW) content of Salsola drummondii. Bars represent mean ± standard errors. Bars having same 1228 letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
Figure 5.7: Seasonal variations in the total soluble sugars (mg g-1 DW) of Salsola drummondii. Bars represent mean ± standard errors. Bars having same 129 letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
Figure 5.8: Seasonal variations in the leaf ion (Na+, K+, Ca++, and Mg++; mg g-1 DW) contents of Salsola drummondii. Bars represent mean ± standard errors. 130 Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
Figure 5.9: Seasonal variations in the activity of the antioxidants enzymes in the leaves of S. drummondii under salt desert. Enzymes activities are expressed in U 132 mg-1Protein. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
IX
List of Abbreviations
A Photosynthetic rate
ANOVA Analyses of variance
APX Ascorbate peroxidase
AsA Ascorbic acid
CAR Carotenoid
CAT Catalase
Chl Chlorophyll
Ci Internal carbon dioxide
D Dead
DAB 3,3'-diaminobenzidine
DRCs Dormancy regulating chemicals
DW Dry weight
EC Electrical conductivity
ECe Electrical conductivity
FW Fresh weight
G Germinated
GA Gibberellins (Gibberelic acid)
GPX Guaiacol peroxidase
GR Glutathione reductase
Gs Stomatal conductance
X
GSH Glutathione
H Hours
H2O2 Hydrogen peroxide
L Light
MDA Malondialdehyde
- O2 Superoxide radical
1 O2 Singlet oxygen
. OH Hydroxyl radical
OC Organic content
Prec Precipitation
R Recovered
RGR Relative growth rate
RH Relative humidity
ROS Reactive oxygen species
S Salt treatment
SOD Superoxide dismutase
T Temperature
Tmax Maximum temperature
Tmin Minimum temperature
TSS Total soluble sugars
TTC 2, 3, 5 - triphenyl tetrazolium chloride
XI
TW Tissue water
V Viable
VPD Vapor pressure deficit
Wr Water uptake
WUE Water use efficiency
XPP Xylem pressure potential
XII
Summary
Salt deserts are unique ecosystems that are characterized by high salinity, temperatures, and low precipitation. Survival in salt desert environment would require synchronization of plant life cycle events with variations in temperature, soil salinity and moisture regimes. Little information is available about the survival mechanisms of perennial xero-halophytes which are naturally found in salt deserts. Keeping this in mind, ecophysiological responses of a salt desert halophyte
Salsola drummondii were studied. Freshly collected seeds were non-dormant and germinated rapidly i.e., within an hour of imbibition in distilled water. Seed germination and rate of germination were progressively inhibited with increasing salinity at all temperatures; however few seeds germinate up to 1000 mM NaCl treatment. Seed germination partially recovered after transfer to non-saline conditions only in the 12-H photoperiod, but not from complete darkness, where most un-germinated seeds remained dormant. Dormancy regulating chemicals particularly, GA4 and fusicoccin alleviated salinity imposed secondary dormancy in all photoperiod regimes only under saline conditions. In general, seeds had higher MDA and antioxidant enzyme activities at sub-optimal conditions of complete darkness and at 25-35oC.
Salsola drummondii seedlings grew optimally at moderate salinity (200 mM NaCl) without undergoing oxidative stress. Whereas, high salinity (400 mM NaCl) severely affected the seedling vitality by accelerating ROS production more than the capacity of antioxidant defense system to detoxify them. Under natural conditions, leaf growth, photosynthetic efficiency and antioxidant defense system of S. drummondii appeared to be correlated with soil moisture, salinity and precipitation. Plants accumulated ash, proline and soluble sugars in summer along
XIII
with decreased photosynthetic rates and higher antioxidant enzyme activities as a possible tradeoff to maintain high water use efficiency. Salsola drummondii thus appears to be well adapted for survival under variable drought and salt stress during seed germination, seedling growth and in its natural population by means of efficient regulation of physiological and biochemical traits.
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XVI
Chapter 1
General introduction
1
Introduction
Salt deserts are hyper-arid habitats with low precipitation, high temperatures and highly
(>40) saline soils (Gul and Khan, 2002; Cooke et al., 2006; Bui, 2013).
Evapotranspiration rates generally exceed the rates of precipitation in these habitats. Soil in salt deserts is aridisol type, which is generally coarse, low in clay content, rocky or gravely and poorly developed (Gates et al., 1956; Cooke et al., 1993; Breckle, 2002). In salt deserts soil is predominantly mineral with low organic content (Horneck et al., 2007;
Al-Nafie, 2008). Calcium carbonate precipitated from solution may cement sand and gravel into hard layers called "calcretes" (Gile et al., 1966). Such calcic horizons are unique features of the soils of arid and semi-arid regions (Cooke et al., 1993; Al-Faifi et al., 2010; Fakhireh et al., 2012). These features make salt deserts unfit for most plants
(Gul and Khan, 2002; Bui, 2013). Few plants called xero-halophytes can however, thrive in these hostile conditions (Al-Dousari et al., 2008; Abd El-Ghani et al., 2011). About 77 species of xero-halophytes are found in different salt deserts of Pakistan, which contribute about 19% of total halophyte diversity of the country (Khan and Qaiser, 2006).
These plants have specialized adaptations to cope with multiple stresses prevailing in salt deserts and their life cycle events appear highly synchronized with changes in habitat conditions (Young et al., 1995; Gul et al., 1999; Xu and Li, 2006; Eggli and Nyffeler,
2009; Ogburn and Edwards, 2010). Despite their natural ability to survive under extreme conditions, xero-halophytes are generally less studied and their tolerance mechanisms are poorly understood.
2
Xero-halophytes are not only important from ecological view point, but a number
of these plants can also be utilized as fodder/forage, biofuel feedstock, medicine and
landscaping (Weber et al., 2007; Qasim et al., 2010; Hameed and Khan, 2011; Ksouri et
al., 2012; Gul et al., 2013). Besides, these naturally stress tolerant plants could also serve
as gene donor for the improvement of conventional crops using molecular studies
(Forster, 1992; Sharma and Goyal, 2003; Flower, 2004). To exploit their economic
potentials, there is need to understand their ecology as well as the mechanisms underlying
their natural stress tolerance (Beatley, 1974; Angert 2006; Salguero-Gomez and de
Kroon, 2010) and such studies on salt desert plants are very limited. This chapter
provides an overview of the information available on xero-halophytes and missing gaps
in this regard.
Seed bank of xero-halophytes
The soil seed bank of an area could be a very valuable tool to provide important insight
about the standing vegetation of the past and represent a pool of regenerative potential
(Templeton and Levin, 1979; Levin, 1990; McGraw et al., 1991; Khan, 1993; Funes et
al., 1999; Mandak et al., 2012). The formation of a soil seed bank starts with seed
dispersal and ends either with germination or death of the seeds. Usually the composition
of seed banks is variable, and is classified as transient (seeds of species with transient
seed banks live for <1 year) or persistent (seeds of species with persistent seed banks live
for >1 year) seed bank (Thompson and Grime, 1979). However, in recent classification
schemes transient and persistent seed-bank categories are further subdivided into 2–3
subtypes, describing them by 1 year to decades (Thompson et al., 1997; Bakker et al.,
3
1996; McDonald, 1999). Although amount of information is limited, the role of
halophytic seed bank in subtropical desert communities is usually different from
temperate salt marshes or salt desert communities (Ungar, 2001; Gul et al., 2013). Some
studies on seed banks of inland salt-desert communities showed that most species
including Cressa cretica, Haloxylon stocksii, Salsola imbricata, and Sporobolus ioclados
had a transient seed bank which depleted gradually in few months after dispersal (Khan,
1990; Zaman and Khan, 1992). However, recruitment from seeds is not common. For
instance, Fakhireh et al. (2012) reported that xero-halophyte Desmostachya bipinnata
propagates mainly vegetatively in a hyper-arid salt desert of Iran. According to Khan
(1993) this is because seeds in salt desert are exposed to intense environmental stress
caused by the high temperatures, low soil moisture and high soil salinity. It is believed
that the water scarcity leading to high salinity coupled with high temperature in
subtropical salt deserts may cause high mortality in seeds of some species (Gul et al.,
2013). However, this information on a number of local xero-halophytes is missing.
Seed germination and seedling growth of xero-halophytes
Germination of seeds at right time and place is of particular importance for halophytes,
which are found in stressful saline environments (Gul et al., 2013). Seed germination of
halophytes is thus regulated by a number of environmental factors such as changes in soil
salinity, moisture, temperature and light (Khan and Gul, 2006). Salinity is reported to be
the major factor influencing germination of halophytes (Hameed et al., 2006; Ahmed et
al., 2012). Under saline conditions, both seed germination, and seedling growth are
inhibited by high osmotic constraint, ion toxicity and/or salt-induced oxidative stress
4
(Xing et al., 2013; Hameed et al., 2014). Non-optimal temperatures and/or photoperiod may further aggravate the inhibitory effects of salinity (Zia and Khan, 2004; Li et al.,
2008; Ahmed and Khan, 2010). Although a large number of such studies are available on halophyte seeds (Gul et al., 2013), amount of information on seed germination of xero- halophytes is comparatively scarce (El-keblawy et al., 2008). In addition, how changes in salinity, temperature and light influence halophyte seeds from physio-chemical view point, is also not fully known (Hameed et al., 2014).
Seedling stage is considered as most vulnerable stage in the life cycle of halophytes and salinity is among major factors limiting seedling survival (Dodd and
Donovan, 1999; Khan and Gul, 2006; Gul et al., 2013). In subtropical saline habitats such as in salt deserts plant propagation via clones/ramets is common, and genets/early seedlings are rarely seen under natural habitats (Fakhireh et al., 2012; Gul et al., 2013).
For instance, a xerohalophytic grass Desmostachya bipinnata in an Iranian salt desert was reported to propagate clonally (Fakhireh et al., 2012). However, in some xero-halophytes seedling emergence was also reported (Shumway and Bertness, 1992; Noe and Zedler,
2001) which could be a function of soil type (Britton et al., 2003). Saline habitats often have salt enriched top/surface soil which is drier and saline then the sub-soils and young seedlings with shallow roots may be subjected to additional harshness of the environmental stresses (Ungar, 1978; Schupp, 1995; Noe and Zedler, 2001). Little is known about the survival mechanisms of early seedlings particularly for the subtropical halophytes. Therefore, deciphering the survival mechanisms of early seedlings is important.
5
Ecological and eco-physiological studies on xero-halophytes
Most studies on xero-halophytes have been conducted in controlled green-house
conditions (Al-Khateeb, 2002; Khan et al., 2005; Heidari-Sharifabad and Mirzaie-
Nodoushan, 2006; English and Colmer, 2011; Koyro et al., 2013) and generally little is
known about their habitat requirements and responses to changes in different
environmental variables under natural conditions (Sayed, 1996; Fakhireh et al., 2012;
Alhdad et al., 2013). The ecological conditions in saline habitats are quite severe and
fluctuate extensively under the influence of numerous abiotic factors, including amount
of annual rainfall received, changes in temperature, irradiance, wind velocity and
direction, poorly formed soils and salinity (Noy-Meir, 1973; Khan, 1993; Buol et al.,
1997; Körner, 2000; Abd El-Ghani et al., 2011). In addition, edaphic factors such as soil
texture, structure, electrical conductivity, pH, and CaCO3 along with the organic content
are also reported to affect the floristic composition and plants distribution patterns in the
saline habitats (Goldberg and Barton, 1992; Khan, 1993; El-Bana et al., 2002; Hedlund et
al., 2003; Jafari, 2004; Wang et al., 2007; Al-Dousari et al., 2008; Cañadas et al., 2010).
Different combinations of these factors result in the complexity and allow very unique
salt tolerant vegetation (Laudadio et al., 2009b). Some studies have shown the changes in
species range, demographic rate, and average population size in response to varying stress
conditions (Hester et al., 2001; Song et al., 2006). Likewise some studies examined ion
and water relations of a few halophytes in response to changes in habitat conditions
(Sayed, 1996; Gul et al., 1999; Aziz et al., 2005; Grigore et al., 2011; Nedjimi, 2012).
These studies indicate that halophytes show different patterns of physiological and
6
biochemical responses under varying natural habitats, which are generally less studied
(Gul et al., 2001; Zia et al., 2007).
Present study
Plant ecologists and eco-physiologists for decades have been concerned about how plants
survive under stressful natural conditions and how to quantify the variations in the
distributional patterns and physiological responses of plants in their natural habitats
(Hester et al., 2001). Although considerable advances have been made into our
understanding and skills of managing wild populations of plants, however our ability to
predict population responses to habitat heterogeneity and environmental stresses remain
inconclusive (Yamada et al., 2007). Even more limited is our understanding about the
plant populations of subtropical salt deserts, which are very hostile due to extreme
temperatures, drought and salinity. Therefore much work is needed before population
management can be viewed as standard procedure and utilization of stress tolerant plants
for sustainable economic benefits for the mankind (Ismail and Ibrahim, 2003; Belligno
and Sardo, 2008; Ladeiro, 2012; Khan, 2014). Keeping this mind, this study was
designed to gain knowledge about ecological requirements and ecophysiological
responses of a medicinally important leaf succulent xero-halophyte Salsola drummondii
to varying environmental conditions of warm salt desert.
Test species and study site
Salsola drummondii Ulbirch. (Amaranthaceae) is a perennial, leaf succulent halophyte
(Figure 1.1) of solonchak (intrazonal saline soil) and saline sandy loam soils. This shrub
7
is common in salt deserts of Balochistan and is also reported from southern parts of
Khyber Pakhtoonkhua and western Punjab (Flora of Pakistan). In addition, this plant is also found in eastern parts of Arabian Peninsula and Southern Iran. There are numerous economic usages of this perennial plant (Figure 1.3). For instance, its semicircular or pear shaped succulent leaves have potential to remove soil salts, are burnt to obtain soda ash by locals, and different plant parts are medicinally important (Khan and Qaiser, 2006;
Gilani et al., 2010). It is also highly palatable to camels (Qureshi et al., 1993). It could be utilized as a sand dune stabilizer and for the improvement of saline soil (Akhani and
Ghorbanli, 1993; Al-Eisawi, 2012). Study area was a salt desert located at Windar,
Balochistan, Pakistan (24o25’07.16” N and 66o37’32.38”E) (Figure 1.2) and is part of saharo-sindian phytogeographical region (Ali and Qaiser, 1986). Similar to most other southern parts of Balochistan, this area has hyper-arid weather with low annual precipitation, high summer temperatures and sparse vegetation (Qasim et al., 2010;
Abuzar et al., 2011; Ahmad et al., 2012). I studied phenological changes in Salsola drummondii population growing in a salt desert of southern Balochistan Pakistan and found that the plants remained in vegetative phase during most parts of the year (Figure
1.4). After monsoon rains plants started flowering (September/October), followed by seed production and maturation during November and December, respectively. Mature seeds had perianth and accumulated initially beneath mother plants, followed by gradual dispersal through wind (anemochory) during late December and January (Figure 1.4) and maintained a transient seed bank (Figure 1.5).
8
Figure 1.1. Salsola drummondii in its natural habitat.
Figure 1.2. Google Earth Image of the study site.
9
Figure 1.3. Economic importance of Salsola drummondii.
10
Figure 1.4. Phenology diagram of Salsola drummondii in salt desert of Balochistan, Pakistan.
11
Figure 1.5. Conceptual flow diagram of seed bank dynamics for Salsola drummondii growing in the salt deserts of Balochistan, Pakistan.
12
Aims, experiments and hypotheses
This study aims to understand the ecological requirements and ecophysiological
responses of a medicinally important leaf succulent xerohalophyte Salsola drummondii to
varying environmental conditions of a warm subtropical salt desert located in southern
Balochistan (Pakistan).
Laboratory studies were combined with field experiments to better understand the
ecology and ecophysiology of test species. Overall three laboratory experiments and one
field study were performed (Figure 1.6). For field study a multi-factorial approach was
followed by investigating combined roles of landscape-scale (e.g. temperature, rainfall,
soil moisture and salinity etc.) and local-scale factors (e.g. neighbor abundance and seed
bank etc.) in eco-physiology of the perennial shrub Salsola drummondii.
Following questions were addressed:
1) What are the effects of different salinity, temperature and light/dark treatments on
germination, recovery and viability of S. drummondii seeds?
2) How do salinity, temperature and light/dark affect seedling growth of test species?
3) Whether non-optimal levels of salinity, temperature and dark cause oxidative
stress in germinating seeds and seedlings? and
4) If variations in habitat conditions lead to changes in plant ecophysiological and
biochemical characteristics?
13
Figure 1.6. Parts of this PhD thesis in which the role of Salsola drummondii under natural habitat conditions and its ecological requirements and ecophysiological responses were studied in the context of 1) Germination ecology, biochemistry and seedling growth and 2) Seasonal eco-physiology.
14
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Chapter 2
Effects of salinity, temperature, light and dormancy regulating chemicals on seed germination of Salsola drummondii
27
Abstract
Salsola drummondii Ulbr. is a perennial halophyte from salt deserts of southern
Balochistan, Pakistan. Experiments were conducted to study the effects of salinity (0,
200, 400, 600, 800 and 1000 mM NaCl), thermoperiod (10/20, 15/25, 20/30 and 25/35
◦C), light (12-H photoperiod and dark) and dormancy regulating chemicals (DRCs) on germination, recovery and viability of the seeds of S. drummondii. Seeds of S. drummondii germinated quickly in distilled water at different temperature regimes and increases in salinity decreased seed germination. Interestingly, few seed germinated in
1000 mM NaCl treatment, which is about twice as high as seawater salinity. Seeds were partially photoblastic and showed relatively higher germination under 12-H photoperiod than in dark. Seeds showed poor recovery of germination from salinity and particularly when germinated in dark. Germination inhibition at high salinity (800 mM NaCl) under
12-H photoperiod was partially alleviated by the exogenous application of different
DRCs, particularly fusicoccin. Moreover, all the DRCs, except GA4+7, ameliorated germination of salt stressed seeds under complete darkness and GA4 and fusicoccin were most effective. This study shows that seeds of S. drummondii are highly tolerant to salinity and variation in temperature but partially photoblastic nature indicate that seeds would not germinate if buried under the soil. Seed germination under saline conditions could improve by the use of DRCs particularly by application of fusicoccin.
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Introduction
Soil salinity is considered an important factor influencing seed germination of halophytes
(Gulzar et al., 2007; El-Keblawy and Shamsi, 2008; El-Keblawy et al., 2011; Gul et al.,
2013; Zeng et al., 2014). Salinity tolerance of halophytes during seed germination varies
considerably among species; however Salsola species are among those with highly salt
tolerance (Khan and Gul, 2006). Wei et al. (2008) reported extremely high salinity
tolerance for S. affinis seeds which could germinate (2%) in 2000 mM NaCl solution.
Seeds of S. nitraria (Chang et al., 2008), S. ferganica (Wang et al., 2013) and S. iberica
(Khan et al., 2002) could germinate up to 1000 mM NaCl concentration, while S.
imbricata germinated up to 800 mM NaCl (Mehrun-Nisa et al., 2007).
Variation in thermoperiod and photoperiod significantly affect seed germination
in halophytes under natural conditions (Khan and Gul, 2006). Seeds of subtropical
halophytes generally germinate optimally at the temperature regime of 20/30 ◦C (Khan
and Ungar, 1997 and 1998; Gulzar et al., 2001). For example, seeds of Limonium stocksii
could germinate in 500 mM NaCl at optimum temperature of 20/30 ◦C but not at 10/20
and 25/35 ◦C (Zia and Khan, 2004). However, seeds of some species preferably
germinate at cooler (10/20 ◦C; e.g. S. imbricata; Mehrun-Nisa et al., 2007) or warmer
(25/35 ◦C; e.g. Desmostachya bipinnata; Gulzar et al., 2007) temperature regimes. Effect
of light on seed germination is also quite variable. Gul et al. (2013) indicated that seeds
of some halophytes either failed to germinate or showed very little germination in dark
while light had little effect on seed germination of some. Similarly, Baskin and Baskin
(1995) reported that out of 41 halophytes, seed germination of 20 species was promoted
29
in presence of light, 10 species germinated better in dark, while 11 species germinated equally well in both light and dark. These reports indicate that the salinity tolerance and germination responses of halophyte seeds greatly depend on combinations of thermoperiod and photoperiod in saline conditions.
Halophyte seeds are reported to remain viable under high temperature and salinity stress and readily germinate when conditions becomes more favorable (Ungar, 1995;
Khan and Ungar, 1997a and 1998; Pujol et al., 2000). However, this recovery response varies from poor recovery in some halophytes such as Zygophyllum simplex (Khan and
Ungar, 1997b) to almost complete recovery in many such as Suaeda fruticosa (Khan and
Ungar, 1998) and Limonium stocksii (Zia and Khan, 2004). In addition, temperature regimes also influence recovery responses of halophytes (Khan and Ungar, 1997; Zia and
Khan, 2004; Gulzar et al., 2007; El-Keblawy and Shamsi, 2008; Ahmed and Khan,
2010). Seed germination under saline condition may also be improved by exogenous application of dormancy regulating chemicals (DRCs) such as gibberellins, cytokinins, thiourea and fusicoccin (Khan and Gul, 2001; Khan and Ungar, 2002; Mehrun-Nisa et al.,
2007).
Salsola drummondii Ulbr. is a leaf succulent perennial xerohalophyte from family
Amaranthaceae, which is commonly found in salt deserts of Saharo-Sindian region specifically eastern parts of Arabian Peninsula, southern Iran and southern to central
Pakistan (http://www.efloras.org/florataxon.aspx?flora_id=5&taxon_id =242100179).
Populations of S. drummondii are found in salt deserts of Balochistan, Pakistan (Khan and Qaiser, 2006), where it grows in association with Suaeda fruticosa, Aerva javanica
30
var. bovei, Prosopis juliflora and several desert grasses. There are several economic
usages of this plant. For instance, cylindrical to terete leaves of S. drummondii are burnt
to obtain soda ash by locals, different plant parts are medicinally important (Gilani et al.,
2010) and leaves could also serve as forage (Qureshi et al., 1993). However, information
about salinity tolerance and germination ecology of S. drummondii seeds is not known. I
proposed to answer following questions regarding the seed germination of S.
drummondii: 1) How tolerant seeds are at germination stage? 2) Whether salinity
tolerance of S. drummondii at seed germination is temperature and light dependant, 3)
Can seeds maintain viability when exposed to high salinity and temperature stress? and 4)
Can salinity tolerance during germination be improved by the application of DRCs?
Materials and methods
Seed collection and study site
Mature seeds of Salsola drummondii were collected from a salt desert located at Winder,
Balochistan (24◦25’07.16” N and 66◦37’32.38”E) during February 2011. Seeds were
separated from perianths, surface sterilized by using 1% commercial bleach (Sodium
hypochlorite), rinsed with distilled water, air dried and stored in clear plastic Petri plates.
Freshly collected seeds were used in germination experiments.
Seed germination responses
Seed germination was carried out in programmed incubators with alternating temperature
regimes of 10/20, 15/25, 20/30 and 25/35 ◦C, where low temperatures represent
temperatures of 12-H dark and higher temperatures with 12-H light period (25 μmol m-2 31
s-1; 400-700 nm, Philips cool-white fluorescent lamps). Germination was carried out in tight fitting plastic Petri plates (5 cm Ф), which were placed in large glass Petri plates to prevent evaporative loss of the test solutions. There was 5 ml of test solution in each Petri plate. Six NaCl (0, 200, 400, 600, 800, and 1000 mM) concentrations were used with four replicates of 25 seeds each per treatment. Germination (Protrusion of embryonic axis;
Bewley and Black, 1994) was recorded every alternate day for 20 days. Rate of germination was calculated according to the method of Khan and Ungar (1984). Seed germination was also carried out to study effect of dark on seed germination in response to increasing NaCl concentrations at various temperature regimes by placing Petri plates in dark-plastic bags. Germination of dark-treated seeds was noted once after 20 d.
Seed recovery responses
Recovery from high salinity stress was recorded by transferring the seeds to distilled water under similar experimental conditions and the germination was recorded for every alternate day. Recovery from dark was also studied by transferring the Petri plates with seeds to 12-H photoperiod keeping other conditions unchanged.
Seed viability responses
All the un-germinated seeds at the end of recovery experiments were tested for their viability using 2, 3, 5 - triphenyl tetrazolium chloride (TTC) test (MacKay, 1972;
Bradbeer, 1998).
32
Seed responses to dormancy regulating chemicals
Effects of different dormancy regulating chemicals (DRCs) for alleviating salinity and light effects on seed germination of S. drummondii were studied under 12-H photoperiod
◦ and in complete dark at 20/30 C. Solutions of GA3 (10 μM), GA4 (10 μM), GA4+7 (10
μM), thiourea (100 μM), kinetin (3 mM) and fusicoccin (5 μM) were added in germination media (0 and 800 mM NaCl). Concentrations of DRCs used in this study were determined in a preliminary study (Data not given). Germination was recorded every alternate day for 20 days in case of 12-H photoperiod treatments, while once after
20 days for dark treatment.
Statistical analyses
The data was transformed using arcsine transformation before the statistical analysis.
Analyses of variance (ANOVA) were performed to determine the significance of the effects of NaCl, temperature and light on seed germination, recovery, viability and mortality data. Bonferroni post-hoc tests (P < 0.05) were carried out to determine significant difference between individual means. Student t-test (P < 0.05) was used to compare DRC treatments with control. Statistical analyses were done by using SPSS for windows version 11 (SPSS, 2001).
33
Results
Seed germination responses
Analysis of variance indicates that NaCl treatments had significant (P < 0.001) effect on
both rate and final seed germination of S. drummondii (Table 2.1). About 80% seeds
germinated in distilled water at all temperature regimes studied indicating little effect of
temperature on seed germination under non-saline conditions. However, higher
temperature regime (25/35 ◦C) delayed the seed germination compared to cooler
thermoperiod (Figure 2.2). Increases in NaCl concentration linearly decreased rate and
final germination and few seeds germinated at 1000 mM NaCl (Figure 2.1 and 2.2). Seed
germination under 12-H photoperiod was significantly higher compared to dark at
favorable temperature regimes and no differences were recorded in other treatments.
Table 2.1 Two-way analysis of variance (ANOVA) indicating significance of the individual and collective effects of various experimental factors [salt (S) and temperature (T)] on the percentage of germinated (G), recovered (R), viable (V) and dead (D) seeds when recovered from salt. Where, numbers represent F-values. * = P < 0.05 and *** = P < 0.001.
Treatments G R V D
T 8.215*** 18.843*** 55.702*** 31.214***
S 202.602*** 82.650*** 95.237*** 46.983***
S x T 1.924* 7.405*** 8.904*** 1.953*
34
Table 2.2 Two-way analysis of variance (ANOVA) indicating significance of the individual and collective effects of various experimental factors [salt (S) and temperature (T)] on the percentage of germinated (G), recovered (R), viable (V) and dead (D) seeds when recovered from temperature. Where, numbers represent F-values. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 and ns = non-significant.
Treatments G R V D
T 5.784** 26.522*** 18.858*** 14.871***
S 41.267*** 7.502*** 23.999*** 22.393***
S x T 0.436ns 6.392*** 3.059* 0.511ns
Table 2.3 Two-way analysis of variance (ANOVA) indicating significance of the individual and collective effects of various experimental factors [salt (S) and temperature (T)] on the percentage of germinated (G), recovered (R), viable (V) and dead (D) seeds when recovered from dark. Where, numbers represent F-values. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 and ns = non-significant.
Treatments G R V D
T 3.759* - 139.178*** 48.030***
S 101.920*** - 72.379*** 30.006***
S x T 1.044ns - 4.866*** 3.210**
35
A 100 Light 80 Dark L = 7.282* 60 S = 109.535*** ns 40 S x L = 1.279 20
0 B 100 L = 34.979*** S = 88.523*** 80 S x L = 2.307* 60 ) %
( 40
n
o 20 i t
a 0
n i 100 C L = 25.943*** m
r S = 93.694*** e 80 S x L = 4.017** G 60 40 20
0 100 D L = 0.009 ns
80 S = 100.874*** S x L = 1.051 ns 60 40 20 0
0 200 400 600 800 1000 NaCl (m M ) Figure 2.1. Effect of salt, light/dark and temperature treatments on the seed germination of Salsola drummondii. A. 10/20 ◦C, B. 15/25 ◦C, C. 20/30 ◦C and D. 25/35 ◦C. Circles represent mean ± standard errors. F- Values were obtained from analysis of variance (ANOVA) by using L (light/dark treatments) and S (NaCl treatments). Where, * = P < 0.05; ** = P < 0.01 *** = P < 0.001 and ns = non-significant. 36
50 A a S = 80.031***
40 30 b bc 20 cd 10 d d 0 50 a B S = 57.918*** 40 30 b
n bc
o 20 i t
a 10 cd
n d
i d
m 0 r e
g a S = 79.266*** 50 C f o
40 e t
a 30 b
R 20 b 10 c c c 0 50 D a S = 67.922*** 40
30 20 b bc 10 c c c 0
0 200 400 600 800 1000 NaCl (mM) Figure 2.2. Effect of salt, light/dark and temperature treatments on the rate of seed germination of Salsola drummondii. A. 10/20 ◦C, B. 15/25 ◦C, C. 20/30 ◦C and D. 25/35 ◦C. Each circle represents mean ± standard errors. Symbols having same letter within each salt treatment are not significantly different (P <0.05) among means (Bonferroni test). F- Values were obtained from Analysis of variance (ANOVA) by using S (NaCl treatments). Where, *** = P < 0.001. 37
Seed recovery responses
Un-germinated seeds of S. drummondii from different NaCl treatments under 12-H photoperiod showed low (~10 to 20%) recovery, when transferred to distilled-water with little or no difference among temperature treatments (Figure 2.3). While, un-germinated seeds from dark treatments did not recover at any of the temperatures (Figure 2.4).
Seed viability responses
Only ~10-30% of the un-germinated from different salinity treatments under 12-hours photoperiod after recovery experiment, remained viable and about 50% were dead
(Figure 2.3). High temperature (25/35 ◦C) and NaCl (> 600 mM) treatments increased the percentage of dead seeds under 12-hour photoperiod. Seed death under 12-H photoperiod was relatively lesser and most un-germinated/un-recovered seeds were viable, especially at 20/30 ◦C (Figure 2.4).
Seed responses to dormancy regulating chemicals
All the DRCs used, inhibited seed germination of S. drummondii in absence of salinity under both 12-H photoperiod and dark (Figure 2.5), however a significant (P < 0.05) improvement in germination was observed at 800 mM NaCl by all DRCs under 12-H photoperiod (Figure 2.5A, B, E and F). Fusicoccin improved germination (> 4 folds) of salt stressed seeds more than any other DRC under 12-H photoperiod (Figure 2.5B and
F). All the DRCs, except GA4+7, improved seed germination of salt stressed seeds under complete darkness and this improvement was highest in case of GA4 and Fusicoccin applications (Figure 2.5C and D).
38
10/20 oC 15/25 oC
100
80
60
40 )
% ( 20 e g
a t
n 0
e o o
c 20/30 C 25/35 C
r
e 100 P
80
60
40
20
0 0 200 400 600 800 1000 0 200 400 600 800 1000 NaCl (mM) Figure 2.3. Percentage of germinated ( ), recovered ( ), viable ( ) and dead ( ) seeds of Salsola drummondii treated with various concentrations of NaCl under 12-H photoperiod.
39
o o 10/20 C 15/25 C 100
80
60
40 ) % ( 20 e g a t
n 0 o o e 20/30 C 25/35 C c r 100 e
P 80
60
40
20
0 0 200 400 600 800 1000 0 200 400 600 800 1000
NaCl (mM)
Figure 2.4. Percentage of germinated ( ), recovered ( ), viable ( ) and dead ( ) seeds of Salsola drummondii treated with different temperatures under 24-H dark.
40
Nacl GA3 GA4 GA4+7 Thiourea Kinetin Fussicoccin A a ** 100 B 4 80 bb 3 bc bc * * * 2 60 c
) 1 40 a % b ( b b 0
d b b n 20 b * * * * o * -1 i
t ** R a e
0 0 800 -2 n l i C D a t
m 100 4 i r v
e a
** e 3 80 ** G b c
h bc bc * 2 * a 60 bc bc n
1 g 40 a a e
0 (
b b f 20 d c * o * * * * -1 l c c ** * d s ) 0 0 800 -2 E F e a ** t 50
a 4 R 40 b b n b bc * o
i * t 30 c 2 a n i 20 a
m b b 0 r d b b b e 10 b * * * * *
G * 0 -2 0 800 0 800 NaCl (mM)
Figure 2.5. Effects of GA3 (10 µM), GA4, (10 µM), GA4+7 (10 µM), thiourea (100 µM), kinetin (3 mM) and fussicoccin (5 µM) on mean final germination of S. drummondii seeds in 12-H photoperiod (A), 24-H dark (C) and rate of germination (E) under control (0 mM NaCl) and saline condition (800 mM NaCl). Relative changes (folds) due to DRCs in mean final germination of S. drummondii seeds in 12-H photoperiod (B), 24-H dark (D) and rate of germination (F) are also given in comparison to respective non-saline and saline controls. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test). Asterisk (*) indicate significant (P < 0.05) difference between a DRC treatment and saline control (t-test).
41
Discussion
Salt desert halophytes of subtropical environment generally lack innate dormancy (Gul et
al., 2013). Similarly, innate dormancy was also absent in seeds of S. drummondii which
germinated well in distilled water at all temperature and light regimes. This finding is in
accordance with the results obtained for other Salsola species such as S. imbricata
(Mehrun-Nisa et al., 2007), S. affinis (Wei et al., 2008), S. nitraria (Chang et al., 2008),
S. ferganica (Wang et al., 2013) and S. iberica (Khan et al., 2002). Lack of innate
dormancy hence appear an adaptation of Salsola species to germinate quickly upon
availability of adequate moisture after rains. Fast germination rate of S. drummondii, as
reported for S. affinis and S. imbricata too, also supports this hypothesis (Mehrun-Nisa et
al., 2007; Wei et al., 2008).
Seeds of S. drummondii were highly salt tolerant and could germinate up to 1000
mM NaCl, which is about twice as saline as seawater. Seeds of other Salsola species such
as S. affinis (Wei et al., 2008), S. nitraria (Chang et al., 2008), S. iberica (Khan et al.,
2002), S. ferganica (Wang et al., 2013) and S. imbricata (Mehrun-Nisa et al., 2007) could
also germinate at or above 800 mM NaCl, indicating that seeds of Salsola species are
highly salt tolerant during germination. However, seeds of co-occurring species such as
Suaeda fruticosa (Khan and Ungar, 1998; Hameed et al., 2006), Desmostachya bipinnata
(Gulzar et al., 2007) and Aerva javanica var. bovei (Khan et al., Unpublished data) failed
to germinate beyond 500 mM NaCl. High salinity tolerance during germination in
comparison to co-occurring species could be advantageous for S. drummondii at least
during early developmental stages to colonize saline gaps of the community.
42
Temperature had little or no effect on seed germination of S. drummondii, however rate of germination at 25/35 ◦C was lower. Wei et al. (2008) also reported that seed germination of S. affinis occurred in a wide range (5 to 30 ◦C) of temperatures, constituting an “opportunistic” germination strategy to produce seedlings whenever conditions are favorable for seedling growth. In contrary, seeds of most subtropical perennial halophytes germinate optimally at 20/30 ◦C (Khan and Gul, 2006). However, some species like S. imbricata prefer cooler (10/20 ◦C; Mehrun-Nisa et al., 2007) temperatures for germination and a few like D. bipinnata germinate better at warmer
(25/35 ◦C; Gulzar et al., 2007) temperatures. Broader “temperature window” for germination as compared to other co-occurring plants, could possibly be an adaptation of
S. drummondii seeds to colonize study site promptly whenever adequate moisture is available.
Light is one of the key factors which determine the timing of germination in seeds of many halophyte species by facilitating the conditional dormancy to protect the seedlings from the extreme environment (Qu et al., 2008; Gul et al., 2012). Light can control the seed germination responses independently as well as in combination with salinity and temperature (Gul et al., 2012). Light requirements of salt desert halophytes for seed germination are quite variable and inconclusive (Khan and Gul, 2006). Some halophytes like Haloxylon recurvum and Zygophyllum simplex germinated better in presence of light (Khan and Ungar, 1997), many such as Lasiurus scindicus and
Panicum turgidum (El Keblawi et al., 2011) germinate better in complete dark and others like Atriplex stocksii (Khan and Ungar, 1997) germinate equally well under light as well as dark. Seeds of Limonium stocksii germinated equally well in light and dark under non- 43
saline conditions but dark was relatively more inhibitory under high salinity treatments
(Zia and Khan, 2004). In contrast, seeds of Suaeda salsa germinated better in dark than light under saline conditions, while were light/dark insensitive under non-saline conditions (Li et al., 2005; Song et al., 2008). It is noteworthy that seeds of S. drummondii germinated better under 12-H photoperiod than in complete dark under no/low (< 400 mM) salinity and moderate temperature (15/25 and 20/30 ◦C) treatments.
This could help recruiting seedlings at or near soil surface under no/low salinity stress conditions, so that their survival chances are maximized (Pons, 1992; El-Keblawy and
Al-Shamsi, 2008). However, difference between germination in light and dark diminished at high (> 600 mM) salinity and extreme (10/20 and 25/35 ◦C) temperatures in this study. This could also be an adaptive strategy of the test species to take advantage of delayed summer monsoon, which may not dilute soil salts to low levels. Such harsh conditions could be an opportunity for salt desert halophytes to produce some seedlings either at soil surface or in low light cracks or litter covers, as even low rains occur once in a year(s) in arid regions.
Most subtropical halophytes show high germination recovery after removal of salt
(Khan and Gul, 2006) as observed in, Arthrocnemum macrostachyum (Khan and Gul,
1998), Suaeda fruticosa (Khan and Ungar, 1997) and Limonium stocksii (Zia and Khan,
2004), indicating salinity enforced dormancy due to osmotic constraint. In contrast, seeds of S. drummondii showed low recovery of germination after alleviation of salt under 12- hours photoperiod at different temperatures. Similarly seeds of S. imbricata also showed poor recovery from salinity when transferred to distilled water (Mehrun-Nisa et al.,
2007). Seed viability testing after recovery experiments indicated that the most un- 44
germinated seeds were dead with < 30% having induced dormancy (un-germinated viable) under 12-H photoperiod condition. It is also noteworthy that there was no germination recovery (enforced dormancy) in un-germinated seeds from complete dark and there was very high percentage of seeds with induced dormancy. Eco-physiological significance of this high induced dormancy and low seed mortality under dark treatment needs to be investigated.
Chemical treatments had differential effect on seed germination of S. drummondii under non-saline and saline conditions. All the DRCs inhibited seed germination under non-saline condition but significantly (P < 0.05) improved germination under highly saline (800 mM NaCl) conditions. Exogenous fusicoccin promoted seed germination of salt stressed seeds of S. drummondii more than any other DRC tested. Fusicoccin, a diterpene glycoside which was initially isolated as a toxin from fungus Fusicoccum amygdali (Ballio et al., 1976), is widely reported to promote seed germination of halophytes (Gul and Weber, 1998; Gul et al., 2000; Khan et al., 2002; Gul and Khan,
2003; El-Keblawy et al., 2005) probably by enhancing cell elongation growth through
ATPase mediated proton extrusion (Galli et al., 1979; Marrè, 1979). Fusicoccin reversed the salinity induced germination inhibition in Zygophyllum simplex seeds completely
(Khan and Ungar, 2002). According to Cocucci et al. (1990) fusicoccin reversed the inhibitory effects of salinity in Raphanus sativus seeds by enhancing H+ extrusion and malic acid synthesis. While, Lutsenko et al. (2005) suggested that fusicoccin affects the
+ + ionic balance particularly the K /Na ratio. Fusicoccin and GA4 could also improve germination of salt stressed S. drummondii seeds under complete darkness. Likewise, fusicoccin also alleviated dark enforced dormancy in Allenrolfea occidentalis (Gul et al., 45
2000). While, GA4 was more effective in stimulating seed germination of Arabidopsis thaliana under dark as compared to GA3 (Fei et al., 2004).
Data indicates that the mature seeds of S. drummondii are non-dormant and partially photoblastic, which can germinate quickly in absence of salinity over a broad range of temperatures, constituting an “opportunistic” germination strategy to fully exploit brief rain periods. Seeds are highly salt tolerant and some seeds can germinate in
1000 mM NaCl solution. Germination inhibition under highly saline conditions can partially be alleviated by the exogenous application of different DRCs, especially fusicoccin. While, fusicoccin and GA4 could ameliorate germination of salt stressed seeds under complete darkness.
46
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53
Chapter 3
Variation in temperature and light but not salinity invoke antioxidant enzyme activities in germinating seeds of Salsola drummondii
54
Abstract
Seeds with efficient antioxidant defense system show higher germination under stress conditions, however, such information is limited for the halophyte seeds. I therefore studied the lipid peroxidation and antioxidant enzyme activities during seed germination of a leaf succulent halophyte Salsola drummondii under different salinity (0, 200 and 800 mM NaCl), temperature (10/20, 20/30 and 25/35 oC) and light regimes. Seeds absorbed water and germinated in less than one hour in non-saline control while increases in salinity decreased rate of water uptake as well as seed germination. Non-optimal temperatures (10/20 and 25/35 oC) and complete dark condition reduced seed germination in comparison to those seeds germinated under optimal (20/30 oC) temperature and 12-h photoperiod, respectively. Generally, higher lipid peroxidation and antioxidant enzyme activities were observed in seeds at non-optimal temperature and in those seeds germinated in dark. These results indicate variation in temperature and light but not salinity enhance antioxidant enzyme activities in germinating seeds of Salsola drummondii.
55
Introduction
Germination is the transition of dry seeds from quiescence to active metabolic state
which requires energy (Nonogaki et al., 2010). Hence, increase in oxygen consumption
for energy production is detected in seeds soon after imbibition (Bewley and Black, 1994;
Rosental et al., 2014). Mitochondrial membranes in dry seeds are damaged and cause
production of reactive oxygen species (ROS) during early imbibitional phase (Crowe and
Crowe, 1992; Tommasi et al., 2001; Noctor et al., 2007; Nonogaki et al., 2010).
Therefore, to prevent oxidative damage, seeds possess a number of antioxidant enzymes
such as superoxide dismutase (SOD) that converts the toxic superoxide radical into
hydrogen peroxide (H2O2; Kliebenstein et al., 1998), catalase (CAT) ascorbate
peroxidase (APX) and glutathione reductase (GR) which detoxify H2O2 (Willekens et al.,
1995; Noctor and Foyer, 1998). Antioxidant compounds like ascorbic acid (AsA) and
glutathione (GSH) also play a role in ROS scavenging (Smirnoff, 2000; Miller et al.,
2010; Foyer and Noctor, 2011). However, AsA is generally absent in dry seeds (De
Tullio and Arrigoni, 2003; Hameed et al., 2014).
Environmental stresses such as salinity, drought and temperature cause ROS
accumulation and decrease in the capacity of cells to detoxify them, hence result in
oxidative damage to different cell components and even cell death (McDonald, 1999;
Tommasi, 2001; Lee et al., 2010). Therefore, the success of germination is strongly
dependent on the quality of antioxidant defense that operates during germination (De
Gara et al., 1997; Bailly, 2004). However, such studies are mostly focused on crops and
other economically important plants (Gidrol et al., 1994; Bailly et al., 1996 and 2001;
56
Oracz et al., 2009). Little is known about halophyte seeds (Khan et al., 2006; Bogdanović
et al., 2008). Furthermore, to the best of our knowledge, no comprehensive study on
effects of different environmental factors such as salinity, temperature and light on
antioxidant enzyme activities during seed germination is available.
Salsola drummondii Ulbr. is a leaf succulent perennial halophyte from family
Amaranthaceae, and is a source of soda ash, medicine and forage for local populations
(Qureshi et al., 1993; Gilani et al., 2010). This species appears to survive vast
- fluctuations in soil moisture (~0.5 to 6%), electrical conductivity (EC1:10; ~10 to 30 dS m
1) and extremly high temperatures (> 45 oC) in natural field conditions and yearly
produces large number of seeds. Seeds in laboratory conditions germinated in up to 1000
mM NaCl at optimal light and temperature conditions (Rasheed et al., unpublished data).
How halophyte seeds respond biochemically to different environmental variables during
their germination is not well understood. Therefore, I investigated the effect of different
environmental factors, like salinity, temperature and light/dark on antioxidant enzyme
activities of S. drummondii seeds during germination.
Materials and methods
Seed collection
Mature seeds of Salsola drummondii were collected during February 2011, from a
population growing in a salt desert located at Winder, Balochistan (Pakistan). Seeds
were separated from perianths, surface sterilized by using 1% commercial bleach, rinsed
with distilled water, air dried and stored in clear plastic Petri plates until used.
57
Seed characteristics
Color, texture, shape, and size (diameter) of the freshly collected seeds were recorded.
Fresh weight of 100 seeds was taken. Seeds were then dried in a forced-draft oven at 105 oC for 48 hours to determine their dry weight. Seed moisture was calculated by subtracting dry weight from fresh weight. Oven-dried seeds were then placed in a furnace at 550 oC for 5 hours to determine ash content. Finally organic weight was calculated by subtracting ash from dry weight.
Seed germination
Seed germination experiments were conducted in programmed incubators (Percival
Scientific, Boone, Iowa, USA). Effects of NaCl treatments (0, 200 and 800 mM NaCl) on seed germination, lipid peroxidation and antioxidant enzyme activities were examined at
20/30 oC, where low temperature coincided with 12-H dark and high temperature with
12-H light period (25 μmol m-2 s-1; 400-700 nm, Philips cool-white fluorescent lamps).
Seeds were germinated in distilled water under alternative temperature regimes of 10/20
(low), 20/30 (moderate) and 25/35 oC (high) both in 12-H light / dark period as well as in dark. There were four replicates of 25 seeds each per treatment. Tight fitting plastic Petri-
Plates (5 cm Ф) with clear lids were used with 5 ml of test solutions. Germination data was recorded on alternate days for a period of 20 days and expressed as described in
Khan and Ungar (1984).
58
Seed water uptake
Seeds were immersed in three NaCl concentrations (0, 200 and 800 mM) and relative increase in fresh weight of seeds (Wr) was recorded (Song et al., 2005) after 50 minutes
(time required for embryo protrusion in distilled water).
Lipid peroxidation
Seeds (0.5 g) were ground fine with mortar and pestle using liquid nitrogen and homogenized in 1% ice-cold trichloroacetic acid. Homogenate was then centrifuged at
12000×g for 20 minutes at 4 oC. Supernatant was used for the determination of malondialdehyde (MDA) (Heath and Packer, 1968).
Antioxidant enzymes
Extraction of antioxidant enzymes was done according to the method of Polle et al.
(1994). While, activities of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC
1.11.1.6) guaiacol peroxidase (GPX, EC 1.11.17) and ascorbate peroxidase (APX, EC
1.11.1.11) were assayed and expressed as described in Hameed et al. (2012).
Statistical analyses
Statistical analyses were conducted by using SPSS version 11.0 for windows (SPSS Inc.
2007). Germination data were arcsine transformed before statistical analysis. Analysis of variance was carried out to determine if different environmental variables affected germination and antioxidant enzyme activities significantly. While t-test and post-hoc
Bonferroni test (P < 0.05) were conducted to compare individual means of treatments.
59
Results
Seed characteristics
Seeds were beige-brown, rough textured, round and small (0.2 mm Ф) with a fresh
weight of about 97 mg per 100 seeds (Table 3.1). There was about 77 % ash, 18 %
organic content and 4.8% moisture in fresh seeds of S. drummondii.
Table 3.1 Seed characteristics of Salsola drummondii.
Parameter
Color RAL1011 Brown Beige *
Texture Rough
Size (mm) 0.2
Fresh weight (g/100 seeds) 0.097
Moisture (%) 4.8
Ash (%) 77.14
Organic Content (%) 18.05
*(HLC = 74-60-36, RGB = 138-102-066 and CYMK (coated) = 30; 50; 80; 0 values are based on various measurements using D65 light with a standard observer according to International Commission on Illumination (CIE 1964). Source: www.ralcolors.com.
60
Seed germination
Analysis of variance (ANOVA) indicated significant (F - value = 113.796; P < 0.001) effect of salinity on final germination percentage of S. drummondii seeds (Table 3.2).
Final seed germination percentage decreased from 91% in distilled water to 55% in 200 mM NaCl and only 8% seeds germinated in high (800 mM NaCl) salinity treatment
(Figure 3.1a). Seeds of S. drummondii germinated (F - value = 3.74; P < 0.05) better at
20/30 oC than at 10/20 oC or 25/35 oC (Figure 3.1b). Furthermore, seed germination of S. drummondii was (F - value = 3.26; P < 0.05) higher (11%) under 12-H photoperiod (12-
H dark: 12-H light) in comparison to dark (Figure 3.1c).
Table 3.2 Analysis of variance showing the effect of different abiotic factors on Percent germination (G), Relative water uptake (Wr), MDA content and antioxidant enzyme activities of Salsola drummondii seeds. Values represent F- values. Where, * = P < 0.05; ** = P < 0.01; *** = P < 0.001 and ns = non-significant.
Parameters Salinity Temperature Light/dark
G 113.796*** 3.745* 6.607*
Wr 119.644*** ------
SOD 0.493ns 54.324** 27.996*
CAT 8.227* 34.916** 285.817***
APX 1.830* 189.297*** 12.039*
GPX 0.479ns 23.463* 22.364**
MDA 0.155ns 7.348* 10.759*
61
a 100 A 80 b 60 40
20 c 0 0 200 800 NaCl (mM)
) B a
% 100 a ( b
n 80 o i t 60 a n i 40 m
r 20 e
G 0
10 :20 20 :30 25 :35 o Temperature ( C)
100 C a b 80
60 40 20 0
Light Dark Treatments
Figure 3.1. Effect of A) salinity, B) temperature and C) light on the percent germination in seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t- test (C) P < 0.05]. 62
Water uptake
Seeds absorbed water (135% weight gain compared to dry seeds) and uncoiled in about
50 (Figure 3.2b). Rate of water absorption and final seed germination decreased with the increase in salinity (Table 3.2; Figure 3.2a) irrespective of light and temperature regimes used.
Lipid peroxidation
ANOVA showed a significant effect of temperature (F - value = 7.35; P < 0.05) and light
(F - value = 10.76; P < 0.05) but not of salinity on lipid peroxidation (Table 3.2; Figure
3.3). There was significantly higher MDA in seeds incubated at 10/20 and 25/35 oC in comparison to 20/30 oC. While, MDA content in complete dark was higher than in 12-H photoperiod (Figure 3.3b and c).
Antioxidant enzymes
After 50 minutes, CAT activity in salinity treated seeds (200 and 800 mM NaCl) was lower (F - value = 8.227; P < 0.05) as compared to water imbibed seeds (Figure 3.5a).
While, activities of SOD, APX and GPX in salinity treated seeds were similar to those in water-imbibed seeds (Figure 3.4a, 3.6a and 3.7a).
Temperature has significant effect on SOD (F - value = 54.324; P < 0.01), CAT
(F - value = 34.916; P < 0.01), APX (F - value = 189.297; P < 0.001) and GPX (F - value
= 23.463; P < 0.05) activities. SOD and GPX activities were comparable between 10/20
63
and 20/30 oC treatments but increased markedly at 25/35 oC (Figure 3.4b and 3.7b). CAT and APX activities were highest at 25/35 and lowest at 20/30 oC (Figure 3.5b and 3.6b).
Light regimes exerted a significant effect on antioxidant enzyme activities (Table
3.2). SOD, CAT, APX and GPX activities were substantially higher in dark compared to
12-h photoperiod (Figures 3.4c. 3.5c, 3.6c, 3.7c).
64
0.25 A
) 0.20 g (
r
W 0.15
0.10
0 15 25 35 50 Time (Minutes) B
150 a b
) 100 % (
c r W 50
0 0 200 800 NaCl (mM)
Figure 3.2. Effect of salinity on the relative water uptake in seeds of Salsola drummondii after 50 minutes of soaking. B) Arrow indicates embryo protrusion. Bars and circles represent mean ± standard error. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05). 65
A 25
20 a 15 a a 10
5
0 0 200 800 NaCl (mM) ) B W 25 F a a 1 - 20 g
l b
o 15
m 10 (
A 5
D 0 M 10 :20 20 :30 25 :35 o Temperature ( C) C 25 a
20 b 15 10 5 0 Light Dark Treatments
Figure 3.3. Effects of A) salinity, B) temperature and C) light on the MDA content in the germinating seeds of S. drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05]. 66
200 A
150
100 a a a 50
0 0 200 800 )
n NaCl (mM)
i
e a t 200 B
o r
P 150
1 - g 100
m b
s b t i 50 n U (
0 D 10 :20 20 :30 25 :30 O S Temperature (oC)
200 C
150 a
100 b 50
0 Light Dark
Treatments Figure 3.4. Effects of A) salinity, B) temperature and C) light on the activity of SOD in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05]. 67
A 40
30 10 a b b
0 0 200 800
) NaCl (m M ) n
i B a e t 40 o r P
30 1 - g 20
m b
s 10 b t i n 0 U (
T 10 :20 20 :30 25 :35
A o
C Tem perature ( C) C 40
a 10 b
0 Light Dark
Treatm ents
Figure 3.5. Effects of A) salinity, B) temperature and C) light on the activity of CAT in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05]. 68
A 120
100
20 ab a b 0 0 200 800 NaCl (m M )
) a n i B
e 120 t
o 100 r
P
80 1 -
g 60 b
m 40 s
t c i 20 n
U 0 (
X 10 :20 20 :30 25 :35 P o A Tem perature ( C) C 120 a 100 80 60 40 b 20 0 Light Dark Treatm ents Figure 3.6. Effects of A) salinity, B) temperature and C) light on the activity of APX in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05]. 69
1.2 A
1.0
0.4 a a a 0.2
0.0 0 200 800
NaCl (m M ) ) n i
e a t 1.2 B o r 1.0 P
1
- 0.8 g 0.6 m
s 0.4 b t
i b
n 0.2 U ( 0.0 X
P 10 :20 20 :30 25 :35 G Tem perature (oC) C 1.2
1.0 a 0.4 b 0.2 0.0 Light Dark
Treatm ents Figure 3.7. Effects of A) salinity, B) temperature and C) light on the activity of GPX in germinating seeds of Salsola drummondii. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other [Bonferroni test (A and B) and t-test (C), P < 0.05].
70
Discussion
Seeds of S. drummondii were small (0.2 mm Ф) and had lower moisture (4.8%) during
the dry state like other perennial halophytes (Song et al., 2005) and germinated rapidly
(50 minutes) on the availability of moisture by uncoiling of embryo. Seeds of S. tragus
(Wallace et al., 1968) and Haloxylon stocksii (Sharma and Sen, 1989) similarly
germinated within 30 minutes. Seed imbibition results in elongation of embryo cells and
uncoiling of the spiral embryo in salsoloideae species which ruptures the seed/fruit coat
quickly and marks germination completion without cell division (Wallace et al., 1968;
Parsons, 2012). Liu et al. (2013) proposed that such fast-germinating seeds are
‘cryptoviviparous-like’, probably to enhance the ability of their seedlings to become
rooted quickly in deeper, moist soil before the upper layer of soil dries in arid desert
environments.
Seeds of S. drummondii germinated optimally in distilled water at 20/30 oC and
under 12-h photoperiod, like most subtropical halophytes (Gul et al., 2013). Reduction in
seed germination of S. drummondii with increases in salinity coincided with decrease in
seeds’ relative water uptake, as reported for Suaeda physophora, Haloxylon
ammodendron and H. persicum (Song et al., 2005). However, reduction in seed
germination at sub-optimal temperatures and in dark is not related to reduce water uptake.
Recently, oxidative stress is ascribed to as a common consequence of environmental
stresses (Grene, 2002; Sharma et al., 2012). Therefore germination inhibition in these
conditions could be linked with oxidative stress.
71
MDA content of germinating seeds of S. drummondii did not change with increases in salinity, which might be indicative of unchanged ROS production or efficient antioxidant system during early phase of germination. However, prolonged exposure to salinity could be detrimental. Likewise, ROS production and MDA content did not change in Salsola ikonnikovii seedlings with increases in salinity from 0 to 300 mM NaCl
(Xing et al., 2013). In contrast, MDA content of germinating seeds of S. drummondii at sub-optimal (10/20 and 25/35 oC) temperatures were higher than at optimum (20/30 oC) temperature. Bhattacharjee, (2013) also showed that high (40 °C) and low (8 °C) temperatures during imbibition enhanced MDA content in germinating rice seeds compared to control (25 °C) treatment. MDA accumulated in dark germinated seeds of S. drummondii, which coincided with reduced germination in dark than in 12-h photoperiod.
Such oxidative damages have been held responsible for poor germination and early seedling establishment in other species under stress conditions (Gong et al., 1997;
Bhattacharjee, 2008).
Enhanced activities of different antioxidant enzymes are reportedly associated with successful completion of germination and stress tolerance of seeds (Puntarulo et al.,
1991; Guan and Scandalios, 1995; Bailly, 2004; Kranner and Seal, 2013; Hameed et al.,
2014). Superoxide dismutase (SOD) acts as first line of defense against ROS in plants
(Alscher et al., 2002). Higher SOD activity was observed in germinating seeds of many plants under salinity (Zheng et al., 2009; Wang et al., 2012), temperature (Mei and Song,
2010) and other stresses (Alscher et al., 2002; Guo et al., 2012). However, such information on halophyte seeds is scanty (Sekmen et al., 2012; Kranner and Seal, 2013).
Although, SOD activity in germinating seeds of S. drummondii did not change with 72
increasing salinity but increased significantly at high temperature (25/35 oC) and in complete darkness compared to optimal (20/30 oC) temperature and 12-h photoperiod respectively.
Catalase (CAT) is a haem-containing antioxidant enzyme that dismutates hydrogen peroxide into oxygen and water (Mhamdi et al., 2010). CAT activity in S. drummondii seeds declined under salinity, which could indicate unaltered ROS levels as indicated by unchanged MDA contents in this study. Apel and Hirt (2004) also indicated that the expression and activities of most antioxidant enzymes are stimulated by ROS accumulation. Similarly, expression of Cat1 and Cat3 genes was also ROS dependant in maize (Polidoros and Scandalios, 1999). In this study, suboptimal temperatures (10/20 and 25/35 oC) and complete darkness resulted in MDA accumulation, which coincided with rise in CAT activity, indicating higher production of ROS in these conditions.
Guaiacol peroxidases (GPXs) are class III peroxidases, which detoxify hydrogen peroxide by utilizing any phenolic compound such as guaiacol (Siegel, 1993; Jouili et al.,
2011). GPX activity although did not change in germinating seeds of S. drummondii under saline conditions but increased at higher temperature (25/35 oC) and in complete darkness. Reports on GPX activities at germination level are very scanty. However, an increase in GPX activity was reported in seedlings of Brassica napus cultivars under drought stress (Abedi and Pakniyat, 2010), in Betula pendula seedlings upon UV exposure (Tegelberg et al., 2008), and in Morus alba plants in response to high temperature treatment (Chaitanya et al., 2001).
73
Ascorbate peroxidase (APX) is another antioxidant enzyme in plants that scavenges hydrogen peroxide by using ascorbate as reductant (Asada, 1992). Its involvement in stress mediated oxidative stress tolerance in both halophytes and non- halophytes is well documented (Bartosz, 1997; Jithesh et al., 2006; Munns and Tester,
2008). However, information on role of APX in stress tolerance of halophyte seeds is scanty. In this study APX activity like most other antioxidant enzymes although remained unaffected by salinity but increased in response to temperature and dark stresses. Over expression of APX along with SOD increased stress tolerance of Tobacco seeds (Lee et al., 2010). Similarly, APX activities increased under drought stress in alfalfa cultivars during germination (Wang et al., 2009).
Salsola drummondii seeds absorbed water and germinated quickly in control, and both water uptake and germination decreased with increases in salinity while MDA and antioxidant activity remained unaffected. Seed germination was reduced at sub-optimal temperatures and in dark with concomitant increase in MDA and antioxidant enzymes.
These data hence show that salinity causes water stress while light and temperature stresses upset ROS balance in seeds of our test species. Further investigations are needed to fully understand these mechanisms.
74
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Chapter 4
Growth, oxidative damage and antioxidant enzyme activities in NaCl-treated seedlings of Salsola drummondii (Amaranthaceae)
84
Abstract
Salsola drummondii Ulbr., is a common leaf succulent halophyte of saline deserts in southern Balochistan (Pakistan) and has several economic usages. Information regarding salinity tolerance mechanisms of this species is not available. I therefore studied growth and oxidative stress management of S. drummondii seedlings in response to NaCl concentrations (0, 200 and 400 mM). Optimal seedling fresh weight, succulene, vitality, lower levels of oxidative damage and unchanged antioxidant enzyme activities are recorded at 200 mM NaCl (modrate salinity). However, growth reduction, increased antioxidant enzyme activities was recorded at 400 mM NaCl (high salinity). However, seedling show higher levels of oxidative damage with decrease vitality indicating that increase in antioxidant enzyme activity was inadequate to manage oxidative stress.
Failure to cope high salinity by S. drummondii seedling may be attributed to the inadequacy of antioxidant defense.
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Introduction
Halophytes complete their life cycle in some of the most challenging conditions like
hyper-aridity coupled with high soil salinity and very warm ambient as well as soil
temperatures (Gul and Khan, 2002; Cooke et al., 2006; Bui, 2013 ). Soil salinity affect
plant due to 1) physiological drought as a result of salt mediated lowering of soil water
potential, 2) ionic toxicity/imbalance caused by excessive Na+, and/or 3) a combination
of these factors (Ramadan and Flowers, 2004; Arzani, 2008; Munns and Tester, 2008;
Attia et al., 2011; Shavrukov, 2013). These direct effects of salinity result in excessive
- production of reactive oxygen species (ROS) such as superoxide radical (O2 ), hydrogen
. peroxide (H2O2) and hydroxyl radical ( OH), which cause oxidative destruction of
important cellular components and if not properly managed may result in plant death
(Zhu et al., 2004; Jithesh et al., 2006; Hameed and Khan, 2011). Plant cells possess an
antioxidant defense system to quench these ROS, which however becomes inadequate
when plants are exposed to high salinity (Smirnoff, 1993; Gosette et al., 1994; Shalata
and Neumann, 2001). Halophytes possess an efficient antioxidant defense which can
protect against ROS over a wider range of salinities compared to glycophytes or sensitive
species (Jithesh et al., 2006; Sekmen et al., 2007; Seckin et al., 2010; Xu et al., 2013).
Seedling is generally the most vulnerable stage in the life history of plants and
salinity is among major factors limiting its survival (Dodd and Donovan, 1999; Khan and
Gul, 2006; Gul et al., 2012). For example, all the early seedlings of highly tolerant
halophyte Kalidium capsicum died when incubated in ~150 mM NaCl solution (Tobe et
al., 2000). Little is known about the survival mechanisms of early seedlings particularly
86
for subtropical halophytes. Therefore, understanding the survival mechanisms of early
seedlings seems important.
Salsola drummondii Ulbr., is a leaf succulent xerohalophyte from family
Amaranthaceae, and is commonly found in the salt deserts of Southern Balochistan
(Pakistan) (Khan and Qaiser, 2006). There are many economic usages of this plant such
as making soda ash by locals, medicinal properties of different plant parts and utility of
leaves as forage (Qureshi et al., 1993; Gilani et al., 2010). Interestingly, under natural
field conditions, S. drummondii appears to tolerate vast fluctuations in soil moisture (~5
-1 to 25 %) and electrical conductivity (ECe1:10; ~10 to30 dS m ) (Rasheed et al.,
unpublished data). However, information on biochemical basis of salt tolerance of this
species is not known. I therefore studied the growth, oxidative stress markers and
antioxidant enzyme activities in early seedlings of S. drummondii in response to
increasing NaCl concentrations.
Materials and methods
Study site and seed collection
Seeds of Salsola drummondii Ulbr. were collected during February 2012, from plants
growing in a salt desert located at Winder, Balochistan (Pakistan). Seeds were separated
from perianth manually, surface sterilized by using 1% commercial bleach, rinsed with
distilled water, air dried and stored in clear plastic Petri-plates until used.
87
Seedling growth
Seeds were placed in clear-lid plastic Petri-plates (5 cm Ф) containing 5 ml of test solution (0, 200 and 400 mM) in a programmed incubator (Percival Scientific, Boone,
Iowa, USA) at 20/30 oC, where low temperature coincided with 12-h dark and high temperature with 12-H light period (25 μmol m-2 s-1; 400-700 nm, Philips cool-white fluorescent lamps) for 20 days. There were four replicates of 10 seedlings per NaCl treatment.
Biomass and succulence
Fresh weight (FW) of seedlings was measured after 20 days of germination. Dry weight
(DW), tissue water (TW), organic content (OC) and ash content of seedlings were determined according to gravimetric methods described in Hameed et al. (2012). While, seedling succulence was estimated by using following formula: Succulence = (FW-
DW)/DW.
Seedling vitality assay
Seedlings were subjected to 2,3,5-Triphenyl-tetrazolium chloride (TTC) test for determination of their vitality (viability) status (MacKay, 1972; Bradbeer, 1998).
Formation of red color due to the reduction of TTC in red colored TPF (1,3,5- triphenylformazan) in living tissues by the activity of various respiratory dehydrogenases, is routinely used as an indicator of viability/vitality. Seedlings were immersed in 0.1%
(w/v) TTC solution for 3-H under complete dark at 25 oC and photographed. Arbitrary
88
values of stain in seedlings were estimated by using ImageJ software (ImageJ/Fiji 1.46,
2012).
Localization of plasma membrane integrity loss
Plasma membrane integrity loss in S. drummondii seedlings was detected according to
Yamamoto et al. (2001) with some modifications. Briefly, seedlings were immersed in 5 ml of staining solution (0.025% (w/v) Evans Blue in 100 μM calcium chloride, pH 5.6) for 15 minutes, followed by washing with100 μM calcium chloride solution (pH 5.6) thrice and photographed. Development of blue color in injured tissues due to the penetration of dye in cells indicates loss of plasma membrane integrity. Arbitrary values of stain in seedlings were estimated by using ImageJ software (ImageJ/Fiji 1.46, 2012).
Localization of Hydrogen peroxide
Hydrogen peroxide (H2O2) was detected in S. drummondii seedlings by following the
3,3'-diaminobenzidine (DAB) staining method as described by Orozco-Cárdenas and
Ryan (1999). Deep brown color formation indicated the presence of H2O2. Arbitrary values of stain in seedlings were estimated by using ImageJ software (ImageJ/Fiji 1.46,
2012).
Extraction and assays of antioxidant enzyme activities
Extraction and assays of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC
1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC
89
1.6.4.2) activities were performed by following methods described in Hameed et al.
(2012).
Statistical analyses
Analysis of variance was used to study if salinity effects on growth and biochemical
parameters were significant. Bonferroni post-hoc test (P < 0.05) was performed to detect
significant differences among means. All statistical analyses were done by using SPSS
for windows version 16 (SPSS, 2007).
Results
Seedling growth
Analysis of variance (ANOVA) indicated significant effect of salinity on growth of S.
drummondii seedlings (Table 4.1). Fresh weight (FW) of S. drummondii seedlings
increased at 200 mM NaCl mainly due to higher tissue water and some reduction
compared to control was seen at 400 mM NaCl (Figure 4.1). Seedlings in absence of
salinity had less while at 400 mM NaCl had highest tissue ash. Succulence was
significantly (F - value = 6.679; P < 0.05) higher at 200 mM NaCl in comparison to
control and 400 mM NaCl treatments (Figure 4.2).
90
Table 4.1 Analysis of variance showing the effect of different NaCl treatments at 20/30 oC in 12-H light on various growth parameters and antioxidant enzyme activities in seedlings of of Salsola drummondii. Values represent F- values. Where, * = P < 0.05; ** = P < 0.01; *** = P < 0.001 and ns = non-significant.
Parameters F-value
Fresh weight 5.233*
Dry weight 0.746ns
Ash content 2.429*
Organic content 6.224*
Succulence 6.679*
Seedling vitality 39.113**
Membrane integrity loss 30.565**
Hydrogen peroxide production 42.258***
Superoxide dismutase 168.352**
Catalase 1.133ns
Ascorbate peroxidase 14.448*
Glutathione reductase 13.799*
91
Ash 0.5 OW DW ) 1 - t s 0.4
TW n s a a l 0.3 P m
6 o i g 0.2 ) B m 1 ( -
0.1 t n a 0.0 a
l 0 200 400
P b NaCl (mM)
g 4 c m (
s s a
m o
i 2 B
a a a 0 0 200 400
NaCl (mM)
Figure 4.1. Biomass of the seedlings of Salsola drummondii growing under different NaCl concentrations expressed as mg plant-1. Stack bars represent mean. Stack bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
92
)
W a D
1
- 10
g b b m 8 O
2 H
g 6
m (
e 4 c n e l
u 2
c c u
S 0 0 200 400 NaCl (mM)
Figure 4.2. Succulence of the seedlings of S. drummondii growing under different -1 concentrations of NaCl expressed as mg H2O mg DW. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
93
Seedling vitality
2,3,5-Triphenyl-tetrazolium chloride (TTC) staining indicated that the seedling vitality was not compromised at 200 mM NaCl, however at 400 mM NaCl substantially low pink coloration was observed as compared to control (Figure 4.3A and B). Analysis of variance (ANOVA) also showed significant variation (F - value = 39.113; P < 0.001) in the color intensity of seedlings treated with different concentration of NaCl (Table 4.1).
Plasma membrane integrity loss
Analysis of variance (ANOVA) indicated significant (F - value = 30.565; P < 0.001) effect of salinity on the plasma membrane integrity of S. drummondii seedlings (Table
4.1). Lowest membrane damage as indicated by Evans Blue staining was observed in seedlings from 200 mM NaCl treatment, while those from 400 mM NaCl treatment showed greater membrane damage (blue color) particularly in leaves and roots in comparison to unstressed seedlings (Figure 4.4A and B).
Hydrogen peroxide production
NaCl treatment had significant (F - value = 42.258; P < 0.0001) effects on the production and accumulation of H2O2 in S. drummondii seedlings (Table 4.1). Lowest H2O2 production (dark brown color) occurred in seedlings at 200 mM NaCl, which was confined to hypocotyl region, while in seedlings from 400 mM NaCl treatment H2O2 production was highest and was distributed to all parts (Figure 4.5A and B).
94
0.5 B
a a 0.4
s t i n 0.3 U
y r a
r 0.2
t i
b b r
A 0.1
0.0
0 200 400 NaCl (mM)
Figure 4.3. Seedlings of S. drummondii growing under different salt stained for vitality by Tetrazolium chloride staining. In (A) the picture was taken 20 days after germination. Scale bars represent 1 cm. (B) Bar represent area in cm (mean ± standard) of the stained regions for the seedlings measured with ImageJ in arbitrary units. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
95
0.25 B a 0.20 s t i
n b
U 0.15
y c r a r
t 0.10 i b r
A 0.05
0.00 0 200 400 NaCl (mM)
Figure 4.4. Seedlings of S. drummondii growing under different salt stained for localization of loss of plasma membrane integrity with Evan blue staining. In (A) the picture was taken 20 days after germination. Scale bars represent 1 cm. (B) Bar represent area in cm (mean ± standard) of the stained regions for the seedlings measured with ImageJ in arbitrary units. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
96
B 0.4 a s
t b i
n 0.3 U
y r
a 0.2 r t i b
r c A 0.1
0.0 0 200 400 NaCl (mM)
Figure 4.5. Seedlings of S. drummondii growing under different salt stained for concentrations histochemical detection of H2O2 with DAB staining. In (A) the picture was taken 20 days after germination. Scale bars represent 1 cm. (B) Bar represent area in cm (mean ± standard) of the stained regions for the seedlings measured with ImageJ in arbitrary units. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05). 97
Antioxidant enzyme activities
Activities of SOD, APX and GR were relatively lower at 200 mM NaCl and substantially
higher at 400 mM NaCl compared to control seedlings (Figure 4.6). Activity of CAT did
not vary significantly among NaCl treatments (Table 4.1, Figure 4.6).
Discussion
Salsola drummondii seedlings had higher FW and succulence when grown in moderate
(200 mM NaCl) salinity treatment compared to control and high (400 mM NaCl) salinity.
Although data on early seedlings is generally lacking, many Amaranthaceae halophytes
of southern Pakistan such as Suaeda fruticosa (Khan et al., 2000a), Arthrocnemum
macrostachyum (Khan et al., 2005), Haloxylon stocksii (Khan et al., 2000b) and Atriplex
stocksii (Khan et al., 2000c) also showed higher FW and succulence under moderately
saline conditions. Similarly many other Salsola species such S. chaudhryi (Al-Khateeb,
2002), S. dendroides, S. richteri and S. orientalis (Heidari-Sharifabad and Mirzaie-
Nodoushan, 2006) also showed higher fresh biomass in low/moderate salinity. Ash
content of the S. drummondii seedlings increased with increases in salinity, which is a
characteristic of succulent halophytes (Khan et al., 2000ab; Khan et al., 2005; English
and Colmer, 2011).
Succulence is an adaptive trait for the regulation of tissue ion concentrations in
many Amaranthaceae halophytes (Sen et al., 2002). For instance, succulence increased in
Suaeda fruticosa (200 mM NaCl, Khan et al., 2000a), Arthrocnemum macrostachyum
(400 mM NaCl, Khan et al., 2005) and Haloxylon stocksii (180 mM NaCl, Khan et al.,
98
80 a 8 SOD CAT a 60 a 6 )
) n n i i e
a e t
40 4 t o o r r P P
b
1
20 2 1 - b - g g
m m
s 0 0 s t t i GR i
n APX 50 a a 30 n U U ( (
y 40 y t t i b i
v 20
ab v
i i
t 30 t c b c A 20 b A 10
10 0 0
0 200 400 0 200 400
NaCl (mM)
Figure 4.6. Activities of different antioxidant enzymes in the seedlings of S. drummondii under different NaCl treatments at 20/30 oC in 12-H light. Bars represent mean ± standard error. Bars with same alphabet are not significantly different from each other (Bonferroni test, P < 0.05).
99
2000b) upon exposure to certain salinity. Similarly in this study I observed higher succulence at 200 mM NaCl compared to other salinity treatments. Higher succulence of
S. drummondii seedlings at 200 mM NaCl coincided with relatively higher ash content at this salinity compared to control. However, high salinity resulted in further increase in ash content but not concomitant rise in succulence, indicating overall higher salt concentration in tissues at 400 mM NaCl that could be toxic. Low vitality of seedlings at
400 mM NaCl treatment also supports this assumption.
Higher tissue Na+ is reported to induce oxidative stress by way of accelerating the
1 -1 production of ROS such as singlet oxygen ( O2), superoxide radical (O2 ), hydrogen
. peroxide (H2O2) and hydroxyl radical ( OH), which are damaging to important cell components including membranes, proteins, nucleic acids and chlorophyll (Zhu, 2001;
Zhu et al., 2004; Botella et al., 2005; Foyer and Noctor, 2005; Hameed and Khan, 2011).
Rise in the levels of H2O2 and membrane damage are widely used indicators of oxidative stress in plants (Gossett et al., 1994; Hernández and Almansa, 2002; Hameed et al., 2012;
Koyro et al., 2013). However, the data indicates that the levels of both H2O2 and membrane integrity loss (membrane damage) were lower in S. drummondii seedlings at
200 mM NaCl. Unchanged antioxidant enzyme activities under moderate NaCl treatment, also indicated absence of oxidative stress in S. drummondii seedlings. In contrast, highest levels of H2O2 and membrane integrity loss were observed in 400 mM NaCl. As discussed above, overall higher cellular salt concentrations due to reduction in tissue moisture might be responsible for higher oxidative stress in S. drummondii seedlings.
100
Membrane damages in Echinacea angustifolia similarly caused by oxidative stress in the presence of high tissue Na+ (Sabra et al., 2012).
High salinity leads the higher SOD activity detoxifing superoxide radicles and generating H2O2 and this observation is supported by results of H2O2 staining data in this experiment. CAT, APX and GR are important H2O2 detoxifying enzymes in plants
(Parida and Das, 2005; Jithesh et al., 2006; Hameed et al., 2012). Activities of APX and
GR but not that of CAT increased significantly in S. drummondii seedlings at 400 mM
NaCl. However this increase was not as much as in SOD activity thus appears inadequate. Greater membrane damage in our test species at high salinity also supports this assumption. In addition, unchanged activity of CAT, an enzyme which can detoxify
H2O2 (Scandalios et al., 1997; Feierabend, 2005), also indicates that the antioxidant defense was inadequate at 400 mM NaCl in S. drummondii seedlings.
In short, these results indicate that the leaf succulent halophyte Salsola drummondii grew optimally at moderate salinity (200 mM NaCl) without undergoing oxidative stress. However, high salinity (400 mM NaCl) severely affected the seedling vitality by accelerating ROS production more than the capacity of antioxidant defense system to detoxify them.
101
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Kalidium capsicum (Chenopodiaceae). Ann. Bot-London. 85, 391−396.
Xu, J., Duan, X., Yang, J., Beeching, J.R., Zhang, P., 2013. Enhanced reactive oxygen
species scavenging by overproduction of superoxide dismutase and catalase
delays postharvest physiological deterioration of Cassava storage roots. Plant
Physiol. 161, 1517−1528.
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Zhang, Z., Li, H., Qiao, S., Zhang, X., Liu, P., Liu, X., 2012. Effect of salinity on seed
germination, seedling growth, and physiological characteristics of Perilla
frutescens, Plant Biosys. 146, 245−251.
Zhu, J.K., 2001. Plant salt tolerance. Trends Plant Sci. 6, 66−71.
Zhu, Z., Wei, G., Li, J., Qian, Q., Yu, J., 2004. Silicon alleviates salt stress and increases
antioxidant enzymes activity in leaves of salt-stressed cucumber (Cucumis sativus
L.). Plant Sci. 167, 527−533.
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Chapter 5
Physiological responses of Salsola drummondii population to seasonal variations
109
Abstract
I investigated the seasonal variations in physiological adaptations of the xero-halophyte
Salsola drummondii in a salt desert population of Balochistan, Pakistan. Measurements of leaf biomass, xylem pressure potential, leaf osmotic potential, ion content, photosynthesis and antioxidant enzyme activities were done in winter (January), summer (April and
July) and Monsoon (August) seasons. Leaf succulence, organic weight, photosynthesis,
WUE, proline and antioxidant enzyme activities correlated well with rhizosphere moisture and ECe which in turn appeared to depend on precipitation. Photosynthetic performance was better during mild winters and monsoon period with higher rate of photosynthesis and higher stomatal conductance due to higher availability of water. A greater vapor pressure deficit was observed during summer showing a negative correlation with the leaf stomatal conductance however, lack of variation in the internal
CO2 concentration was suggestive of stomatal and biochemical co-limitation of photosynthetic rate during summer. A reduction in leaf succulence, xylem pressure potential and leaf osmolality by late summer was also observed. The increase of water use efficiency, activities of superoxide dismutase, catalase, ascorbate peroxidase, and the accumulation of proline during periods of low water availability and high salinity in the soil suggest that S. drummondii efficiently regulates its metabolic functions to survive under drought and saline conditions. This study highlights the importance of physiological and biochemical adaptations of S. drummondii, which could be critical for its persistence in a salt desert population.
110
Introduction
Salinity limits the growth and biomass production of plants by limiting water uptake
through roots, ionic toxicity and photosynthesis (Maricle et al., 2007; Munns and Tester,
2008; Cheeseman, 2013; Shavrukov, 2013). Toxic effects of salinity are a consequence of
excessive production of reactive oxygen species (ROS) such as singlet oxygen,
superoxide radicle, hydrogen peroxide and hydroxyl radicle (Zhu, 2001; Foyer and
Noctor, 2005; Foyer and Shigeoka, 2011) due to high light intensity, temperature and
salinity and/or low moisture availability. Most ecophysiological studies on halophytes
have been carried out under controlled laboratory conditions (Peleg et al., 2011) but little
is known about the plant responses in their natural environments (Bui, 2013). Salt deserts
represent very hostile environments for plant growth, however, halophytes and
xerohalophytes are well adapted to these conditions (Khan and Qaiser, 2006) and
ecophysiological investigations could indicate the level of environmental stress faced by
them (Larcher, 2003).
Decline in photosynthetic gas exchange is often considered as an adaptation to
increase water use efficiency of the plants under stress conditions (Koyro, 2006; Koyro et
al., 2013). Reduction in photosynthesis is often related to slow plant growth, which could
be beneficial for survival of plants under stress (Hameed et al., 2012). According to
Neumann (2011) growth reduction under salt stress might be an adaptation to increase
chances of survival long enough to produce some seeds. Vapor pressure deficit of the air
(VPD) is amongst important abiotic factors which has significant impact on the
physiology and morphology of plants (Grange and Hand, 1987; Grantz, 1990; Leuschner,
111
2002). Photosynthetic gas exchange in many plants is highly sensitive to VPD, and high
VPD limits net Photosynthesis (A) and stomatal conductance (Gs). Pathre et al. (1998) found that VPD was a more important factor than temperature or photosynthetic photon flux density (PPFD) in causing midday reduction in net photosynthesis (A) and stomatal conductance (Gs).
Reduction in photosynthetic CO2 fixation under stress conditions could also lead to increased production of reactive oxygen species (ROS) such as singlet oxygen, superoxide radicle, hydrogen peroxide and hydroxyl radicle (Hameed et al., 2012).
Accumulation of these ROS can damage cell membranes, proteins and nucleic acids (Gill and Tuteja, 2010), hence may cause plant death if are not scavenged by plants intrinsic antioxidant defense system, which is composed of both enzymes and substrates.
Superoxide dismutase (SOD), catalase (CAT) and ascorbate peoxidase (APX) are frequently used indicators of plants antioxidant responses (Jithesh et al., 2006; Hameed and Khan, 2011).
Salsola drummondii Ulbirch. is a perennial leaf succulent xerohalophyte from the family Amaranthaceae (Khan and Qaiser, 2006). It is also reported from eastern part of
Arabian Peninsula and Southern Iran (Flora of Pakistan) and usually co-occurs with
Suaeda fruticosa, Aerva javanica var. bovei, Suaeda monoica, Prosopis juliflora,
Prosopis cineraria, Heliotropium subulatum, Euphorbia caducifolia and some halophytic grasses. Salsola drummondii is used by locals to make soda ash, medicine (Gilani et al.,
2010) and as forage (Qureshi et al., 1993). Beside, its economic importance no such
112
information available about the physiological adaptations and survival of S. drummondii
under harsh environmental conditions.
This study evaluated the physiological adaptations of a Salsola drummondii
population to salt desert conditions in southwestern Pakistan. Following questions were
addressed: 1) How seasonal variations in habitat conditions affect water relations of test
species? 2) Which seasonal conditions facilitate optimal photosynthesis in S.
drummondii? 3) How osmoprotectants’ (proline and soluble sugars) accumulation is
affected by seasonal variations in habitat conditions? and 4) if higher antioxidant enzyme
activities coincide with higher temperatures, salinity and drought period? .
Materials and method
Study site
This study was carried out in a salt desert located at Windar, Balochistan, Pakistan
(24o25’07.16” N and 66o37’32.38”E). This region is characterized by warm summers
(41-44oC), mild winters (6-11oC) with low (<20 mm) rainfall mostly in December. The S.
drummondii population under study is located intermediate in position between
Mediterranean and sub-tropical climate zones within the Saharo-Sindian phytogeographic
region.
Soil analyses
Soil samples were collected from three different depths (20, 60 and 180 cm). Soil pH,
electrical conductivity (ECe), moisture and ions (Na+, K+, Ca+2 and Mg+2) were measured
113
in distilled water dilutions of soil extracts against known standards on an atomic absorption spectrometer (Perkin Elmer AA-700).
Plant analyses
Growth parameters
Leaves were collected from S. drummondii plants growing in ten randomly chosen permanent quadrates (4'x4') during the four study periods (Table 5.1). Leaf succulence, ash and organic contents were determined gravimetrically as indicators of leaf growth.
Table 5.1 Description of sampling time.
Weather Conditions Month Season o o Prec. (mm) Tmax ( C) Tmin ( C) RH (%)
January Winter 13.6 25.5 11.8 65.0
April Early Summer 5.7 38.7 20.3 50.0
June Late Summer 9.8 42.0 27.2 64.0
August Post-monsoon 3.1 36.0 27.0 69.0
114
Water relations
Leaf sap osmolality (mosmol Kg-1) was measured using a vapor pressure osmometer
(5520 VAPRO, Wescor Inc, Logan, UT, USA). Xylem pressure potential of freshly excised twigs was measured with a plant water status console (Model 1000, PMS
Instrument Company).
Proline and total soluble sugars
Hot water extracts of oven dried leaves were used to determine proline by acid ninhydrin method (Bates et al., 1973) and total soluble sugars (TSS) by the anthrone reagent method (Yemm and Willis, 1954) on a UV/VIS spectrophotometer (BECKMAN
COULTER Du 530) in the laboratory.
Leaf cations
Fine- ground dry leaves (0.5 g) were boiled in 10 ml of deionized water for 2 hours at
100 oC. These hot water extracts were cooled and then filtered with Whatman No. 42 filter paper. Filtered extracts were then used for the analysis of various cations namely
Na+, K+, Mg++, and Ca++ with help of Atomic Absorption Spectrometer (Perkin Elmer
AA-700).
Photosynthetic gas exchange and pigments
Gas exchange measurements were performed four times during the study period. The gas exchange parameters were measures by using LI-COR 4600XT portable photosynthesis system (LI-COR Biosciences, Lincoln, Nebraska 68504). The following variables were
115
evaluated: rate of photosynthesis (A), stomatal conductance (Gs), intracellular CO2 concentration (Ci). The ratio of intrinsic water use efficiency (A/Gs) was also calculated by using the measured values of A and Gs. For the extraction of photosynthetic pigments, leaves were homogenized in ice-chilled 80% acetone and absorbance of the centrifuged extracts were recorded at 663.2, 646.8 and 470 nm against the 80% acetone blank by using a UV/VIS spectrophotometer (BECKMAN COULTER Du 530). Contents of the photosynthetic pigments were estimated according to the method of Lichtenthaler (1987).
Antioxidant enzymes
To assess the antioxidant defense system in the leaves of S. drummondii were collected randomly in each study season and carried to the laboratory in liquid nitrogen.
Antioxidant enzymes were extracted following Hameed et al., (2012). Superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activity were assayed according to the methods of Beyer and Fridovich (1987), Abey (1984) and Nakano and
Asada (1981) respectively. Protein contents of the extracts were estimated by Bradford
(1976).
Statistical Analysis
Data were statistically analyzed by using SPSS version 11 (SPSS, 2007). Analyses of variance (ANOVA) and post-hoc Bonferroni test were used for the data.
116
Results
Weather conditions
During 2009 high precipitation (15-20 mm) was mainly observed during winters
(December to January) due to rain and heavy night time dew, followed by another high
precipitation episode in July (~13 mm) in the form of monsoon rains (Figure 5.1).
However, mean annual rainfall was ~8.63 mm, indicating hyper-arid nature of the study
site. Hottest (>40 oC; mean maximum temperature) period of the year was observed
during April to June, while December to February constituted the coldest (<15 oC; mean
minimum temperature) period (Figure 5.1).
Edaphic conditions
Soil moisture was in agreement with precipitation data and was highest in the moist
months. Soil moisture varied greatly with depth with lower values in surface soil (20 cm)
and higher in root zones (60 and 180 cm) (Figure 5.2). While, soil ECe1:5 was higher for
surface (20 cm) soil and lower for the deeper zones (60 and 180 cm). Soil ECe1:5 values
were significantly (P < 0.05) higher during the dry months of April and June (Figure 5.2).
Soil Na+ contents varied significantly among seasons and depths (ANOVA; Table 5.2)
with higher values in top soil during summer (Table 5.3). Na+ was also the dominant
cation of the study site, followed by Ca++ and Mg++, while the K+ ion is the least
dominant. Salt accumulation (Na+ and Ca++ salts particularly) was significantly higher in
the dryer months (April and June) regardless of soil depth (Table 5.3).
117
25 50 Mean Maximum Mean Minimum T ) 20 40 e
m m m p (
15 30 e n r o a
i * t t u a
t r i
10 20 e p
i ( c o
C e r
* ) P 5 * 10 *
0 0 l t r r r r i y y y y h e l r s r r e e e e
a c n u p u a a r b b b b u J M g u u a o A J m t r m m u n c M e b e e a A t c v e J O p e o F e N D Months S Figure 5.1. Mean monthly precipitation (bars), maximum (red symbols) and minimum (blue symbols) temperatures (oC) in the study site during 2009. Asterisks (green symbols) indicates the sampling months.
118
Table 5.2 Two-way analysis of variance (ANOVA) of soil cation content due to seasons (S), soil depth (D) and S x D interactions on the soil mineral content of the Salsola drummondii community.
Source of Na+ K+ Ca++ Mg++ variation
S 11.157*** 3.258* 4.484* 10.857***
D 21.016*** 7.358** 15.295*** 18.661***
S x D 6.731*** 0.591ns 0.807ns 2.342*
Numbers indicate F-values at * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and ns = non- significant
119
Table 5.3 Seasonal variations in the soil cations (Na+, K+, Ca++ and Mg++; mg Kg-1 DW) contents drawn from different depths of the Salsola drummondii community.
Soil Months Na+ K+ Ca++ Mg++ depth 20 cm 1112.33±107.94a 124.67±12.72a 1682.67±192.12a 164.05±31.08a January 60 cm 994.13±97.37a 109.93±44.78ab 1039.33±370.59a 70.87±7.28a 180 cm 510.67±53.17b 68.27±14.55b 458.00±168.59a 66.62±20.47a 20 cm 4641.33±884.59a 171.33±18.70a 1466.00±298.59a 273.33±21.33a April 60 cm 1579.50±6.70ab 146.00±24.00ab 1486.00±256.00a 190.00±72.00a 180 cm 1043.20±51.80b 86.50±11.50b 819.50±64.50a 88.02±11.52a 20 cm 1544.67±376.02a 136.00±6.11a 2816.00±249.48a 210.00±16.43a June 60 cm 1857.00±112.43a 121.67±13.87ab 667.33±308.07ab 176.00±13.00ab 180 cm 1129.67±94.37a 55.20±10.77b 818.00±66.00b 83.24±11.29b 20 cm 1875.33±201.67a 244.59±65.29a 1451.33±310.73a 77.49±10.47a August 60 cm 1091.40±14.80ab 131.50±8.30a 878.00±48.00a 89.69±9.81a 180 cm 647.00±144.57b 135.67±17.70a 81.67±10.84a 53.63±7.47a
120
20 cm 6 40 5
30 4
3 20 2 10 1 ) 1 - 0 0
m
6 S 60 cm d 40 ( M 5 y o t i i v s
i 30
t 4 t u
c r u e
d 3 20 ( n %
o
c 2 )
l
a 10
c 1
i r t
c 0 0 e l E 180 cm 6 40 5
30 4
3 20
2 10 1
0 0 January April June August
Months
Figure 5.2. Seasonal variations in the soil electrical conductivity (ECe 1:5; Bars) and moisture (%) content (circles) at 20, 60 and 180 cm depth from the soil surface.
121
Plant Analyses
Plant Growth
Analyses of variance (ANOVA) indicated that leaf succulence, ash and organic matter
varied significantly among seasons (Table 5.4). Pattern of variations in the leaf
succulence of S. drummondii was in accordance with the data of soil moisture and found
higher in the months of August and January (post-monsoon and winter period) (Figure
5.3). Ash content was higher in the months of April and June (early summer and pre-
monsoon). However, it was vice versa for the leaf organic content (Figure 5.3).
Leaf sap osmolality and stem XPP
Seasonal variation in the leaf osmolality was not high throughout the year. However,
osmolality was higher during the month of June (pre-monsoon) and remained unchanged
during rest of the study period (Figure 5.4). The stem XPP was also higher during the dry
periods of April and June (Early summer and pre-monsoon) (Figure 5.5).
Proline and total soluble sugars
Proline content increased linearly from January to June and dropped in August (Figure
5.6). Whereas, leaf TSS contents were lowest in April and were similar in other sampling
periods (Figure 5.7).
122
Leaf cations
Leaf Na+ and K+ contents peaked during June and coincided with lower Ca++ levels
(Table 5.4; Figure 5.8). While, leaf Mg++ contents showed little variations among seasons.
Photosynthetic gas exchange and pigments
The differences in gas exchange parameters of S. drummondii (Table 5.4 and 5.5) reflected the variations in the atmospheric conditions of the local environment. The photosynthetic performance of S. drummondii leaves strongly reduced during the dry and hot periods (months of April and June) of the year (Table 5.5). However, the water use efficiency, as assessed by A/Gs, was enhanced markedly during this period (Table 5.5).
Analyses of variance (ANOVA) showed significant seasonal variations in the photosynthetic pigments in the leaves of S. drummondii among seasons (Table 5.4). Leaf
Chl a and b contents decreased in the month of April (early summer). However, the extreme hot and dry conditions of early summer resulted in greater reduction of Chl b than Chl a and so Chl a:b increased in the leaves of Salsola drummondii (Table 5.5).
Antioxidant enzymes
Activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase
(APX) activity were generally higher during summer (April and June) (Figure 5.9).
123
Table 5.4 Summary of F-values from one way analysis of variance (ANOVA) in growth and physiological parameters of Salsola drummondii during the study year.
Plant parameters F- Value
Succulence (g/g dw) 248.35*** Ash content (Ash; %DW) 117.66*** Organic content (OC; %DW) 110.66*** FW : DW 248.35***
Ash : OC 66.70*** Chl a (mg g-1 DW) 2.71* Chl b (mg g-1 DW) 11.21*** Total Chl (mg g-1 DW) 5.87** CAR (mg g-1 DW) 15.23*** Chl a / b 12.21***
Chl / CAR 0.40ns -1 -1 A (μmol g DW S ) 6.16** Gs (μmol g-1 DW S-1) 7.84** Ci (μmol g-1 DW S-1) 1.72ns
WUE 7.00** Total soluble sugars (mg g-1 DW) 22.54** Proline (mg g-1 DW) 12.28** Na+ (mg g-1 DW) 10.12**
K+ (mg g-1 DW) 13.49** ++ -1 Ca (mg g DW) 6.95* Mg++ (mg g-1 DW) 4.59* Na+/K+ 15.42** Na+/Ca++ 35.34***
Na+/Mg++ 6.96* Leaf osmolality (mOsmol kg-1 plant water) 4.73* Xylem pressure potential (-MPa) 5.23**
Numbers indicate F-values at * = P < 0.05, ** = P < 0.001, *** = P < 0.0001 and ns = non-significant
124
) 2.5 a W D
1 - 2.0 b g
O 2 1.5 c H ( d e
c 1.0 n e l
u 0.5 c c u
S 0.0
) a W a 60 D b % (
t c
n 40 e t n
o C
20 h s A 0 ) a W 60 D b %
( c
c t
n 40 e t n o C
c 20 i
n a g r O 0 January April June August Months Figure 5.3. Seasonal variations in leaf succulence, inorganic, and organic contents of Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test). 125
) r
e b t a 1500 w a a y t
t a i n l a a l l o P 1000 m 1 - s g O
K f
l a o
e 500 L m s O m (
0 January April June August Months
Figure 5.4. Seasonal variations in the leaf osmolality (mOsmol kg-1 plant water) of Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
126
0
-1
) a -2 P M
- (
-3 P
P X -4 c bc
-5 ab a
January April June August
Months
Figure 5.5. Seasonal variations in the xylem pressure potential (-MPa) of shoot in Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
127
a 8 ) ab W D
1 - 6 g
g m ( 4 b b e
n i l o r
P 2
0 January April June August
Months
Figure 5.6. Seasonal variations in the leaf proline (mg g-1 DW) content of Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
128
35 a a 30 s r b a g ) 25 u W s
l D
20
a 1 -
b c g u
l 15 g o s m
l (
10 a t
o T 5
0 January April June August Months
Figure 5.7. Seasonal variations in the total soluble sugars (mg g-1 DW) of Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
129
a 60 Na + K+ a 25 ab 50 20 b b 40 15 ) 30 b b W 10
D b 20 1 -
g 5
10 g 5
m ++ ( ++ ab
Mg a Ca a ab y
t 0.6 4 i l a b a
u 3 0.4 Q ab 2 0.2 b 1
January April June August January April June August Months
Figure 5.8. Seasonal variations in the leaf ion (Na+, K+, Ca++, and Mg++; mg g-1 DW) contents of Salsola drummondii. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test).
130
Table 5.5 Seasonal variations in the photosynthetic pigments and gas exchange parameters of Salsola drummondii.
Parameter January April June August
Chl a (mg g-1 DW) 0.34±0.05ab 0.31±0.03b 0.36±0.04a 0.42±0.01a
Chl b (mg g-1 DW) 0.13±0.05bc 0.09±0.01c 0.19±0.01a 0.17±0.02ab
Chl a+b (mg g-1 DW) 0.46±0.05bc 0.40±0.04b 0.55±0.05ac 0.59±0.03a
Car (mg g-1 DW) 0.13±0.00c 0.11±0.01c 0.15±0.01b 0.18±0.01a
Chl a:b 2.73±0.13b 3.53±0.17a 1.92±0.18c 2.58±0.29bc
Chl:Car 3.57±0.06a 3.72±0.21a 3.61±0.15a 3.44±0.23a
A (μmol g-1 DW S-1) 42.71±3.78a 13.49±2.32b 27.50±1.46c 45.45±1.82a
Gs (μmol g-1 DW S-1) 1.16±0.15a 0.75±0.04b 0.35±0.04c 0.83±0.28ab
Ci (μmol g-1 DW S-1) 238.12±9.16a 229.05±11.87a 201.15±16.84a 244.16±7.91a
WUE 2.49±0.09b 4.35±0.62a 4.44±0.24a 2.63±0.55ab
131
ab SOD 60
40 a
bc 20 c
)
n 0 i
e a t CAT o r 0.6 P
1 -
g ab
m 0.4
s t
i b n b U 0.2 (
y t i v i t 0.0 c A APX a 15 ab bc c 10
5
0 January April June August
Months Figure 5.9. Seasonal variations in the activity of the antioxidants enzymes in the leaves of S. drummondii under salt desert. Enzymes activities are expressed in Units mg-1 Protein. Bars represent mean ± standard errors. Bars having same letter within each salt treatment are not significantly different (P < 0.05) among means (Bonferroni test). 132
Discussion
Water stress caused by both soil and atmospheric water deficits, is one of the limiting environmental factors to plant productivity worldwide (Ghanoum, 2009) and in arid regions it is usually associated with salinity stress. I found that the changes in the ambient conditions like low precipitation and high ambient temperatures of summer coincided
+ with high soil salinity (measured as ECe1:5 and Na accumulation) and creating stressful conditions for plant growth (Aziz, 2007; Aziz et al., 2011). Salt tolerant plants employ a combination of morphological, physiological and biochemical tactics to cope with deleterious effects of excess salts (Munns, 2002; Aziz et al., 2011; Nedjimi, 2012).
A number of plants in the family Amaranthaceae characteristically utilize leaf or stem succulence to endure water scarcity and high salinity (Munns, 2002; Eggli and
Nyffeler, 2009; Ogburn and Edwards, 2012; Griffiths, 2013). Leaf succulence in S. drummondii was high in August and January and declined as the soil water decreased, indicating that the plants utilized this stored tissue water when faced with drought in following months. Leaf succulence also declined in the sub-tropical halophyte Limonium stocksii with decreasing soil moisture (Zia et al., 2008). The decline in leaf succulence and xylem pressure potential of S. drummondii coincided with increase in total ash contents, Na+, K+, soluble sugars, proline and leaf sap osmolality indicating osmotic adjustment during the drier late summer months (June). Accumulation of inorganic solutes (e.g. ash and Na+ contents) and organic osmoprotectants (e.g. sugars and proline) are commonly used indicators of osmotic adjustment in plants. These results are in agreement with earlier studies on local halophytes (Gul et al., 2001; Khan and Naqvi,
133
2002; Aziz et al., 2005) indicating the importance of osmotic adjustment for plant survival during dry parts of the year when soil salinity is also high. Seasonal changes in proline contents were found to increase in relation to variations in drought stress rather than soil salinity (Boscaiu et al., 2012). In the present study, increase in proline during late summer (June) could be related to low soil moisture availability and/or high temperature but proline content was not enough to serve as osmotica but rather suggests its role in cellular protection.
Photosynthetic dynamics are sensitive to changes in environmental factors
(Chaves et al., 2002; Lawlor and Tezara, 2009; Yu et al., 2009; Santos et al., 2013). The present research also showed strong influence of changes in climatic and edaphic factors on photosynthesis of S. drummondii. Photosynthetic performance of S. drummondii leaves decreased during the warm and dry summer with low moisture availability and high salinity. High light intensity, summer temperatures and water deficit could be the possible reasons for this decreased photosynthesis in S. drummondii during summer
(Chaves et al., 2002; Lawlor and Tezara, 2009; Yu et al., 2009; Santos et al., 2013).
Photosynthetic rates of S. drummondii dipped in early summer (April) due to a sharp rise in temperature resulting in decreased total soluble carbohydrate content comparable to other parts of the year. Intercellular CO2 concentrations (Ci) is known to decrease
(stomatal limitation), increase (metabolic or biochemical limitation) or remain unchanged with decreasing photosynthetic rates and stomatal conductance. No increase in Ci was registered throughout the year which suggested that environmental conditions were not too extreme to cause distinct biochemical inhibition of photosynthesis in S. drummondii during the summer months. 134
High Na+ is also reported to inhibit photosynthesis in a number of ways such as by reducing leaf chlorophyll content under salt stress with consequent decline in photosynthesis (Koyro, 2006; Li et al., 2010). Chlorophyll contents of the sub-tropical halophytes Suaeda fruticosa, Heliotropium curassavicum, Haloxylon stocksii and
Atriplex stocksii decreased during summer (Aziz et al., 2011). Lower chlorophyll b resulted in a higher chlorophyll a/b ratio in early summer (April) which recovered in late summer (June). Better photosynthetic efficiency in the monsoon period along with higher chlorophyll a/b ratio (higher chl a) could be due to reduced light stress (fewer sunshine hours) which allowed the plant to accumulate sufficient energy reserves for subsequent flowering and fruit production later on in December and January, respectively. Little variation in carotenoid content and in the chlorophyll/carotenoid ratios throughout the year suggested little role of the xanthophyll cycle in quenching excess excitation energy however, further investigations into the epoxidation / de-epoxidation states of the violaxanthin –antheraxanthin - zeaxanthin pigment system would be needed to confirm this hypothesis (Taiz and Zeiger, 2010; Demmig-Adams et al., 2012).
Abiotic stresses such as salinity, drought and high temperatures are known to cause increased production of ROS in plants and tolerant species possess efficient antioxidant defense to minimize oxidative damages (Jithesh et al., 2006; De Gara et al.,
2010; Sharma et al., 2012; Ozgur et al., 2013). Most of the published research deals with plants of cold environments (Anderson et al., 1992; Wang et al., 2009; Zhang et al.,
2009), but information on antioxidant responses of warm subtropical plants is scanty.
Higher leaf antioxidant enzyme activities indicated that higher production of ROS during
135
summer in both the chloroplast (SOD and APX) and cytoplasm (CAT) was efficiently counterbalanced by S. drummondii.
In conclusion, climate driven conditions appeared to influence plant performance under natural conditions. During this work it became evident that precipitation events had the most impact in rhizosphere moisture and salinity also which appeared to have important roles in modulation of CO2/H2O gas exchange traits. In addition, vapor deficit appeared to be more important than its components (relative humidity and temperature) in influencing net photosynthetic rates. Reduction in photosynthesis under stress conditions is known to cause enhanced production of ROS in plants. S. drummondii displayed higher activities of antioxidant enzymes in summer probably to counter higher
ROS produced during summer. Considering the potential economic usages and local abundance of this species in its peculiar environment it is important to understand the ecophysiological responses of S. drummondii to abiotic conditions.
136
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Chapter 6
General conclusions
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General conclusions
Salsola drummondii Ulbr. is a leaf succulent perennial halophyte from the salt deserts of southern Balochistan, Pakistan. This plant is a source of soda ash, medicines and forage for local populations. It may also be utilized to minimize the soil erosion in barren saline lands and for the improvement of saline soil. Information about the ecology and stress tolerance mechanisms of this important plant is limited. Therefore, I conducted this research study to understand the survival mechanisms of Salsola drummondii under natural habitat conditions using laboratory and field studies. Vegetative growth of this plant was slow due to the water scarcity, soil salinity and extreme temperatures.
Flowering occurred during September-October and fruits appeared in December. Seed dispersal started in January with the help of perianth (anemochory), thus a transient seed bank was observed.
The mature non-dormant seeds of S. drummondii were partially photoblastic and showed quick germination (within ~50 minutes) in absence of salinity over a broad range of temperatures under laboratory conditions. Seeds were highly salt tolerant and some seeds germinated in 1000 mM NaCl solution. Exogenous application of different chemicals, especially fusicoccin, improved seed germination under saline conditions, while fusicoccin and GA4 alleviated dark-enforced dormancy. Rate of seeds water uptake decreased with increase in salinity which coincided with decrease in seed germination but not with MDA accumulation and induction of antioxidant enzyme activities. Non-optimal temperatures (10/20 and 25/35 oC) and absence of light although reduced seed germination but not the water uptake compared to optimal (20/30 oC) temperature and
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12-h photoperiod, respectively. Generally higher MDA and antioxidant enzyme activities were evident in seeds at non-optimal temperature and in dark. Salsola drummondii seedlings grew optimally at moderate salinity (200 mM NaCl) with no oxidative stress.
However, high salinity (400 mM NaCl) severely affected the seedling growth and vitality by accelerating ROS production beyond the capacity of antioxidant defense system to detoxify them.
Field studies revealed that the Salsola drummondii at the study site was stable and well adapted for hyper-arid saline conditions. Variations in phenological and physiological traits coincided with the temporal changes in ambient and edaphic parameters. Summer (April to June) was unfavorable period when there were high ambient temperatures and soil salinity along with low precipitation and soil moisture.
Plants accumulated more inorganic and organic solutes in their leaves for osmotic adjustment in summer. Decreased photosynthesis in summer appears a tradeoff to increase water use efficiency. Reduction in photosynthesis in summer coincided with higher antioxidant enzyme activities to cope with oxidative stress. Changes in different eco-physiological parameters, as observed in this study appear important for survival of
S. drummondii plants under field conditions.
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