Up regulation in transcript abundance of plastidic isoforms of antioxidant enzymes and accumulation of compatible osmolytes impart tissue tolerance to salinity stressed wheat plants

The response of salt tolerant wheat genotype (Kharchia 65), and sensitive cultivars (HD2687, HD2009, WL711) to vegetative stage salinity stress (for 4 weeks) were studied at 1.1 (control), 9.1 (S1) and 14.2 (S2) dSm salinity levels. Based on relative change in Membrane stability, PSII efficiency, retention of chlorophyll and carotenoid contents, Kharchia 65 showed better tolerance to salinity than other genotypes considered. To understand the role of different component mechanisms, expression of genes involved in ion exclusion, antioxidant defence and compatible osmolyte synthesis were analysed. Expression of SOS1 (plasma membrane Na/H antiporter), NHX (vacuolar Na/H antiporter), Ionic (sodium exclusion) and tissue tolerance (Sodium compartmentation, compatible solute accumulation and antioxidant defence) mechanisms were analysed in leaves of the genotypes after 4 weeks of salinity stress. Expression assay and the content of respective constituents indicated that apart from the wellknown ion exclusion ability, Kharchia 65 also showed high level of tissue tolerance resulting in high early vigour and maintenance of growth rate afterwards. In Kharchia 65, sensing of salinity stress at plasma membrane activates NADPH Oxidase (RBOH) genes and generate ROS at apoplast. Apoplastic ROS triggers calcium influx and activates calcium signaling genes of SOS pathway (SOS1 and NHX). ROS generated from organelles chloroplast, peroxisome and mitochondria triggers cellular oxidative burst. ROS and calcium activates MAPK genes and downstream transcription factors, NAC and bZIP. MAPK signaling induces cellular antioxidant and compatible osmolyte biosynthesis and imparts tissue tolerance to salinity.


Introduction
Soil salinity is wide-spread in approximately 100 countries around the world with a significant diversity in nature, property and extent of salinisation [1]. The global area of saltaffected soils is around 831 million hectares [2]. A soil is regarded as saline if the electrical conductivity of the soil saturation extract (ECe) surpasses 4 dS m -1 [3]. Soil salinity imposes a range of adjustments in plants, viz. alterations in metabolism, nutrient uptake and retardation of growth and development [4]. Despite the well-watered status of soil, salinity induces water deficit by reducing soil water potential making it cumbersome for the roots to extract rhizospheric water [5].
The concomitant water deficit and ion toxicity in saline soils augments generation of cellular reactive oxygen species (ROS) such as superoxide radical (SOR), hydrogen peroxide (H2O2), and hydroxyl radical (OH . ) [6]. As an artefact of photosynthetic electron transport, chloroplasts are oxygen enriched organelles and under stressful situations PSI and PSII become sites of ROS production [7]. The SOR is disimutated to less toxic H2O2 by the enzyme superoxide dismutase (SOD). Environmental stresses also promote oxygenase activity of RUBISCO and thus photorespiratory pathway involving chloroplast, peroxisome and mitochondria is activated and generates H2O2. ROS are also produced in mitochondria and by plasma membrane bound NADPH oxidase or respiratory burst oxidase homologues (RBOH)in apoplasm. Thus the stress perceived by chloroplast is transmitted to other organelles and result in cellular oxidative burst and the organellar mechanisms to combat ROS are activated [8]. The emergence. Scheduled routine of irrigation was practiced for both control and treated pots throughout the crop growth period. Each treatment was replicated 10 times in the form of pots.
Soil samples were collected at weekly intervals from each variety and treatment. From these samples ECe values of soil were estimated, and average of all the values were taken as the mean level of soil salinity. Actual salinity levels expressed as electrical conductivity of soil extract (ECe) were 1.1, 9.1 and 14.2dS m -1 for control, 100 and 200 mM NaCl, respectively.

Physiological parameters
Membrane stability index was estimated as described earlier by measuring electrical conductivity of leaf samples incubated at [18]

Superoxide radical, hydrogen peroxide and TBARS contents estimations
Superoxide radical content was determined by nitroblue tetrazolium chloride (NBT) reduction assay. Hydrogen peroxide was measured as titanium-hydro peroxide complex [21].
The tissue localisation of Superoxide radical, and H2O2 were determined as described earlier [22].The lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) as described earlier [23]. The membrane injury was also estimated by Evans blue staining followed by spectrophotometric estimation of tissue bound dye [24].
Qualitative estimation of superoxide radicals was done by NBT assay. Localisation of hydrogen peroxide (H2O2) was determined by DAB assay [25,26]. The second leaf of wheat seedlings was cut into pieces of approx. 1 cm and dipped in NBT/ DAB solution. Leaf segments were viewed under stereomicroscope after removal of chlorophyll from leaf tissue.

Antioxidant enzymes assay
Samples from uppermost expanded leaves were collected from all the treatments and were frozen in liquid N. Frozen samples were extracted using extraction buffer (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA and 1 mM ascorbic acid). The crude extract filtered through 4 layers of muslin cloth and the filtrate was centrifuged at 4ºC for 20 min at 15,000g. The supernatant was aliquoted and stored in -20ºC freezer [27]. Activity of antioxidant enzymes were assayed using the frozen extract. Total SOD activity was estimated by the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) by the enzyme [28]. APX was assayed by recording the decrease in optical density due to ascorbic acid at 290 nm [29]. CAT was assayed by measuring the disappearance of H2O2 according to Aebi [30].
GR was assayed as per the method of Smith et al. [23].

Estimation of compatible solutes content
For extraction of soluble sugars, dried and powdered leaf samples were boiled with of 80% (v/v) ethanol three times followed by boiling with required amount of distilled water [33]. For estimation of sugars, sample aliquot was mixed anthrone regent and was incubated in a boiling water bath for 8 min. The absorbance of the mixture was recorded at 630 nm after cooling to room temperature in a UV-visible spectrophotometer (model Specord Bio-200, AnalytikJena, Germany).
Trehalose was determined by using the trehalose assay kit from Megazyme (Megazyme International Ltd, Bray, Co. Wicklow, Ireland). Finely ground dry plant material was mechanically shaken with 40 ml of hot (~ 80°C) deionised water on a magnetic stirrer for 15 min. After cooling to room temperature, samples were diluted and filtered. Pre-existing glucose in the filtrate was determined in a control reaction without added trehalase and absorbance was taken at 340nm (A1). Then, the sample was mixed with trehalase, and incubated for 5 min and absorbance was again recorded at 340 nm (A2) against the reagent blank to determine the trehalose content. Difference of A2-A1 was used for obtaining ΔA trehalose (NADPH/2). The concentration of trehalose was determined from the extinction coefficient of NADPH, i.e., 6.3 mM -1 cm -1 . Trehalose content was obtained by dividing the value of NADPH by 2. Proline content was estimated according to the method of Bates et al. [34]. Glycine-betaine content was estimated according to the protocol of Grieve and Grattan [35].

Assay of enzymes associated with compatible osmolyte biosynthesis
Samples from uppermost expanded leaves were collected from all the treatments and were frozen in liquid N. Frozen samples were extracted using extraction buffer containing 0.1M Na-citrate, pH 3.7, 1 mM PMSF, 2 mM EDTA and insoluble polyvinylpyrrolidone (10 mg/g dry weight). The homogenate was then filtered through 2 layers of muslin cloth and centrifuged at 31,500g for 30 minutes at 4°C. The supernatant was used for the TPS enzyme assay [36].
Assay mixture contained 0.05 M Hepes-KOH, pH 7.1, 5 mM UDPG, 10 mM glucose 6phosphate, and 12.5 mM MgCl2, enzyme extract, and water in a total volume of 0.4 ml. Assay mixture was incubated at 35 °C for 30 min; the reaction was stopped by heating at 100°C for 5 min. Thereafter the samples were stored on ice for 10 min and centrifuged at 2000g. UDP formed was determined in the supernatant by the decrease in absorbance at 340 nm in a mixture containing 0.14M Hepes-KOH, pH 7.6, 2 mM phosphoenol pyruvate, 0.3 mM NADH, 5U lactic dehydrogenase, 5U pyruvate kinase, and the sample in a total volume of 0.5 ml.
The reaction was started by addition of pyruvate kinase. Controls omitting either glucose-6phosphate, UDP-glucose or both, were run to eliminate possible interfering reactions, which could produce ADP, UDP or pyruvate. Each determination was made in triplicate. One unit of TPS was defined as the amount of enzyme, which produces 1.0 µmol of NAD + per minute at 37 °C and pH 7.0.
For the assay of BADH, plant tissue was homogenized into a fine powder in liquid nitrogen, and then suspended in 10 ml of extraction buffer (50 mM Hepes/KOH pH 8. 0.1 mM EDTA, 5 mM DTT) at room temperature, and centrifuged at 10000g at 4°C for 10 min. Reaction mixture contained 50 mM Hepes/KOH (pH 8.01) 5 mM DTT, 1 mM EDTA, 1 mM betaine aldehyde, 1 mM NAD and 1.0 mg protein from enzyme extract, with a total volume of 1.0 ml [37]. Reaction was carried out at 37 °C for 10 min. Absorbance was recorded in a UV-visible spectrophotometer at 340 nm. One unit of the enzyme activity was defined as the formation of 1 µmol NADH per min under the above condition.
Δ-Pyrroline-5-carboxylate synthetase activity was assayed by recoding the decrease in absorbance due to NADPH at 340 nm [38]. For extraction of enzyme, leaf tissue was ground with liquid nitrogen and in extraction buffer containing 50 mM tris-HCl (pH 7.2), 10 mM MgCl2, 0.6 M KCl, 3 mM EDTA, 1 mM DTT, 5% PVPP and 1 mM β-mercapto ethanol. After grinding, the homogenate was filtered through 2 layers of muslin cloth. The filtrate was then centrifuged at 10000g for 20 minutes at 4 ºC. The supernatant was taken as enzyme extract.
Reaction mixture contained 100 mM Tris-HCl (pH 7.2), 25 mM MgCl2, 75 mM sodium glutamate, 5 mM ATP, 0.4 mM NADPH and distilled water to make the volume up to 1 ml.
The reaction was started by addition of enzyme extract. The reaction velocity was measured as the rate of consumption of NADPH, monitored as decrease in absorbance at 340 nm as a  qRT-PCR products were also visualised by agarose gel electrophoresis to confirm the single specific band. Normalization of the data for each transcript was carried out using TaActin as an internal control and level of expression were analyzed using 2 -DDCt method [40].

Statistical analyses
Values are means of 3 observations (n = 3), and data was subjected to analysis of variance by CRD. F-test was carried out to test the significance of the treatment differences and the least significant differences (LSD) were computed to test the significance of the different treatments at 5% level of probability by the SPSS 16.0. Mean separation was done using Sidak's multiple comparisons test following one-way ANOVA. Graphs and heatmaps were made using MS Excel and GraphPad Prism version 8 (La Jolla, California, USA).

Salinity stress negatively affects physiological parameters
Salinity stress imposition decreased the total chlorophyll and carotenoid content of all genotypes, The relative reduction in pigments was less in Kharchia 65 (Fig 1a, b). Stress induced injury was more pronounced in genotypes WL 711 and HD2687, HD 2009 showed only a moderate drop in chlorophyll content. There was decline in MSI with increase in salinity in all the four genotypes ( Fig.1c) with a lower average decline in Kharchia 65 than in other genotypes. Results on photosystem II efficiency is presented in Fig. 1d Photosystem II efficiency showed reduction in all the genotypes under salinity treatment.

Variation in ROS accumulation and associated enzymes
Exposure to soil salinity significantly increased the H2O2 accumulation in all the genotypes than that that observed in control (Fig. 2a

Variation in osmolyte accumulation and associated enzymes
Total soluble sugar content escalated under salinity treatments noticeably in all the genotypes (Fig. 4a). Sugar accumulation was highest in S1 treatment in all the genotypes. Sugar content was significantly higher in Kharchia 65 under S1 and S2 salinity levels, while lowest content was observed in HD 2687. Trehalose content (Fig. 4b) increased with the increase in salinity treatments in all the genotypes and Kharchia 65 showed 6 and 8 folds increases in trehalose content under S1 and S2 salinity levels, respectively. Glycine-betaine content was also augmented by salinity in all the genotypes (Fig. 4c). In S1 treatment all the genotypes showed significant increase, however, highest content was observed in Kharchia 65. Under S2 treatment, only Kharchia 65 maintained significantly higher stress induced glycine-betaine content and all other genotypes showed decline over S1 treatment. Proline content (Fig. 4d) increased in all the four genotypes with increasing salinity levels. Significantly greater increases in proline content were observed in HD 2009 and HD 2687 than Kharchia 65, and lowest in WL 711 under both the salinity levels. The increases were 5.71, 6.97, 6.07 and 4.78; 6.59, 7.84, 6.72 and 5.69 times over control in case of Kharchia 65, HD 2009, HD 2687 and WL 711 under S1 and S2 treatments, respectively at vegetative stage.
Stress induced up regulation in the TPS activity in all the genotypes was greater under S1 treatment (Fig. 5a). Under S2 treatment, TPS activity declined compared to S1 treatment, though gene under both S1 and S2 (Supplementary File 3, Fig 6a, b). WL 711 showed slight expression only in the case of S1 treatment. Partial nucleotide sequences for APX of Kharchia 65, HD RT-PCR for osmolyte biosynthesis enzymes were performed with gene specific primers and amplicons were obtained in all the four genotypes under all the three treatments ( Supplementary Fig 4). In the case of TPS gene, RT-PCR amplicons of size 590 bp were amplified from the four genotypes. Very little expression was observed in control plants of all the four genotypes (Supplementary File 4, Fig 6a, b). Salinity induced over expression was observed in all the four genotypes. However, under S1 and S2 treatments very prominent

qRT-PCR analysis of genes associated with tissue tolerance and stress signaling
Genes associated with tissue tolerance and stress signaling were analyzed by qPCR.
The genes associated with sodium exclusion (SOS1) and Vacuolar partitioning (NHX) were both upregulated by salinity. As the intensity of salinity was increased from S1 to S2, expression of the genes was also upregulated. Expression of RBOH-F gene was upregulated by salinity stress. In S2 treatment there was approximately 5-10-fold upregulation in expression (Fig 6a, b). The relative expression of MAPK gene was increased by both S1 and S2 treatments.
Expression of stress regulated transcription factors, bZIP and NAC were also upregulated by salinity stress in all the genotypes. NAC4 expression in S2 treatment was significantly different among all the genotypes, while in the case of bZIP, Kharchia 65 maintained the highest stress induced expression in S2 treatment.

DISCUSSION
The present study unveiled that salinity stress negatively affects various physiological traits in wheat.  [66] reported that the expression of TabZIP1 was induced by salt, low temperature, and wounding treatments. Thus TabZIP1 may be a key player in wheat's response to various environmental stresses. The underlying molecular mechanisms can be complicated as the expression of TabZIP1 was maximum at 2 h and 24 h after treatment with ABA [66]. Several transcriptomic studies have revealed that many of the NAC genes are controlled by diverse biotic and abiotic stresses, suggesting that they have vital role in stress signaling. Our results revealed that salinity treatment also upregulated TaNAC gene expression in in leaves of wheat genotypes. Xia et al. [67] reported that the TaNAC8 transcript abundance was up-regulated by salt stress, osmotic stress (PEG treatment) and low-temperature treatment, suggesting the role of TaNAC8 in responses to these environmental stresses.

Conclusion
Salinity induced ROS production has been suggested as the primary cause for (by activating SOS1) and tissue tolerance mechanisms: vacuolar sodium compartmentation (by NHX1) osmolyte accumulation and antioxidant defense. (Fig 7).    Tissue tolerance to vegetative stage salinity stress in tolerant genotype(s). Sensing of salinity stress at plasma membrane activates NADPH Oxidase (RHOH) genes and generate reactive Oxygen Species (ROS) at apoplast. Apoplastic ROS triggers calcium influx and activates calcium signaling genes of SOS pathway. ROS generated from organelles chloroplast, peroxisome and mitochondria triggers cellular oxidative burst. ROS and Calcium activates MAPK genes and downstream transcription factors , NAC and bZIP. MAPK signaling induces cellular antioxidant and compatible osmolyte biosynthesis and imparts tissue tolerance to salinity. ROS burst mediated triggering of putative RCDs and calcium signaling improves sodium exclusion and vacuolar sodium compartmentation by activating SOS1, VP1 and NHX1.