Deciphering Biochemical Responses, Metabolome Analysis and Key Genes Controlling Sorghum [Sorghum Bicolor (L.) Moench] Ion Transport in Responses to Salt Stress

Background: Crops usually encounter several abiotic stresses, and salt stress is one of the major constraints affecting plant growth and agricultural productivity globally. Sorghum is not only a valuable source of food but also a potential model for studying and better understanding the salt stress mechanics in the cereals and obtain a better knowledge of their cellular mechanism. Herein, we examined the effects of salt stress on physio-biochemical and molecular responses of sorghum genotypes to determine their tolerance. Summary: A pot experiment was conducted in a climate-controlled greenhouse with different salt stress treatments (60, 80, 100, and 120 mM NaCl). The results indicated that exposure to NaCl-induced salinity reduces growth of sorghum cultivars by damaging chlorophyll pigment and chloroplast, particularly at a higher stress level. SSG 59-3 alleviated adverse effects of salinity by suppressing oxidative stress (H 2 O 2 ) and stimulating enzymatic and non-enzymatic antioxidant activities (SOD, APX, CAT, POD, GR, GST, DHAR, MDHAR, GSH, ASC, proline, GB), as well as protecting cell membrane integrity (MDA, electrolyte status, photosynthetic rate and antioxidant potential and upregulation of ion transporter genes, thereby alleviating stress, which may be augmented as genetic resources to establish sorghum cultivars with improved quality in saline soils. in and in by . Polyamines. Poylamines in in 5 ml of 5% cold perchloric acid 108 . The supernatant, free polyamines at -20 0 C for further benzoylation reaction using 500 µl of 1 ml 2N NaOH, 10 µl of 99% ( v/v ) benzoyl chloride, incubated for 30 min and then, saturated with 2 ml NaCl. Benzoylated polyamines were extracted with diethyl ether, incubated at -20°C for h and were separated with the HPLC fitted with C 18 ODS 2 analytical column which was pre-equilibrated and run isocratically at a flow rate of 1 ml min -1 with acetonitrile: water (50: 50 v/v ) for 40 min. The injected volume was 10 µl and UV- detector was used to measure the absorbance at 224 nm. Polyamines were quantified in the eluent by comparing the integration areas obtained for the samples with those obtained for the standards and the results were express as nmol g -1 fresh weight of the sample.


Introduction
Salt stress is one of the major abiotic stresses, drastically affecting global agricultural productivity 1,2 . Salt-affected soils are estimated to comprise 23 % of the cultivated land, approximately 3.5108 ha, the global extent of saline soils to be 412 Mha, which closely agrees with the FAO 3 . The annual loss caused by soil salinization in agricultural productivity is estimated to be US$ 31 million, and the production potential of up to 46 million ha per year. Soil salinization alone has rendered significant chunks of land unproductive or less productive 4 . It is especially worrisome as urban expansion drives farming into more or less dry land, 30 % of the cultivable soils will become unusable due to salt stress, and global food needs expecting to rise by 70 % to feed over 9.7 billion people by 2050 (http://www.fao.org/wsfs/world-summit/en), requiring agricultural productivity gains on smaller land areas and lower water resources 5 .
Salinity causes multiple responses in plants, particularly physiological, morphological, biochemical and molecular alterations 6 . It induces ion imbalance, leading to ion toxicity, osmotic stress and generation of reactive oxygen production (ROS), cellular damage, oxidative damage of membrane lipids, proteins and nucleic acids 7 . The retention of excess of sodium ions in proteins competes with potassium ions, inhibiting synthesis of metabolic enzymes and thereby protein synthesis 8 . Concentrations of NaCl higher outside the roots impede water absorption, make water extraction more difficult for plants leading to the osmotic stress 9 . High levels of salt in the leaves induce stomata shutting, electron transport impairment and photosynthesis contributing to reduced photosynthetic activity and productivity [10][11][12] .
Sorghum [Sorghum bicolor (L.) Moench] belongs to the family Poaceae and physiologically classified as C4 plants and ranked fifth among the top five economically valuable cereal crops globally 13 . It is grown worldwide in over 41.14 million hectares of area and accounts for 58.72 million metric tonnes of grains with an average yield of 1.43 metric tonnes per hectare 14 . Sorghum plays a vital role in global food production and is a dietary staple for more than 500 million people from several rural communities and food-insecure people in many parts of the world 15 . It is well known for its adaptability in arid and semi-arid regimes, moderately drought tolerant, and highly biomass productive 16,17 . In the current global climate change scenario, the crop is likely to become more critical due to its more extensive adaptability to high temperature, water scarcity, and saline conditions, making it a valuable feed resource 13 . Sorghum possesses a diverse range of morphological, anatomical and physiological, characteristics that enables it to survive under stress conditions. Plants emerging in saline soils regulate ion accumulation so that ion toxicity is avoided and appropriate osmotic solutes are retained 18 . This osmotic adjustment retains the volume and turgor pressure of the cells and organelles of the growing plant. The roots of the constantly transpiring plants must exclude the excess of cations and anions in saline soils that also depends on the type of root and plant species to avoid the ion toxicity 19 . Plants respond to the effects of salt stress via a complex network of mechanisms aimed at sustaining cellular metabolism under the prevailing conditions 20 . To scavenge high ROS levels, an efficient system of and enzymatic and non-enzymatic antioxidants is involved 17 . The enzymatic antioxidants include superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), as well as the enzymes of the ascorbate (ASC)-glutathione (GSH) cycle [GSH reductase (GR), ASC peroxidase (APX), monodehydroascorbate dehydrogenase (MDHAR), and dehydroascorbate reductase (DHAR)] that detoxify ROS 7,21 , while non-enzymatic antioxidants include phenolics, flavonoids, tocopherols, ascorbic acid and glutathione 22,23 whose up-regulation suggested a pertinent role of theses antioxidants in alleviating salt stress-induced oxidative damage. For example, the intake, partitioning or removal of ions from specialized cells under salinity is necessary for normal growth and development 22 .
Roots are the first organs experiencing high Na + ions which display a greater reduction in growth than shoots. Interestingly, age-related differences in responses in plant cells, tissues and organs have been observed 24 . Variations were however considerably less studied in oxidative stress and antioxidant protection in the different organs and stages of development. Sorghum is an economically important climate resilient cereal crop with wider ecological adaptability. In the current global climate change scenario, the crop is likely to become more critical due to its more extensive adaptability to high temperature, water scarcity, and saline conditions, making it a valuable feed resource. Therefore, this work aims to elucidate the effects of salinity on antioxidant potential, oxidative stress, and expression of stress-inducible genes in roots, young and mature leaves harvested from vegetative and physiological maturity in salt-treated and non-treated sorghum genotypes.

Biomass accumulation.
To assess the effect of high salinity (NaCl) on sorghum growth, biomass (fresh and dry weight) of plants, under different salt concentrations was measured. With the increasing levels of salt stress from control to 120mM NaCl, a significant reduction in plant fresh and weight was observed among all the genotypes at p<0.05 ( Fig. 1A and 1B). At 35 DAS, PC-5 had maximum reduction (79.6 %) in fresh weight while minimum in SSG 59-3 (29.9 %) at 100mM NaCl, while at 120mM, the decline was more, 87.6 % in PC-5 and 41.9 % in SSG 59-3. At two sampling stages, SSG 59-3 had more biomass production over PC-5. Root length decreased significantly with increasing concentration of NaCl in all the genotypes at 35 and 95 DAS (Fig. 1C). At 35 DAS, minimum percent reduction of plant height was observed in SSG 59-3 (26 %), and a higher reduction in PC-5 (69 %) at 100 mM NaCl. While at 120 mM, the percent decrease was more, PC-5 had a maximum reduction of 78 %, while SSG 59-3 had only 36 % reduction over their respective controls. A similar trend was noticed for shoot length (Fig. 1D). SSG 59-3 (108.6 cm) acquired maximum shoot length compared to PC-5 (48.3 cm). A significant interaction was found at both sampling stages, i.e., 35 and 95 DAS.
Morpho-physiological analysis. RWC of leaves was relatively low under salt stress as compared to control conditions in all the sorghum genotypes ( Fig. 2A). At 35 DAS, the reduction in RWC under salt stress was much higher in PC-5 (5.2 %) as compared to SSG 59-3 (17 %) at 100 mM NaCl. SSG 59-3 maintained RWC under increasing salt concentrations. The osmotic potential of leaves was measured in terms of -MPa at two developmental stages (Fig. 2B). The osmotic potential of leaf declined progressively with the increasing level of salt from 0 to 120 mM NaCl. At 35 DAS, values of osmotic potential became more negative in PC-5 than SSG 59-3, i.e.,-1.13 MPa and -0.71 MPa, respectively, at 120 mM NaCl. At 95 DAS, trends were similar at 35 DAS. A significant difference in chlorophyll fluorescence/photochemical quantum yield was observed within the genotypes and treatments (Fig. 2C). At 35 DAS, SSG 59-3 showed more quantum yield than PC-5. Percent reduction was higher in PC-5, i.e., 54 % and 62 %, and lower in SSG 59-3, i.e., 20 % and 28 % at 100 and 120 mM NaCl, respectively. Up to 100 mM, both sorghum genotypes performed better, but at 120 mM, the reduction was more, which drastically reduced the photosynthetic rate, and conferred that 100 mM was under the physiological tolerance range of the crop. After attaining physiological maturity (95 DAS), the quantum yield reduced significantly. Salt stress significantly reduced the chlorophyll stability index (CSI) (%) of sorghum genotypes at p< 0.05 (Fig. 2D). At 35 DAS, the reduction was more eminent in genotype PC-5 (65.3 %) trailed by genotype SSG 59-3 (22.9 %). The decline in chlorophyll stability in terms of loss of chlorophyll was higher in PC-5 (85.3 %) and lower in SSG 59-3 (30.2 %) at 120 mM NaCl, suggesting the tolerance behavior of SSG 59-3 genotype. At physiological maturity, PC-5 exhibited the highest percent reduction (81.3%) at 120 mM NaCl, indicating its susceptibility towards salinity due to severe loss in chlorophyll content, while SSG 59-3 had 46.8 percent reduction, indicating its stability towards chlorophyll content  During the vegetative stage, the photosynthetic rate was low and hence, PC-5 (52.5 %) had a decrease in chlorophyll a content as compared to SSG 59-3 (22.5 %) at 100 mM (Fig. 3A). The analysis of 3B demonstrates that as the level of NaCl decreased from control to 120 mM NaCl, a parallel reduction was recorded in the chlorophyll b content. A considerable difference was observed within the genotypes and treatments. Total chlorophyll content followed the similar pattern (Fig. 3C). The overall mean value of chlorophyll a, chlorophyll b, and total chlorophyll content was maximum at 35 DAS compared to 95 DAS.

Figure 3
Effect of salt stress on chlorophyll a (A), chlorophyll B (B), and total chlorophyll (C) of sorghum genotypes at 35 and 95 DAS. Values are means of at least three replicates and significant differences between means, as determined by Tuckey's test (P < 0.05).

Na + : K + and Cl − Content
High soil salinity causes over-accumulation of Na + inside the cell, which may result in ionic imbalances and metabolic toxicity. Na + : K + ratio (Fig. 4A) increased significantly with the increase of applied NaCl concentration in leaves and in roots, Na + accumulation was more significant in roots only under 120 mM treatment (Fig. 4B). As with the effect of salt on biomass, there was an apparent gradient, with highest Na + accumulation in roots. SSG 59-3 maintained lower Na + ions. It imported more K + ions, so had a lower Na + /K + ratio, while PC-5 excluded fewer Na + ions and had a higher Na + /K + ratio as Na + competes with K + for uptake through a common transport system. In accord with trends observed in the Na + content in roots, the Na + in leaves also increased, and K + content decreased with increasing salt stress levels. 6 ASC/GSH cycle components. Mild or severe salinity stress increased SOD activity in l e a v e s a n d root in a concentration independent manner (Fig. 5A). At 35 DAS, the percent increase in SOD activity in leaves was maximum in SSG 59-3 (51 %) considering it as salt-tolerant genotype. In contrast, the increase was less in PC-5 (19 %), suggesting it as salt-susceptible genotype at 100 mM. A similar trend was found in roots in all the genotypes; however, SOD activity in roots was lower than leaf tissue at both the growth stages. Differential response of CAT activity (Units mg -1 protein) in leaves and roots is shown in Fig. 5B. At vegetative stage, a significant increase in CAT activity was observed in the leaves of SSG 59-3 (46 %) with a higher decrease in PC-5 (22 %). A similar trend was observed in roots of all the genotypes; however, CAT activity in roots was lower than leaf tissue at both growth stages. A significant increase in POX activity was observed in all the sorghum genotypes (Fig. 5C). In SSG 59-3, the increase in activity was 38 % and 44 % at 100 mM and 120 mM NaCl, whereas PC-5 had lower POX activity (22 % and 29 %). Roots also had a similar basal level of POX activity as observed in the leaves at both developmental stages.
APX activity increased significantly in SSG 59-3 (55 %), whereas its activity declined in PC-5 (26 %) at 100 mM NaCl (Fig. 5D). Further increase in salt concentration enhanced the APX activity, more in SSG 59-3 (65 %) and less in PC-5 (33 %). The same trend was observed in roots. Differential response of GPX activity (Units mg -1 protein) showed a significant increase, SSG 59-3 (45 %), with decrease in the leaves of salt-sensitive genotype PC-5 (24 %) (Fig. 6A). Salt stress increased the GR activity in both the tolerant and susceptible sorghum genotypes (Fig. 6B). However, the increase was found to be higher in tolerant genotypes. MDHAR activity increased significantly at 35 DAS in the sorghum genotypes under salt stress (Fig. 6C). At 35 DAS, the activity increased by 53 % in SSG 9-3 while it reduced in PC-5 by 28 % at 100 mM NaCl. Among the studied genotypes, PC-5 showed the lower, while SSG 59-3 had the higher DHAR activity under salinity conditions (Fig. 6D). Enzymatic antioxidants. Salt stress resulted in a significant increase in total glutathione content in leaves at both stages ( Fig. 7A). At 100 mM, total glutathione content in SSG 59-3 was 54 % as compared to PC-5, where the percent increase was only 21 %. A higher concentration of Na + ions had a pronounced effect on total glutathione content, which further increased the glutathione content by 69 % in SSG 59-3 and 32 % in PC-5. A higher concentration of Na + ions has a pronounced effect on total glutathione content, further increased the GSH content ( Fig. 7B) by 56 % in SSG 59-3 and 31 % in PC-5. In oxidized glutathione (GSSG), the percent increase was more in the case of SSG 59-3 (52 %) than PC-5 (27 %) (Fig. 7C). In roots, the values of total glutathione, GSSG, and GSH were not higher as compared to leaves.
In all plant parts, there was a significant increase of ascorbic acid (ASC) under salinity stress ( Fig. 8A and 8B). At 35 DAS, ASC content increased by 52.51 % in SSG 59-3 as compared to PC-5 where percent increase was only 23 % at 100 mM NaCl. A significant decline in carotenoid content of all the genotypes under stress conditions was observed with more decrease in salt susceptible genotype (PC-5) as compared to the tolerant one (SSG 59-3) at both the developmental stages (Fig. 8C). Higher percent reduction was recorded in PC-5, i.e., 40 % and 52 %, and lower percent reduction in SSG 59-3, i.e., 18 % and 24 % at 100 mM and 120 mM NaCl, respectively.
Antioxidant capacity and polyphenolic compounds. Antioxidant activities viz. FRAP, DPPH and ABTS were evaluated to exploit the antioxidant potential of two sorghum genotypes at p< 0.05 (Table 1). The red coloured SSG 59-3 grains showed high FRAP and DPPH activities as compared to the PC-5. In ABTS assay, SSG 59-3 exhibited highest antioxidant activity at 43.76±0.23 mg/100g and lowest was exhibited by white PC-5 grains at 20.24±0.15 mg/100g. In the FRAP assay, SSG 59-3 exhibited highest antioxidant activity at 16.64±0.10 mg/g TE, while lower was showed by white grains PC-5. A significant difference in total phenolic content was observed between different sorghum varieties. The red pericarp SSG 59-3 had    Compatible solutes. Accumulation of compatible solutes viz. proline (Fig. 9A) and glycine betaine (Fig. 9B) increased considerably in leaves and roots of sorghum genotypes under salt stress. For proline, the percent increase was maximum in SSG 59-3 (50 %) while PC-5 had minimum of 23 %. For glycine betaine, the percent increase was maximum in SSG 59-3 (58 %) while PC-5 had by 23 % at 100 mM. In roots, glycine betaine level increased significantly, but the accumulation level was less from leaves in all the genotypes. Salt stress resulted in a significant increase in total soluble sugars (TSS) (Fig. 9C). At 100 mM, SSG 59-3 accumulated 52 % TSS as compared to PC-5, where the percent increase was only 31 %. A higher concentration of Na + ions had a pronounced effect on TSS, which further increased the total soluble sugar content by 68 % in SSG 59-3 and 48 % in PC-5. In roots, the values of total soluble sugar content were less as compared to leaves. Oxidative Stress Markers. Salt stress also leads to higher accumulation of ROS, which disturb cellular redox homeostasis, and result in oxidative damage. H2O2 content and markers of oxidative damage of cell membranes, i.e., relatives stress index (RSI) and malondialdehyde (MDA) were measured (Fig. 10A). Increase in the NaCl concentration resulted in a significant increase in the accumulation of H2O2 content, more particularly in roots than leaves. Following the trend of H2O2, RSI (Fig. 10B), and MDA ( Fig. 10C) also increased significantly (p < 0.05) in leaves and root, more particularly at 120 mM NaCl in PC-5. Thus, oxidative stress parameters indicate that root tissues suffered the most from the salinity-induced oxidative stress, followed by leaves. SSG 59-3 accumulated less stress markers due to oxidative damage.
Polyamines. During plant maturation the, total polyamine concentration remains constant, but under stressed conditions, their level of accumulation enhanced (Fig. 11). A typical chromatogram of benzoylated polyamine standards separated by a C18 reverse phase HPLC column is illustrated in Fig. 12A. The identities of the peaks were confirmed by adding polyamine standards to the extract of the plant tissue (  Figure  2B shows the separation of naturally occurring polyamines from leaf tissue of sorghum plant including the hexanediamine internal standard. Each peak represents the increased levels of Put, Spd, and Spm at 100 mM NaCl concentration in sorghum genotypes.

Figure 11
Chemical structures of the polyamines discussed in this study Gene expression analysis of ion transprter genes. Semi-quantitative RT-PCR has now become the most common approach for quantification and comparison of gene expression levels due to its robustness, precision and accuracy. The PCR product using specific primer of ion transporter genes viz. SOS1, SOS2, NHX, and H + -PPase indicated that the appearance of single band ( Fig. 13 and 14) and their up regulation during salinity indicated the antioxidative cellular defense mechanism to combat the toxic effects of salt accumulation inside the cytoplasm. In SSG 59-3, the higher expression of SOS1 (Fig. 13A) and SOS2 (Fig.  13B) under high Na + ions indicated its tolerance behavior to exclude the toxic effects of Na + ions, while in PC-5, the up-regulation of these genes was lower ( Fig. 13A and 13B). The expression was more at 120 mM NaCl salt concentration. The expression level of NHX (Fig. 14A) and H + -PPase (Fig. 14B) peaked at 100 and 120mM NaCl salinity. Actin constitutively expressed stably under all salt treatments (Fig. 14C). The higher expression of ion transporter genes under high salinity indicate that SSG 59-3 may be utilized as salt-tolerant crop due to its better genetic and agronomical traits. The detection of salt tolerant genes could be adding a molecular base to the application of identification of salt tolerant genotypes in sorghum.   Yield traits. SSG 59-3 was found significantly superior in retaining the maximum number of leaves per plant as compared to other genotypes at different salinity levels ( Table 2). Plant height decreased significantly with increasing salinity levels in sorghum genotypes at p< 0.05 (Table 2). At 35 DAS, the percent decline was higher in PC-5 (76.8 %) while lower in SSG 59-3 (26 %) over their respective controls. The number of tillers per plant was more pronounced at physiological maturity due to increased biomass and dry matter ( Table 3). During physiological maturity, the percent decrease in the number of tillers was observed more in PC-5 at 100 and 120 mM i.e., 19 % and 59 %, respectively, while in SSG 59-3, the percent decline was less i.e., 8 % and 19 %, respectively. At physiological maturity, the harvested seeds showed a higher percentage reduction in seed yield in PC-5 (42 %) while lower was noted in SSG 59-3 (13 %) ( Table 3). SSG 59-3 was found superior in retaining seed yield at different salinity levels.

Discussion
Soil salinization has emerged as a serious threat affecting crop productivity as well as geographical distribution of crop plants 25 . To contribute to our understanding of the biochemical and molecular mechanisms underlying salinity, we exposed sorghum genotypes to four different salinity levels, and compared responses in leaves and roots under two different developmental stages. With the increasing levels of salt stress from control to 120 mM, a significant reduction in plant fresh and dry weight was observed among all the genotypes (Fig. 1A and 1B). Increased accumulation of fresh biomass in tolerant genotype might be due to an increase in protoplasmic components and rapid cell division and elongation, leading to the luxury of vegetative growth, resulting in increased production of fresh biomass and dry matter 26 . While the decrease in biomass might be a reason for higher Na + ions, which resulted in delayed physiological maturity of the crop 27 . The detrimental effect of NaCl on dry weight may be due to a direct impact on the photosynthetic rate. The decreased root and shoot length (Fig. 1C and 1D) leads to high NDF content, which in turn reduces IVDMD under osmotic stress in sorghum. The decrease in root and shoot length could be due to increased accumulation of Na + ions, which in turn may cause root tip cell damage and decreased essential micronutrient uptake by plants under salt stress 28 .
The physiological characteristics of crop plants might be adversely affected owing to reduced leaf expansion, photosynthetic impairment, premature leaf senescence, and alterations to the protein and pigment structure 29 . The key factors that govern the relationship between plant waters, productivity and growth during salt stress include relative water content, osmotic potentials, stomatal conductance, and total chlorophyll. RWC is regarded as a plant water status indicator. A perusal of the data on RWC ( Fig.  2A) in leaves showed that salt treatments had a progressively decreasing effect in sorghum genotypes. It is also proposed that high RWCs might enable tolerant genotypes conduct more efficiently physiobiological activities under stress than susceptible chickpea genotypes 30 . Osmosis in plant cells can act as a mechanism of osmotic adjustment to decrease the cellular osmotic potential and thus maintain water absorption and turgor. A decrease in osmotic potential (ψs) (Fig. 2B) under stress conditions has been proposed to play an important role in osmotic adjustment and plants' survival mechanism under dry conditions 31,32 . The primary causes of an elevated osmotic potential under saline conditions include hydrolysis of biomolecules into smaller molecules such as mono and oligosaccharides, and proline among amino acids etc. Photochemical quantum yield (Fv/Fm) revelations into plant capability for abiotic stress tolerations and how the photosynthetic machinery has been affected under these stressful conditions. 33,34 . In the present investigation, chlorophyll fluorescence (Fv/Fm) (Figure 4.15) of leaf reduced with the increasing salinity levels (Fig. 2C). In photosystem II (Fv/Fm), the maximum quantum yield reduced significantly, whereas in saline conditions, non-photochemical quenching (qN) in sorghum increased substantially 34 . Values of chlorophyll content decreased with the progression of salt concentration (Fig.  3). Chlorophyll degradation in leaves under high Na + ions may also be accompanied with ROS generation, resulting in pigment-protein complex instability, chlorophyll oxidation and degeneration of other chloroplastic pigments 2 . A decline in concentration of K + and Mg 2+ can lead to the overall drop in chlorophyll concentration under salinity stress concentrations 35 .
Under salt stress, the ratio of Na + /K + ions in leaves and roots significantly increased in all genotypes ( Fig. 4A and 4B). Salt stress maintained abnormally high Na + /K + ratio, inactivated the enzymes used in the various developmental processes, and inhibited protein synthesis 33 . Another interesting point is that sorghum can also accumulate higher Na + concentration in the roots and limit the transportation of Na + up to shoots under salt stress, which is called salt exclusion 36 , an important salt-tolerance-related process. Growth inhibition in maize organs was correlated linearly with increasing Na + accumulation within tissues 9 .
In the present study, attempts were made to establish a correlation between antioxidative defense mechanism and salinity induced changes in leaves and roots of sorghum genotypes at two growth stages viz. vegetative stage and physiological maturity. During oxidative stress, the excess production of ROS is scavenged by a complex enzymatic antioxidative system which controls ROS production and ultimately protects the plant against oxidative damage 37 . This salinity-induced defense mechanism is differential and mostly dependent on differential activities of antioxidant enzymes, salinity extent, and exposure time 38 . SOD is the most effective intracellular enzymatic antioxidant, ubiquitous in all aerobic organisms and all subcellular compartments prone to ROS-mediated oxidative stress 21 . The percent increase was more in tolerant genotype (SSG 59-3) at both stages, suggesting that salt-tolerant genotypes have a better superoxide radical scavenging ability (Fig. 5A). Lee et al. 39 reported that transgenic tobacco plants overexpressing Cu/Zn-SOD showed tolerance to salt and water stresses. The induction of catalase activity (Fig. 5B) was reported on accumulation of H2O2 and seemingly consistent with this enzyme's role in scavenging enhanced H2O2 level 40,41 . Salinity-induced stimulation in POX activity in the present study was more pronounced in tolerant genotypes than in sensitive ones in both the tissues (Fig. 5C), suggesting their possible role inefficient removal of H2O2 in tolerant genotypes. Bhattacharjee and Mukherjee 42 reported a gradual decline in POX activity with increasing heat shock and salinity stress in A. lividus. Enhanced activity of peroxidase under various stresses was linked to protection from oxidative damage, lignification, and cross-linking of the cell wall. APX and GPX are one of the specific enzymes that scavenge chloroplastic H2O2 using ascorbate as an electron donor in the first step of the ascorbate-glutathione cycle and is considered the most important plant peroxidases in H2O2 detoxification 43 . The enhanced activity of APX (Fig. 5D) and GPX (Fig. 6A) concomitant with an enhanced content of ascorbic acid and glutathione may help to quench ROS in SSG 59-3 than PC-5 genotype. GR is essential to recycle GSH in the ascorbateglutathione cycle in an NADPH-dependent reaction. GR activity enhanced significantly in leaves and roots of sorghum genotypes (Fig. 6B). Similarly, higher induction in GR activity in tolerant varieties than sensitive varieties was reported in Macrotyloma uniflorum 44 and chickpea 45 . A significant interaction effect of MDHAR (Fig. 6C) and DHAR (Fig. 6D) activities implicated notable increases in their activities under all salt in sorghum genotypes. Similar results were also reported in maize 9 , and wheat 46,47

differing in salt tolerance
Non-enzymatic antioxidants include ascorbic acid, phenolics, glutathione, and carotenoids 22,23 . Glutathione is a major non-protein thiol in plants, which plays a pivotal role in protecting plants from environmental stress. The concentration of total glutathione (GSH+GSSG) (Fig. 7A), GSH (Fig. 7B) and GSSG (Fig. 7C) increased in leaves and roots of sorghum genotypes at 35 and 95 DAS. At the level of organ specificity, GSH level increased in response to high salinity, particularly in roots. This could be ascribed to the increased demand and metabolism of sulfur under stress for antioxidants' biosynthesis such as GSH 48 . Salt stress resulted in a significant increase in total ascorbic acid content in leaves of sorghum genotypes at both stages ( Fig. 8A and 8B). The diminished ascorbate pool under various stresses may be due to changes in glutathione pool that have been implicated in the recycling of ascorbate or failure to maintain ascorbate level may be indicative of an overall decline in the capacity to withstand oxidative stress 46 . The increased absorption of sulfate affects GSH levels in tissues under high salinity 49 . The increased content of AsA and GSH, accompanied by the reduced AsA and GSH redox status, indicated the crucial role of the AsA-GSH cycle for scavenging ROS 9 . Reduction in the levels of photosynthetic pigments, including chlorophyll a and b and accessory pigments such as carotenoids, on exposure to abiotic stresses have been reported by several researchers [50][51][52] .
FRAP evaluation measure the capacity of a Fe 3+ /tripyridyl-s-triazine complex to be reduced in the presence of an antioxidant. The formation of non-specific radicals is the function of ABTS and DPPH assay. DPPH activity in the red pericarp sorghum was reported to be higher [53][54][55][56] . Hence, the present results indicated that sorghum grains with colored pericarp had high antioxidant activities. Significant variations in total phenols, flavonoids and flavanols were observed among different sorghum genotypes (Table 1). This could be due to the absence of tannins, which indicate that red colored pericarp sorghum is of Type I 54,57 .
Plants maintain their cell turgor and osmoregulatory mechanisms by accumulating various compatible solutes such as proline, glycine betaine (GB), polyamines and proteins 58,59 under different abiotic stresses that decreased the cytoplasmic osmotic potential, enabling water absorption 60 . Proline is considered the only osmolyte, which has been shown to scavenge singlet oxygen and free radicals, including hydroxyl ions, and stabilize proteins, DNA, as well as the membrane 61 . Proline content increased significantly under salt stress in both leaves and roots of sorghum genotypes (Fig. 9A). Glycine betaine (GB) is one of the osmoregulatory solutes that naturally accumulates in the plants 62 . Accumulation of glycine betaine increased considerably in leaves and roots of sorghum genotypes because of salt stress (Fig. 9B). More specifically, GB can protect the O2 evolving machinery of chloroplasts when exposed to high NaCl concentrations 63 . Saneoka et al. 64 reported that the salt-induced accumulation of betaine and BADH mRNA coincides with ABA in sorghum. Accumulation of soluble carbohydrates increases the resistance against various stresses. Total soluble carbohydrates act as osmolytes inside the cell (Fig. 9D). The imposition of water or salt stress in sorghum has been demonstrated to be accompanied by an increase in the sugar levels of embryos, which may help in osmoregulation under stress conditions 65 .
H2O2 is a natural plant toxic cellular metabolite; it is a toxic only at high concentrations and causes thylakoid degradation 66,67 . In this study, although H2O2 accumulation increased during salt stress in tolerant and susceptible genotypes (Fig. 10A), there was a lower ROS level in tolerant genotypes, which might be related to higher antioxidant activity and also higher proline and total soluble sugar content. Forghani et al. 68 observed that H2O2 content was about 54 % more in the salt-stressed as compared to control plants. The relative stress index (RSI) in terms of leakage of electrolytes increased significantly in leaves and roots of sorghum genotypes (Fig. 10B). Increased electrolyte leakage from tissues is usually an expression of modification in the physical properties of cell membranes. Malondialdehyde (MDA) is one of the final products of peroxidation of unsaturated fatty acids in phospholipids and is responsible for cell membrane damage. Salt stress-induced higher production of MDA content was reported in present work (Fig. 10B) which is in agreement with those reported by several workers 69,70 . Salt stress-induced membrane lipid peroxidation results in membrane fluidity, leading to enhanced electrolytic leakage in wild type and transgenic lines in sorghum 71 . During stress conditions, the total polyamine concentration enhanced ( Fig.  11 and 12). The chromatogram in Figure 12 shows the separation of naturally occurring polyamines (Put, Spd and Spm) from leaf tissue of sorghum plant with their enhanced concentration. At 100 and 120mM NaCl, their concentration enhanced progressively in SG 59-3 [72][73][74] .
Analysis of yield components is an essential tool for assessing crop development and productivity 75 . Salt stress significantly reduced the yield attributes and yield among the sorghum genotypes, i.e., plant height, number of leaves/plant ( Table 2), number of tillers per plant, and seed yield per plant (Table 3). Reduction in yield attributes was more at a higher level of salt (120 mM) because of the adverse effect on the physiological process and dry matter accumulation and ultimately reduced the plant's seed yield. The reduction in the number of seeds per plant was due to the reduced flower production and fertilization. Seed yield was affected due to pollen sterility, abortion, pollen germination, and in-compatible fertilization, which directly reduced yield attributes and yield under salt stress 76 .
The differential expression of ion transporter genes under different salinity levels indicated their adaptive behavior for Na + ion exclusion (Fig. 15). Salt stress elicits a cytosolic calcium signal 77 . Increased intracellular concentration of Ca 2+ ions are sensed by SOS3 which interacts and activates SOS2, a serine/threonine protein kinase. Both SOS2 and SOS3 regulate the expression level of SOS1, a salt tolerance gene which encodes a sodium proton antiporter (NHX) 78 . Co-expression of SOS1 (Fig. 13A) and SOS2 (Fig. 13B) together with NHX (Fig. 14A), dramatically enhance the salt tolerance in SSG 59-3. Expression of a constitutively expressed H + -PPase (Fig. 14B) remains stable at 100 and 120mM NaCl. Qiu et al. 79 showed that constitutively active SOS2 kinase could enhance a NHX exchange activity in purified plasma membrane vesicles from wild-type but not sos1-1 mutant plants 80 . In sos2-2 and sos3-1 mutants 81 , the plasma membrane NHX exchange activity is much lower but can be recovered to near wild-type levels by addition of activated SOS2 in vitro to the membrane vesicle preparations 79 .

Figure 15
Regulation of Na + and K + ion homeostasis by the SOS pathway. Excess of Na + ions elicits a calcium signaling pathway which activates the SOS1-SOS3 protein kinase complex, which in turn stimulates the SOS1 and sodium proton antiporter (NHX) exchange activity and thus, regulates the expression of genes encoding them.

Conclusion
Thresholds of sorghum-specific antioxidant defense mechanisms are stimulated at all developmental stages and in all tissues. However, each organ utilizes the most effective and in accordance with the other metabolic networks in that particular organ at its unique age in order to cope with salinity and the associated oxidative stress from the arsenal of alternate antioxidant mechanisms available in sorghum tissue. Deciphering these specific alternatives could help developing more efficient metabolic engineering mechanisms specific to different organs and ages to cope with specific stress conditions. This research clearly establishes SSG 59-3 as a potential sorghum line with naturally occurring antioxidant potential, and highlights the need to further understand and explore the fine-tuning plant sensing and signaling systems. Hence, SSG 59-3 seems physiologically and biochemically promising and may be exploited in plant breeding programs aimed at developing salt-tolerant sorghum genotypes. Plant material and growth conditions. Seeds of the two sorghum genotypes viz. SSG 59-3 (salttolerant) and PC-5 (salt susceptible) were procured from Forage section, Department of Genetics & Plant Breeding, CCS Haryana Agricultural University Hisar, India. A screen-house experiment was conducted where sorghum seeds were sown directly in plastic pots lined with polyethylene bags filled with 10 kg sandy loam soil at 2-3 cm depth with standard conventional cultivation practices. All pots were rinsed with an equal volume of water and nutrient solution as per the recommended package of practices (POP). The physicochemical properties of the soil used were determined before sowing (Table 4) and the pots were saturated with desired salt levels, i.e., 60, 80, 100, and 120 Mm NaCl. Controls were run simultaneously in two genotypes.

Methods
Treatments. Saline solutions of required molarity were prepared by mixing NaCl, CaCl2.2H2O, MgCl2.6H2O, and MgSO4.7H2O in the appropriate amount ( Table 5). The soil's electrical conductivity was checked at an interval of 15 days and at sampling stage to maintain the desired salt concentration. Three replications of each treatment were taken.
Raising of the crop. The seeds (10) of uniform size were selected and surface sterilized with 0.01 % mercuric chloride (HgCl2) solution to prevent the fungal attack and then thoroughly washed with distilled water and air-dried before sowing. The seeds were sown at a depth of 5 cm and after seedling emergence, thinning was done up to five seedlings per pot.
The studies are complied with relevant institutional, national, and international guidelines and legislation.
Sampling. Samples were collected at the pre-flowering (35 days after sowing, DAS) (Plate 1) and postflowering (95 days after sowing, DAS) (Plate 2) stage and immediately frozen in liquid nitrogen and stored at 80°C, for further biochemical and molecular analysis. The morpho-physiological traits were determined from the flag leaf. Dry weight (DW) of the plant organs was measured after drying the tissues for 5 days at 80°C. Analysis for salt responsive protein profiling and mRNA expression profiling of genes was carried at the early vegetative stage. Data on yield and yield attributes were recorded at the maturity stage.  Physiological indices. The fresh weight of samples was determined from one whole plant and the sample was kept in a hot air oven at 70 °C until the constant weight was achieved for determination of dry weight per plant from each replication. Root and shoot length was calculated from the base to the top growing bud at the vegetative stage and from bottom to the topmost portion of plant excluding arrow length, at maturity. RWC was measured from fresh leaf samples, excised, weighed immediately, and placed in distilled water at a constant temperature in diffused light for 6 h. Then, leaf discs were dried in an oven at 80 ºC for 72 h for dry weight was recorded and calculated as 82 : The osmotic potential (OP) was determined using a psychrometric technique (Model 5199-B vapor Pressure Osmometer, Wescor Inc. Logan, Utah, USA) in the third leaf from the top. The reading (mmole kg -1 ) displayed on osmometer was recorded using osmolarity reference standards of sodium chloride (Wescor Inc, USA). Photochemical quantum yield (Fv/Fm) was recorded in intact plants using a chlorophyll fluorometer (OS-30p, Opti-Science, Inc., Hudson, USA) at mid-day. Initial (F0) and maximum (Fm) fluorescence were recorded and variable fluorescence (Fv), derived by subtracting F0 from Fm. Chlorophyll stability index (CSI) was estimated as the method describe 83 . Chlorophyll a, chlorophyll band total chlorophyll estimation was done by incubating 50 mg of leaf material in 10 ml of DMSO for 3 h at 60 C and other sets of the same amount of leaf were heated at 32 C for one h in a water bath. After cooling, 10 ml of DMSO was added, and then the absorbance of the solvent was recorded at 663 and 645 nm. The CSI was calculated using the formula given as under: CSI (%) = 1-[Total chlorophyll of heated samples/ Total chlorophyll of nonheated sample] x 100. Na + : K + determination . Na + and K + content was determined in 50 mg of dried and well-ground plant material. Samples were digested in concentrated H2SO4/ HClO4 (9:1) and the clear supernatant was analyzed by mass spectrometry (ICP-MS, Finnigan Element XR, Scientific, Bremen, Germany).
Total antioxidant capacity. One hundred milligram fresh plant tissue was homogenized in liquid nitrogen, and extracted in 2 ml of ice-cold 80% ethanol 95 . Ferric reducing antioxidant power (FRAP) reagent constituting 300 mM acetate buffer (pH 3.6), 0.01 mM 2,4,6-tripirydylo-S-triazine (TPTZ)) dissolved in 0.04 mM HCl and 20 mM FeCl3.6H2O was mixed with the ethanolic extract and absorbance was read at 600 nm using trolox was used as a standard. For DPPH antioxidant activity, a suitable aliquot (3.5 ml) of DPPH solution in methanol was added to 0.1mL of sorghum extract followed by incubation period of 40 min at room temperature in dark, absorbance was recorded at 515 nm and expressed in milligram per gram Trolox equivalents (TE) 96 . ABTS activity was assayed with slight modifications 97 . The ABTS radical cation solution was prepared by mixing 7 mM ABTS and 2.45 mM potassium persulfate solution followed by incubation of 16 h at room in the dark. After incubation, the mixture was diluted with buffer (pH 7.4) and absorbance was read at 734 nm.
Polyphenolic compounds. Extraction was done in 1% HCl/methanol (v/v) for 2 h with shaking, centrifuged, and supernatants were pooled. Total phenols were estimated using gallic acid was used as standard for quantification of the samples containing phenolic content and data is expressed as mg/g gallic acid equivalents (GAE) 98 . Total flavanoids were estimated using catechin as a standard 99 . o-dihydroxy phenols were quantified using chlorogenic acid as the standard 100 . Flavanols were estimated using rutin as a standard 101 .
Compatible osmolytes. The proline content in the sample tissue was analyzed by homogenizing 500 mg tissue in 5 ml of 3 % aqueous sulphosalicylic acid, centrifugation at 5000 rpm for 20 min and extracted using ninhydrin reagent and toluene extraction 102 . The absorbance was read at 520 nm. Glycine-betaine (GB) estimation was done in finely powdered plant material (500 mg) 103 . Dried material was mechanically shaken with deionized water and extract was diluted with 2 N sulfuric acid (1:1), centrifuged at 10,000 × g for 15 min and absorbance was measured at 365 nm. Total soluble carbohydrates (TSC) were extracted using 2 % phenol and conc. H2SO4 and the absorbance of solution was measured at 490 nm 104 .
Oxidative stress markers. Malondialdehyde content was assayed in 250 mg of tissue from control and stressed plants were ground in 2 ml of chilled 1 % TCA and centrifuged at 10,000 rpm for 20 min 105 . After centrifugation, the supernatant reacted with 20 % (w/v) trichloroacetic acid (TCA) containing 0.5 % thiobarbituric acid to produce pinkish red chromogen, thiobarbituric acid-malondialdehyde (TBA-MDA). Absorbance was measured at 600 nm using the extinction coefficient of 155 mM -1 cm -1 . Membrane injury index was measured as the percent proportion of ion leakage into the external aqueous medium to the total ion concentration of the stressed tissue as measured by the external medium EC 106 . Two hundred mg of leaf and root tissues were boiled in de-ionized water at 27 °C and solution's electrical conductivity (EC) was measured. The membrane injury index was calculated as follows: Membrane injury (%) = EC1 / EC2 x 100. Hydrogen peroxide (H2O2) concentration was measured by homogenizing one gram of fresh leaf and root tissue in ice-cold 0.1 M phosphate buffer (pH 7.0) and 40 µl was used in the assay based on the peroxidemediated oxidation of Fe 2+ , followed by reaction of Fe 2+ with xylenol orange at 570 nm 107 .
Polyamines. Poylamines in fresh tissue (1 g) was homogenized in 5 ml of 5% cold perchloric acid 108 . The supernatant, containing the free polyamines was frozen at -20 0 C for further benzoylation reaction using 500 µl of extract, 1 ml of 2N NaOH, 10 µl of 99% (v/v) benzoyl chloride, incubated for 30 min and then, saturated with 2 ml NaCl. Benzoylated polyamines were extracted with diethyl ether, incubated at -20°C for 1 h and were separated with the HPLC fitted with C18 ODS 2 analytical column which was pre-equilibrated and run isocratically at a flow rate of 1 ml min -1 with acetonitrile: water (50: 50 v/v) for 40 min. The injected volume was 10 µl and UV-detector was used to measure the absorbance at 224 nm. Polyamines were quantified in the eluent by comparing the integration areas obtained for the samples with those obtained for the standards and the results were express as nmol g -1 fresh weight of the sample.            Effect of salt stress on hydrogen peroxide (H2O2, A), relatives stress index (RSI, B), and malondialdehyde (MDA, C) of sorghum genotypes at 35 and 95 DAS. Values are means of at least three replicates and signi cant differences between means, as determined by Tuckey's test (P < 0.05).

Figure 11
Chemical structures of the polyamines discussed in this study    Regulation of Na+ and K+ ion homeostasis by the SOS pathway. Excess of Na+ ions elicits a calcium signaling pathway which activates the SOS1-SOS3 protein kinase complex, which in turn stimulates the SOS1 and sodium proton antiporter (NHX) exchange activity and thus, regulates the expression of genes encoding them.

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