Varietal Variation in Physiological and Biochemical Traits of Durum Wheat Genotypes under Salinity Stress 

The world human population is increasing day by day and estimated to reach 8.0 billion by 2025 and 8.9 billion by 2050. To meet the demand of increasing population, there is need to double world food production by 2025 in order to feed the world (UNFPA 2015). Abiotic stresses mainly drought, salinity, heat, chilling and other factors adversely affected germination, growth, crop productivity and reported potential yield loss were 17, 20, 40, 15 and 8% respectively (Ashraf and Harris 2004). Salinity is a very detrimental stress limiting plant performance and productivity in most arid and semi-arid areas of the world. Salinity stress interferes with the normal physiological processes causing membrane damage, nutrient imbalance, altered levels of growth regulators, enzymatic inhibition and metabolic dysfunction, including photosynthesis which ultimately leads to plant death (Mahajan and Tuteja 2005; Kumar et al., 2018a; Mann et al., 2019a). In many arid and semi-arid areas of the world where sustainability of agriculture is limited by salinity, use of biological potential may be a key component of sustainable plant production (Yadav et al., 2020; Sheoran et al., 2021). Wheat is an important source of energy and protein and generally considered as moderately tolerant crop to salinity stress with threshold without yield loss at 6 dS m^-1 (Mass and Hoffmann 1977; Munns et al., 2006). Durum wheat is always considered as a less tolerant to bread wheat (Munns and James 2003; Munns and Tester 2008) and It has also been reported in literature that there is no yield reduction of durum wheat with irrigation of up to 5.7 dS m^-1 (Ayers and Westcot 1985; Royo and Abio 2003). Thus, understanding the effects of salinity on physiological and biochemical traits becomes indispensable for wheat improvement programs which have depended mainly on the genetic variations present in the wheat genome through conventional breeding. Taking this into consideration the present study was carried out to assess the variability for physico-biochemical traits on five durum wheat genotypes.

To evaluate the salinity tolerance of durum wheat genotypes, an experiment involving five durum wheat genotypes viz; HI 8737, HD 4728, HD 4730, MACS 3972 and HI 8708 and two levels of salinity i.e. normal water (Control) and saline water (EC_iw -10.0 dSm^-1) was conducted in randomized block design with three replications during 2018-2019 and 2019-2020. The experiment was conducted in net house of Division of Crop Improvement, ICAR-Central Soil Salinity Research Institute, Karnal, Haryana, India. The net house was covered with a high quality transparent polythene sheet to avoid the rain water entry and to maintain the desired salinity as per treatments. The observations on different physico-biochemical parameters such as chlorophyll content, osmolytes, Na⁺/K⁺ and antioxidative enzymes were recorded in roots as well as shoots at the vegetative stage. Chlorophyll content was estimated according to the method of Hiscox and Israelstam (1979) using dimethyl sulfoxide (DMSO). Chlorophyll content was expressed in mg g^-1 FW according to Welburn (1994) formula. Osmolytes particularly total soluble sugars, proteins and proline were assessed in roots as well as shoots. Total soluble sugars were determined using anthrone reagent by the method of Yemn and Willis (1954). Bradford reagent was used for estimation of total soluble proteins (Bradford 1976). Proline content (mg g^-1 fresh weight) was estimated by using the method of Bates et al., (1973) using 3% sulphosalicylic acid. For ionic analysis, the plants were uprooted and washed with distilled water to remove dust and salt particles. Oven dried and finely ground root and shoot (100 mg each) were digested separately with 10 ml of HNO₃:HClO₄ (3:1) di-acid mixture and measurements were taken on flame photometer (Systronics Flame Photometer 128) using standard NaCl and KCl. The activities of different antioxidative enzymes i.e. CAT (catalase), APX (ascorbate peroxidase), SOD (superoxide dismutase) and POX (peroxidase) were recorded in roots and shoots by following the standardize methods of Aebi (1984) for CAT, Nishikimi et al., (1972) for SOD, Nikano and Asada (1981) for APX and Shannon et al., (1966) for POX. Data was analyzed using factorial RBD for two factors. For critical difference (CD), treatments and genotypes were compared using at 5% level of significance with the help of OPSTAT software (CCS HAU, Hisar).

Significant variability was observed in the response of durum wheat genotypes for physiological and biochemical traits (Table 1). The highest and significant genotypic variability for chlorophyll content was recorded in genotype HD 4728 (1.36 mg g^-1) followed by HD 4730 (1.29 mg g^-1), HI 8737 (0.99 mg g^-1) and MACS 3972/HI 8708 (0.98 mg g^-1). Salinity of 10 dS m^-1 water caused 26.36% reduction in the chlorophyll content in comparison to control. This reduction in the content might be due to inhibition in the activity of ALA synthase or due to increased chlorophyllase activity (Singh et al., 2016, Mann et al., 2019a). Roots are actually the first important organ that has sense the salt stress and lead to alteration in plant development, mineral distribution and membrane variability resulting from calcium dislocation by sodium and membrane permeability (Lata et al., 2019a). For assessing response of the plant to abiotic stresses, osmolyte accumulation is a vital physiological index (Pooja et al., 2019). Among osmolytes, total soluble sugars, proteins and proline are the important ones. Among five genotypes, HI 4730 had accumulated higher TSS (4.68 mg g^-1) in shoot while HI 8737 in roots (1.40 mg g^-1). Salinity stress limits the accumulation of TSS in roots whereas it enhanced the TSS accumulation in shoot to avoid the negative effects on the plant performance as soluble sugar plays a key role in osmoregulation, controlling water potential and osmotic potential and acting as a key component of the source-sink partitioning between different organs in the plant cells (Kumar et al., 2015; Pooja et al., 2019 and Lata et al., 2019b). Salinity stress reduced root TSS content by 53.8% while enhanced shoots TSS by 15.9%. Proteins may also contribute to osmotic adjustment (Lata et al., 2017) as these are the potential source of nitrogen. The present results revealed significant differences in the accumulation pattern of root and shoot proteins, genotype HI 8737 had highest protein content in root (2.92 mg g^-1) as well as shoot (6.73 mg g^-1), while HD 4728 had the lowest content (2.37 and 5.86 mg g^-1), respectively. Salinity stress caused dual response i.e. increased accumulation of proteins in root portion by 45.6% and decreased shoot proteins by 33.9% (Table 1). Decreased protein biosynthesis is common phenomenon under the stress conditions which could also commenced with the synthesis of preferential specific stress proteins necessary for tolerating the effect of salinity (Kumar et al., 2015; Pooja et al., 2017; Mann et al., 2019b). Another important osmolyte, proline that could act as a signalling molecule which activates the adaptation response under the stress conditions (Mann et al., 2015; Kumar et al., 2017). Variability was recorded among the genotypes but stress condition enhanced the accumulation of proline in both root (104.2%) and shoot (121.3%). Increased accumulation of proline in roots as well as shoot portion indicated the response of wheat genotypes for counteracting the adverse effects of toxic salt ions in cell vacuoles (Kumar et al., 2016). Accumulation of these compatible solutes in the cytoplasm is regarded as a key strategy for osmotic adjustment by the plants to endure the salt stress. One of the deleterious effects of high salinity stress is manifested as nutrient imbalance like high soil Na⁺ concentrations reduce the amounts of available K⁺, Mg⁺⁺ and Ca⁺⁺ for plants resulting in Na⁺ toxicity on one hand and deficiencies of essential cations on the other (Kumar et al., 2018). Present results revealed that wheat genotypes had higher shoot Na⁺/K⁺ than root Na⁺/K⁺ (Table 1). Root Na⁺/K⁺ was lowest in HD 4728 followed by HI8737> MACS3972 > HD 4730 and HI 8708, whereas shoot Na⁺/K⁺ showed this pattern i.e. HI8737<MACS3972<HI 8708<HD 4728<HD 4730 (Table 1). Salinity stress significantly enhanced Na⁺/K⁺ in both root and shoot (0.73 and 1.82) in comparison to their control (0.13 and 0.27). Salinity enhanced Na⁺ content in plants which normally lead to a reduction of K⁺ levels, since the two cations compete for the same binding sites and Na⁺ interferes with K⁺ uptake by block K⁺ specific transporters (Mann et al., 2015). Leaves are the last sink and the most sensitive part of the plant in contact with the atmosphere where salt accumulates and the mechanisms by which these genotypes maintained relatively low cellular Na⁺/K⁺ seem to be important for salt tolerance (Kumar et al., 2015; 2017).
 

       
Salinity stress employs several symptoms, such as production of reactive oxygen species (ROS), limited growth and yield, similar to those observed under other abiotic stresses. For maintenance of normal growth of plants, the ROS need to be scavenged (Mann et al., 2015; Rani et al., 2018). Various antioxidative enzymes i.e. SOD (superoxide dismutase), CAT (catalase), POX (peroxidase) and APX (ascorbate peroxidase), play an important role in scavenging of ROS. The first enzyme which act against ROS is SOD which converts O₂- (ROS) ­to H₂O₂. In present study, the SOD activity of all genotypes increased significantly with salinity as compared to control in leaf as well as in root tissue (Table 2). In leaf tissue, the highest SOD activity was observed in genotype MACS 3972 (284.0 units mg^-1 protein) and lowest in genotype HD 4728 (228.5 units mg^-1 protein). In case of root tissue, the highest SOD activity was observed in genotype HD 4728 (134.89 units mg^-1 protein) and lowest was in genotype MACS 3972 (68.36 units mg^-1 protein). Overall, the SOD activity of leaf tissue in all genotypes was higher as compared to root tissue. The CAT and APX activity were also significantly increased under salinity stress in leaf and root tissue of all genotypes. In leaf tissue, the higher activity for APX was found for genotypes - HI 8737, HD 4728 and MACS 3972 and lower for genotypes - HD 4730 and HI 8708. Similarly, highest activity of CAT was observed in genotypes - MACS 3972 and lowest CAT activity was found in genotype - HI 8738 (Table 2). In root tissue, the CAT and APX activity was detected higher in genotypes - MACS 3972 and HD 4730. Similarly, the lower activity of these enzymes was observed in the HI 8708 genotype.
 

       
Another important enzyme i.e. peroxidase, activity was also varied significantly among the genotypes (Table 2). The POX activity in leaf tissue of all genotypes was higher as compared to root tissue. It was observed that in leaf tissue, the POX activity was higher for two genotypes i.e. HD 4728 and HD 4730, as there was no significant difference. In similar manner, the POX activity in root tissue of genotype HD 4730 was found higher among other genotypes of wheat. The lower POX activity was found for the genotype HI 8708 in both leaf as well as root tissue. In general, the POX activity was found increased in higher salinity as compared to control. The increase in the activity of plant antioxidant system has positive relation with the decrease in oxidative damage and enhancement in tolerance to salinity (Sharma et al., 2013; Mann et al., 2019b; Elkelish et al., 2019; Sheng et al., 2019; Pooja et al., 2020).
 
Co-relation coefficient analysis
 
Results from correlation studies revealed significant association of root and shoot traits under salinity stress (Table 3). Among shoot traits, highest positive correlation was observed for SOD activity between proline content (0.954**) and Na⁺/K⁺ (0.95**) whereas proline content depicted highest negative correlation with protein content (-0.918**). Among root traits, highest positive correlation was noted between Na⁺/K⁺ and POX activity (0.941**), proline content and protein content (0.901**). TSS content showed negative correlation with all the studied root traits. Significant positive correlation was also observed between Na⁺/K⁺ and antioxidative enzymes which illustrated the mechanism to protect the plant from salinity stress induced oxidative damage.
 

       
In present study, the increased activities of antioxidative enzymes in shoot and root tissues of all wheat genotypes showed the salinity tolerance. Wheat genotypes exhibited the variable response to salinity. The genotype which showed the higher antioxidative enzyme activities with higher osmolytes accumulation might have higher salt tolerance potential and vice-versa. Based on the activities of antioxidative enzymes in shoot and root tissues, it was observed that the genotype MACS 3972 had most efficient antioxidant system against oxidative stress and genotype HI 8708 consisted of less. Hence, it can be concluded that the genotype MACS 3972 has potential to grow in saline conditions and it could be valuable for crop improvement programme.

The authors are thankful to Head, Division of Crop Improvement and Director, CSSRI, Karnal for providing necessary facilities for the research work.