INTRODUCTION
Abiotic stress is a major cause of crop loss worldwide. Drought is one of the greatest abiotic stress conditions limiting crop productivity (Ashraf, Reference Ashraf2010), particularly in arid and semi-arid regions of the world (Blum, Reference Blum2005). Water stress adversely affects a variety of vital physiological and biochemical processes in plants such as reducing stomatal conductance, photosynthesis and enhancing pigment degradation (Hasanuzzaman et al., Reference Hasanuzzaman, Hossain, da Silva, Fujita, Bandi, Shanker, Shanker and Mandapaka2012). Extensive studies on stress have demonstrated that exposure of plants to water stress induces overproduction of reactive oxygen species (ROS), such as the superoxide radical, hydrogen peroxide and hydroxyl radical in plant cells (Farooq et al., Reference Farooq, Wahid, Kobayashi, Fujita and Basra2009). ROS are highly reactive to membrane lipids, protein and DNA, causing oxidative damage and impairing normal functions of cells. These are believed to be the major contributing factors to stress injuries and to cause rapid cellular damage. To minimize adverse effects of oxidative stress, plants have evolved complex enzymatic and non-enzymatic antioxidant systems such as low molecular mass antioxidants (glutathione, ascorbate, carotenoids) and ROS scavenging enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APOX)) (Apel and Hirt, Reference Apel and Hirt2004). Balance between ROS production and activities of antioxidant enzymes determines the occurrence of oxidative signalling and/or damage (Moller et al., Reference Moller, Jensen and Hansson2007). The enhancement of antioxidant defence mechanisms is considered to be an adaptive mechanism of plants to drought stress, and the strengthening of these defense mechanisms through the enhanced functions of antioxidant components may reduce or prevent oxidative damage and improve the drought resistance of plants (Hasanuzzaman et al., Reference Hasanuzzaman, Hossain, da Silva, Fujita, Bandi, Shanker, Shanker and Mandapaka2012).
Exogenous application of stress alleviating agents is an important means to improve stress tolerance in field crops (Farooq et al., Reference Farooq, Wahid, Kobayashi, Fujita and Basra2009). The -SH containing compounds, such as thiourea (TU) and thioglycolic acid (TGA), are an important group of stress alleviating substances. Several studies support the major role of -SH compounds in modulating plant response to various abiotic stress conditions. These are reported to decrease denaturation of membrane protein (Rikin et al., Reference Rikin, Atsmon and Gitler1979), improve translocation of sucrose from source to sink via phloem loading (Srivastava et al., Reference Srivastava, Nathawat, Ramaswamy, Sahu, Singh, Nair, Paladi and D’Souza2008), enhance photosynthetic pigments (D’Souza et al., Reference D’Souza, Nathawat, Nair, Radhakrishna, Ramaswamy, Singh and Sahu2009), net photosynthetic rate (Burman et al., Reference Burman, Garg and Kathju2004) and N-metabolism (Garg et al., Reference Garg, Burman and Kathju2006) and decrease stress-induced drop in photo-system I (PS I) and photo-system II (PS II) (Ramaswamy et al., Reference Ramaswamy, Nathawat, Nair, Sharma, Kumawat, Singh, Sahu and D’Souza2007) in plants under stress conditions.
Clusterbean (Cyamopsis tetragonoloba L. Taub) is an annual legume crop belonging to family Leguminosae. It is mainly cultivated in north-western India, Pakistan, Sudan and the southern USA (Texas and Oklahoma). It is a major source of galactomannans, which are used as thickening, gelling and suspending agents, viscosifier and emulsion stabilizers in paper, textile, paint, drilling, oil operation, civil engineering, agrochemistry, food, cosmetics and pharmaceutical industries (Gresta et al., Reference Gresta, Sortino, Santonoceto, Issi, Formantici and Galante2013). India is a major producer of clusterbean, and contributes to 80% of the world's total production of clusterbean (Pathak et al., Reference Pathak, Singh and Henry2011), which is 0.75 to 1.0 million tonne annually. In India, Rajasthan alone contributes 70% of India's total production. The bulk of clusterbean production is generated from the north-western hot arid region. There the crops are frequently prone to water deficit stress due to low and erratic rainfall (Garg et al., Reference Garg, Burman and Kathju2006).
To date, very little information is available regarding the effects of -SH compounds on antioxidant enzyme activities, lipid peroxidation, gas exchange parameters and yield of clusterbean under water limiting conditions. Therefore, the present field experiments were conducted with objectives to assess growth, yield, gas exchange parameters and antioxidant enzyme activities under moisture limiting conditions in clusterbean. This paper reports the results of two experiments that tested the hypothesis that exogenous application of -SH compounds could improve growth, yield, gas exchange parameters and activities of antioxidant enzymes of clusterbean under a water limiting environment.
MATERIALS AND METHODS
Study environment
Field experiments were conducted during two kharif seasons (July–November) in 2010 and 2011 at the Central Arid Zone Research Institute, Regional Research Station, Bikaner (28°4′ N; 74°3′ E; 238.3 m above mean sea level), Rajasthan, India. The climate of the experimental site is hot arid with an average annual precipitation of 287 mm. More than 85% of the total annual rainfall is received during the south-west monsoon season (July to September). The weather data for the crop growing seasons during the two-year experiment are presented in Table 1. The soil was Torripssamentes typic with the following key properties for the 0–20-cm layer: pH (soil/H2O, 1:2.5): 8.5, organic carbon: 1.5 g kg−1 (Walkley–Black), available P: 0.004 g kg−1 (Olsen) and available K: 0.107 g kg−1 (1 N NH4-acetate). Texture was loamy sand, with sand (2000–50 μm), silt (50–2 μm) and clay (<2 μm) content of 864, 61 and 85 g kg −1 respectively.
Table 1. Weather data for the cropping period at Bikaner, Rajasthan, India.
Treatments and experimental design
The treatments included two -SH compounds and their concentrations, namely TGA (200, 300 and 400 mg L−1) and TU (500, 750 and 1000 mg L−1). An additional untreated control (water spray) was also included in the study. Thus, there were seven treatments. The experiment was laid out in a randomized complete block design (RCBD) with three replications. Two foliar sprays of -SH compounds were applied at vegetative and pre-flowering stages of crop. Size of each plot was 5.0 × 3.5 m with 2-m gap in-between. Each plot was bordered with an earth dike of 30 cm in height. Overall, there were 21 plots for the whole experiment.
Crop management practices
The land was cleared manually with minimal soil disturbance in both years of the study. Prior to the pre-planting tillage operations during the growing seasons of the two years, thoroughly mixed farm yard manure (containing 0.33% N, 0.21% P and 0.39% K) was applied uniformly over the entire field at 2.5 M g manure ha−1. After receiving adequate monsoon rain, the land was prepared by a tractor-drawn disc harrow. Clusterbean (cultivar RGC-1003) was sown on 1 August and 30 July in 2010 and 2011, respectively. In each plot, 20-cm deep furrows were created with the help of manual drawn plow and seeds were sown uniformly at ~10-cm depth. A basal dose of 10 kg N ha−1 (as urea) and 20 kg P ha−1 (as single superphosphate) was applied at sowing. Fluchloralin (45 EC) at a rate of 1 L ha−1 was applied before the sowing of the crop to control weeds.
Leaf sampling and determination of physio-biochemical traits
Leaf samples were taken at flowering stage (50 days after sowing (DAS)) from two plants per plot. Completely developed third and fourth leaves from the top of the plant were used for all measurements. Leaves were collected on ice between 0930–1030 h, and taken to the laboratory, washed with distilled water, and excess water was removed.
Determination of antioxidant enzyme activities
Leaf samples (0.5 g fresh weight) were homogenized in ice-cold 50-mM potassium phosphate buffer (pH 7.0) containing 0.1-mM ethylenediaminetetraaceatic acid (EDTA) and 1% (w/v) polyvinylpolypyrolidone. The homogenate was filtered through four layers of cheese cloth, and centrifuged at 4 °C for 20 min at 15,000 × g. The supernatant was collected and an appropriate aliquot dilution of the crude extract was used for enzyme assays. Complete operation of enzyme assays was carried out at room temperature (23 ± 1 °C) unless otherwise stated. The enzyme assay was performed for three replications.
The CAT activity was assessed following the method described by Chance and Maehly (Reference Chance and Maehly1955). CAT activity was measured following the decomposition of H2O2 at 240 nm (coefficient of absorbance, ɛ = 39.44 mM−1 cm−1) in a reaction mixture (1 ml) contained 680 μL of 50-mM phosphate buffer (pH 7.0), 300 μL of 200-mM H2O2 and 20 μL of enzyme extract. CAT enzyme specific activity is expressed as μmol H2O2 decomposed mg−1 (protein) min−1.
The SOD activity was determined by the method of Becana et al. (Reference Becana, Aparicio-Tejo, Irigoyen and Sanchez-Diaz1986) based on the photochemical inhibition of p-nitro blue tetrazolium chloride (NBT). Reaction mixture (3.0 ml) contained 50-mM phosphate buffer (pH 7.8), 0.1-mM EDTA, 14.3-mM methionine, 82.5-μM NBT, 2.2-μM riboflavin and 50 μL of enzyme extract. Test tubes were irradiated under six 15-W florescent tubes for 30 min. The photo-reduction of NBT was measured at 560 nm with a UV/visible spectrophotometer. Blanks and controls were run in the same manner but without illumination and enzyme respectively. One unit of SOD activity was defined as extract volume that caused 50% inhibition of the photo-reduction of NBT.
The APOX activity was assayed by following decrease in absorbance at 290 due to ascorbate oxidation (ɛ = 2.8 mM−1 cm−1) in a reaction mixture (1 ml) contained 530 μL of 50-mM phosphate buffer (pH 7.0), 200 μL of 0.5-mM ascorbic acid, 200 μL of 0.1-mM H2O2, 50 μL of 0.1-mM EDTA and 20 μL of enzyme extract for 1 min following the method of Nakano and Asada (Reference Nakano and Asada1981). APOX enzyme-specific activity is expressed as μmol ascorbate oxidized mg−1 (protein) min−1.
For guaiacol peroxidase (GPOX), the oxidation of guaiacol was measured by following the increase in absorbance at 470 nm (ɛ = 26.6 mM−1 cm−1) for 1 min. The assay mixture (1 ml) contained 530 μL of 50-mM phosphate buffer (pH 7.0), 50 μL of 0.1-mM EDTA, 200 μL of 10-mM guaiacol and 200 μL of 10-mM H2O2, and 20 μL of enzyme extract for 1 min as described by Chance and Maehly (Reference Chance and Maehly1955). The GPOX activity is expressed as μmol (tetraguaiacol formed) mg−1 (protein) min−1.
The glutathione reductase (GR) activity was measured as described by Shaedle and Bassham (Reference Shaedle and Bassham1977) by following decrease in absorbance at 340 nm (ɛ = 6.2 mM−1 cm−1) for 1 min. The reaction mixture (1 ml) contained 655 μL of 50-mM Tris-HCl buffer (pH 7.5), 125 μL of 0.5-mM oxidised glutathione (GSSG), 50 μL of 0.1-mM EDTA, 50 μL of 3-mM MgCl2, 100 μL of 0.15-mM NADPH and 20 μL of enzyme extract. The GR activity was expressed as μmol (NADPH oxidized) mg−1 (protein) min−1.
Determination of lipid peroxidation
The method of Cakmak and Horst (Reference Cakmak and Horst1991) was followed for the measurement of lipid peroxidation products in terms of 2-thio-barbituric acid reactive substances (TBARS), and expressed as equivalents of malondialdehyde (MDA). Fresh leaf sample (0.5 g) was ground in 10 ml of 10% tri-chloroaceatic acid (TCA) at 4 °C and centrifuged at 10,000 × g for 5 min. The 1-ml aliquot was taken and added to 4 ml of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA. It was then cooled in an ice bath and centrifuged at 10,000 × g for 5 min. The absorbance was then recorded at 532 and 600 nm. The MDA content was determined using the following formula:
$$\begin{equation*}
{\rm MDA }( {{\rm \mu mol g}^{-1} {\rm FW}})\; = \;\left[ {(A532 - A600)\; \times \;V\; \times \;1000/\varepsilon } \right]\; \times \;W,
\end{equation*}$$
where ɛ is the specific extinction coefficient (ɛ = 155 mM−1 cm−1), V is the 10-ml volume of grinding medium, W is the fresh weight of leaf, and A600 and A532 are the absorbance at 600- and 532-nm wavelengths respectively.
Determination of membrane stability index (MSI)
The MSI of leaf samples was determined following the procedure described by Sairam et al. (Reference Sairam, Rao and Srivastava2002). Leaf samples (0.1 g) were cut into discs of uniform size and placed in 10 ml of double-distilled water in two sets. One set was kept at 40 °C for 30 min and its conductivity (C 1) was recorded. The second set was kept in boiling water bath (100 °C) for 10 min and its conductivity (C 2) was recorded. The conductivity was measured with a conductivity meter. MSI was calculated using the following formula: MSI = [1 – (C 1/C 2)] × 100.
Determination of photosynthetic pigments
Chlorophyll and carotenoid contents were extracted by the non-maceration method (Hiscox and Israelstam, Reference Hiscox and Israelstam1979). Fresh leaves (0.05 g) were extracted in 10-ml dimethyl sulfoxide (DMSO) for 65 °C for 4 h. The amount of chlorophyll a, b and carotenoids was determined spectrophotometrically by reading the absorbance at 645, 663 and 470 nm respectively (Arnon, Reference Arnon1949).
Determination of gas exchange parameters
Gas exchange parameters, i.e. net photosynthetic rate (P N), stomatal conductance (g s) and transpiration rate (E) were measured in vivo with a portable photosynthesis system (TPS-2 CO2 Gas Analyzers, USA). Gas exchange measurements were made on fully expanded leaves located in the upper third part of the canopy. Three plants were selected randomly from the central 1 × 1-m area from each plot to measure gas exchange parameters. Measurements were done in sunny and clear weather 0900 and 1100 h. The instantaneous water use efficiency (WUE) was determined as P N/E (Nogueira et al., Reference Nogueira, Martinez, Ferreira and Prado2004).
Determination of growth, yield attributes and yield
Ten plants were randomly selected from the central 2 × 2-m area of each plot to measure leaf area (LA) and dry matter (DM) accumulation per plant at 50, 60 and 70 days after sowing. Leaf area index (LAI) was calculated as the ratio of leaf area to ground area. Yield components, i.e. number of pods plant−1 (NP), number of seeds pod−1 (NS) and 1000-seed weight (SW) were recorded for all the plants from the central 1 × 1-m area of each plot at harvest. At the harvest maturity stage of the crop (on 4 November in 2010, and 29 October in 2011), seed yield (SY) and total above-ground biomass (ABY) were determined on an area of 2 × 2 m from each plot by manual harvesting of plants 3 cm above the ground and allowed to dry in the field. DM of seed and straw was determined by drying sub-samples in a convection oven at 65 °C to a constant weight.
Statistical analysis
All measured parameters were tested for significant differences between treatments using analysis of variance (ANOVA) for a randomized complete block design. Wherever significant, separation of treatment means was achieved by the procedure of least significant difference (LSD) as described by Gomez and Gomez (Reference Gomez and Gomez1984); p ≤ 0.05 was used as a critical limit for distinguishing the degree of variance between means. Pearson correlation coefficients were used to evaluate relationships between different parameters. The treatment × year interaction was not significant for any trait measured.
RESULTS
Climatic condition
The weather data during the cropping periods are presented in Table 1. The rainfall received after sowing of crops was 244 mm and 200 mm during 2010 and 2011 respectively. During both years, there was no rainfall for 45 days of sowing, and thus crops experienced prolonged terminal water deficit stress. The average monthly maximum temperatures from July to October were 34.3 °C and 33.7 °C for 2010 and 2011 respectively.
Antioxidant enzyme activities
The activities of antioxidant enzymes (CAT, APOX, GPOX, SOD and GR) measured at 50 DAS are presented in Figure 1. Exogenous application of -SH compounds significantly (p ≤ 0.05) enhanced the activities of antioxidant enzymes. Compared with non-treated plants, the -SH-treated plants showed 17–42, 21–51, 23–46, 19–44, 32–57% higher activities for CAT, APOX, GPOX, SOD and GR respectively. The effects of -SH application on antioxidant enzyme activities was chemical- and concentration-specific. For instance, the highest activity of CAT, APOX and GPOX were recorded for TGA 400 mg L−1-treated plants, whereas TU 1000 mg L−1-treated plants showed the highest activity of SOD and GR enzymes.
Figure 1. Effects of foliar application of -SH compounds on (a) SOD, (b) APOX, (c) GPOX, (d) GR and (e) CAT antioxidant enzyme activities. Columns with different letters indicate that they differ significantly (p ≤ 0.05).
MDA content and MSI
The MDA content and MSI varied significantly (p ≤ 0.05) among tested treatments (Figures 2a and b). Application of exogenous -SH compounds significantly (p ≤ 0.05) decreased MDA content and increased MSI. Averaged across the years, the mean MDA contents for -SH-treated plants were 10 to 19% lower, and MSI was 11 to 18% higher than the untreated plants. The TGA 400 mg L−1-treated plants had the highest MSI and the lowest MDA contents in both the years.
Figure 2. Effects of foliar application of -SH compounds on (a) MSI, (b) MDA content, (c) chlorophyll a, (d) chlorophyll b, (e) total chlorophyll and (f) carotenoid content. Columns with different letters indicate that they differ significantly (p ≤ 0.05).
Photosynthetic pigments
The effects of application of -SH compounds on chlorophyll contents and carotenoids are presented in Figures 2c–f. Application of -SH compounds significantly (p ≤ 0.05) improved the contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid during both the years. The -SH-treated plants showed greater increment in chlorophyll content relative to carotenoid content, and for chlorophyll a than chlorophyll b. Averaged across both the years, -SH application had 23–38, 11–17, 18–30 and 9–16% greater chlorophyll a, chlorophyll b, total chlorophyll and carotenoid contents, respectively, compared with untreated plants. The greatest increments in the contents of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoid) were recorded for 1000 mg L−1 TU.
Gas exchange parameters
Effects of application of -SH compounds on gas exchange parameters are presented in Figure 3. The g s had a range of 394–561 mmol m−2 s−1. Averaged across the years, the -SH-treated plants had 17 to 42% greater g s (Figure 3a) and 11–24% higher E values (Figure 3b) than untreated plants. Averaged across the years, the P N value varied from 11 to 15 μmol m−2 s−1. Exogenous -SH compounds significantly (p ≤ 0.05) increased P N, and -SH-treated plants had 1.2 to 1.4 times greater P N value compared with untreated plants. Among -SH compounds tested, TGA-treated plants showed greater increments in P N relative to TU-treated plants (Figure 3c). The WUE, measured as the ratio between P N and E, showed significant (p ≤ 0.05) response to -SH application, and -SH-treated plants exhibited 10–14% higher values of WUE than that of untreated plants (Figure 3d). The plants treated with 400 mg L−1 TGA had the greatest P N, g s and E values followed by TU 1000 mg L−1.
Figure 3. Effects of foliar application of -SH compounds on (a) g s, (b) E, (c) P N and (d) WUE. Columns with different letters indicate that they differ significantly (p ≤ 0.05).
Growth attributes
Foliar application of -SH compounds had significant effects (p ≤ 0.05) on LAI and DM measured at 50, 60 and 70 DAS during both the years (Table 2). The LAI varied from 1.5 to 2.0, 2.2 to 3.0 and 2.4 to 3.2 at 50, 60 and 70 DAS respectively. Compared with untreated plants, the -SH-treated plants had 16–28, 20–34 and 21–33% higher LAI at 50, 60 and 70 DAS respectively. The -SH application significantly (p ≤ 0.05) improved DM accumulation. Averaged across both the years, the -SH-treated plants had 12–19, 21–32 and 25–38% higher DM production than untreated plants measured at 50, 60 and 70 DAS respectively.
Table 2. Effects of foliar application of -SH compounds on leaf area index (LAI) and dry matter (DM) accumulation of clusterbean grown during 2010 and 2011 at Bikaner, Rajasthan, India.
Values followed by different letters in each column are significantly different at p ≤ 0.05 according to least significant difference (LSD).
TGA: thioglycolic acid; TU: thiourea.
Yield components and yields
Effects of application of -SH compounds on yield components (NP, NS and 1000-seed weight) and yields (SY and ABY) are presented in Table 3. Application of -SH compounds had significant effects on NP and NS during both the years. The NP varied from 87 to 114 plant−1 and SN ranged from 6.9 to 8.2 per pod. Averaged across both the years, -SH compounds-treated plants had 16–23% higher NP and 8–14% higher NS than untreated plants. The -SH application failed to effect significant difference in 1000-seed weight during either year.
Table 3. Effects of foliar application of -SH compounds on yield components and clusterbean yield grown during 2010 and 2011 at Bikaner, Rajasthan, India.
Values followed by different letters in each column are significantly different at p ≤ 0.05 according to least significant difference (LSD).
TGA: Thioglycolic acid; TU: Thiourea; NP: pod number per plant; NS: seed number per plant; SY: seed yield; ABY: aboveground biomass yield.
Seed yield varied from 1006–1247 kg ha−1 and 968–1151 kg ha−1 during 2010 and 2011 respectively. Application of -SH compounds significantly (p ≤ 0.05) increased SY in both the years (Table 3). Averaged across the years, application of -SH compounds gave 14 to 22% higher SY than control (untreated) plants. Compared with untreated plants, TGA 400 mg L−1-, TU 1000 mg L−1-, TGA 300 mg L−1-, TU 750 mg L−1-, TU 500 mg L−1- and TGA 200 mg L−1-treated plants gave 212, 202, 190, 188, 154 and 142 kg ha−1 higher SY respectively. ABY varied from 3652 to 4818 kg ha−1. Averaged across both the years, the -SH-treated plants had 495 to 871 kg ha−1 greater ABY than control.
Correlation among measured parameters
Correlation coefficients among DM (at 70 DAS), MDA content, MSI, total chlorophyll content, antioxidant enzyme activities and gas exchange parameters are shown in Table 4. DM was positively correlated with the activities of antioxidant enzymes, TCHL content, gas exchange parameters, and negatively correlated with MDA content. MDA content was negatively correlated with most of the measured parameters. P N had significant positive correlation with the activities of CAT, APOX, GPOX and GR. Overall, the results indicate that activities of antioxidant enzymes are positively correlated with MSI, total chlorophyll content and gas exchange parameters, and negatively correlated with MDA content.
Table 4. Correlations between dry matter accumulation at 70 DAS (DM70 DAS), malondialdehyde (MDA) content, membrane stability index (MSI), total chlorophyll content (TCHL), activity of antioxidant enzymes (CAT, SOD, APOX, GPOX and GR) and gas exchange parameters.
†Pearson correlation coefficient.
NS: non-significant; *significant at p ≤ 0.05; **significant at p ≤ 0.01.
DM (70 DAS): dry matter at 70 DAS; CAT: catalase; SOD: superoxide dismutase; APOX: ascorbate peroxidase; GPOX: guaiacol peroxidase; GR: glutathione reductase; P N: net photosynthetic rate; g s: stomatal conductance; E: transpiration rate.
DISCUSSION
Drought is one of the most serious production constraints for world agriculture (Mir et al., Reference Mir, Zaman-Allah, Sreenivasulu, Trethowan and Varshney2012). The exogenous application of stress alleviating substances has been advocated as an effective option to achieve sustainable yields under water deficit conditions. An important requirement for determining efficacy of stress alleviating substances in plants is a proper comparison of biochemical parameters among the plants treated with these chemicals.
Exposure of plants to environmental stress conditions leads to the generation of ROS, which causes oxidative damage. The equilibrium between production and scavenging of ROS may be perturbed by a number of adverse abiotic stress factors (Apel and Hirt, Reference Apel and Hirt2004). Our results showed significant enhancement in the activity of antioxidant enzymes with application of exogenous -SH compounds (Figure 1).
Enhancement of antioxidant enzymes with the application of exogenous -SH compounds might be attributed to increased synthesis of enzymes (i.e., glutathione is a thiol containing compound, which is an essential cofactor for GPOX) due to supply of thiol (-SH) containing compounds (Loggini et al., Reference Loggini, Scartazza, Brugnoli and Navari-Izzo1999). Furthermore, higher photosynthetic activity (Figure 3) for -SH-treated plants increases soluble sugar content, which could stabilize the structure and hence result in higher activity of antioxidant enzymes (Chaves et al., Reference Chaves, Maroco and Pereira2003). The observed positive correlations among the activities of SOD, CAT, APOX, GPOX and GR in our study (Table 4) suggested that increase in SOD activity was accompanied by increase in the activities of other enzymes as a result of high demand of quenching H2O2. This increased antioxidant production, lowered the ROS-based damages in plant system, as is evident from the negative correlation between antioxidant enzymes activities and MDA content (Table 4).
Decrease in MDA content (Figure 2b) and increase in MSI (Figure 2a) observed under -SH treatments in the present study could be attributed to decreased lipid peroxidation due to enhanced activities of antioxidant enzymes (Figure 1). An increased activity of antioxidant enzymes has been correlated with decreased lipid peroxidation under stress conditions (Anjum et al., Reference Anjum, Saleem, Wang, Bilal and Saeed2012), and this relationship was confirmed by our correlation analysis (Table 4). Besides antioxidant protection, the intracellular and extracellular redox states of thiols play a critical role in the stabilization of protein structure and function, regulation of enzyme activity and control of transcription factor activity (Ramaswamy et al., Reference Ramaswamy, Nathawat, Nair, Sharma, Kumawat, Singh, Sahu and D’Souza2007).
Decrease in chlorophyll content under water deficit stress is attributed to the oxidation of chlorophyll and damaged ultra-structure of chloroplasts (Anjum et al., Reference Anjum, Xie, Wang, Saleem, Man and Lei2011). Our results indicated that application of -SH compounds significantly enhanced chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content in leaves of clusterbean under water deficit condition (Figures 2c–f). These results are in agreement with the findings of Liu et al. (Reference Liu, Li, Shang and Qi2002), who observed that application of TU reduced the rate of chlorophyll degradation during senescence; this may be responsible for the maintenance of higher chlorophyll concentration as reported in wheat (Sahu and Singh, Reference Sahu and Singh1995), and in clusterbean (Burman et al., Reference Burman, Garg and Kathju2004). This appears logical because -SH compounds (i.e., thiourea) exhibit cytokinin-like activity, and are known to delay leaf senescence (Vassilev and Mashev, Reference Vassilev and Mashev1974).
Recent studies have suggested that both diffusive limitation through stomatal closure and non-stomatal limitation (such as oxidative damage to chloroplast) are responsible for decline in photosynthesis under drought stress (Prasad, Reference Prasad, Staggenborg, Ristic, Ahuja, Reddy, Saseendran and Yu2008). Our results indicated that -SH application improved photosynthetic parameters (P N, g s and E) compared with non-treated plants. Among the studied parameters, the -SH application had the greatest improvements in P N followed by g s and E (Figure 3). Higher P N for -SH-treated plants might be explained by the higher content of photosynthetic pigments (Figure 2) and less water deficit-induced oxidative damage to metabolic activity reflected by higher activity of antioxidant enzymes (Figure 1) along with increasing flow of CO2 via higher g s (Figure 3a). This is confirmed by a correlation study, which indicated that P N was positively correlated with photosynthetic pigment contents, antioxidant enzymes activity, chlorophyll content and g s (Table 4). D’Souza et al. (Reference D’Souza, Nathawat, Nair, Radhakrishna, Ramaswamy, Singh and Sahu2009) found that -SH compounds help plants to maintain photosynthetic efficiency and delay chlorophyll degradation, besides providing antioxidant protection and stability in protein structure.
Plants treated with foliar application of -SH compounds had significantly greater growth measured in terms of LAI and DMA (Table 2). LAI, which denotes extent of radiation capture by crops under field conditions, decreases under drought. Broadly, Drought-induced reduction in LAI is ascribed to reduction in cell wall turgor and photosynthesis. Reduction in DM under drought is mainly ascribed to reduce LAI, which results in reduced light interception (Nam et al., Reference Nam, Subbaroa, Chauhan and Johansen1998). The better LAI of clusterbean treated with -SH compounds may be explained by their ability to maintain higher cell turgor and photosynthesis under moisture deficit conditions. Burman et al. (Reference Burman, Garg and Kathju2004) and Garg et al. (Reference Garg, Burman and Kathju2006) postulated that -SH compounds enhanced photosynthesis, which increased sap production in the leaf lamella, and resulted in the maintenance of relative water content (RWC) and water potential (WP) in the leaf, and hence higher cell turgor. The higher LAI coupled with higher P N might be responsible for enhanced DM accumulation (DMA) of -SH compound-treated plants.
In the present study, exogenous application of -SH compounds led to significantly higher numbers of pods and seeds per pod compared with untreated plants (Table 3). The -SH compounds can protect cell membrane and negative effects of ROS during stress, which can lead to greater production of assimilates (indicated by greater DM) and improved translocation of assimilates and apportioning to sinks (Srivastava et al., Reference Srivastava, Nathawat, Ramaswamy, Sahu, Singh, Nair, Paladi and D’Souza2008). Thus, improvement in yield components with -SH compounds may be explained by the ability of -SH compounds to increase DM production, better translocation of assimilates and apportioning of DM to sinks.
Drought mainly influences yield by limiting seed numbers by either influencing the amount of DM produced by the time of flowering or by directly influencing pollen or ovule function, which leads to decreased seed-set. Secondarily, drought influences seed filling mainly by limiting the assimilate supply, leading to smaller seed size and lower yields (Prasad, Reference Prasad, Staggenborg, Ristic, Ahuja, Reddy, Saseendran and Yu2008). Significant improvement in seed yields with -SH application in the present study could be attributed to significant improvement in yield attributes (pod number and seed per pod) due to greater photosynthesis and better partitioning of photosynthates. The enhancement of photosynthesis (Nathawat et al., Reference Nathawat, Nair, Kumawat, Yadava, Singh, Ramaswamy, Sahu and D’Souza2007; Ramaswamy et al., Reference Ramaswamy, Nathawat, Nair, Sharma, Kumawat, Singh, Sahu and D’Souza2007) and better partitioning of photosynthates to sink for various crop species treated with -SH compounds have been reported earlier under abiotic stress conditions (Sahu and Singh, Reference Sahu and Singh1995).
CONCLUSIONS
In conclusion, our results indicate that exogenous application of -SH compounds ameliorate clusterbean crop performance in a water deficit environment. This ameliorative effect was associated with the alteration of physio-biochemical processes such as increase in antioxidant enzyme activities, photosynthetic pigment content, gas exchange parameters and membrane stability, and decrease in lipid peroxidation. The study shows that foliar spray of -SH compounds (TU and TGA) can help to increase clusterbean yield under a water deficit environment. The -SH compounds can be considered as a potential growth regulator for improving crop growth and yield under limited soil moisture conditions.
Acknowledgements. This study was conducted under Institute project T 03-55 funded by CAZRI, Jodhpur. We wish to thank Director, CAZRI for providing funds and necessary facilities to carry out the study, and B.M. Yadav and Bharu Singh for their assistance in data collection and laboratory work.
