Comparative analysis of physiological and biochemical responses to salt-stress reveals key mechanisms of salt-tolerance in some Saudi Wheat Cultivars

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Wheat has been widely considered to be a moderately salt tolerant plant and its tolerance and responses to salinity stress vary among different tissues and cultivars. This study was conducted to investigate the impact of salt stress on growth and yield of three Saudi bread wheat cultivars, Najran, Mebiah and Qiadh, and characterize the differential responses of the roots and shoots to reveal different underlying mechanisms for salt tolerance. One-month old plants grown under control and salinity conditions were harvested to measure growth parameters (including fresh weight, dry weight and plant length), biochemical response (i.e. proline, soluble sugars, starch and organic acids contents) and antioxidant activity (phenolics content). A distinctive variation was observed between the three cultivars, Najran was the most tolerant to salt stress while Qiadh was the most susceptible cultivar. Under salt stress, a dramatic decline in growth parameters was noticed across all cultivars however, Qiadh exhibited the most conspicuous reduction in growth as well as in yield. In contrast, a pronounced increase of metabolite contents was shown in the three cultivars under salinity stress and was different not only between these cultivars but also between root and shoot tissues. The obtained results confirm that different wheat cultivars employ various mechanisms to alleviate the harmful effects of salt stress. The diversity in salt stress responses among different wheat cultivars can offer a promising avenue for enhancing crop productivity. In this study, the salt-tolerant Najran cultivar can serve as genetic source for breeding programs aimed at developing new varieties with enhanced salt tolerance. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In recent years, climate change has had a direct impact on agricultural production and quality of yields by increasing the frequency and severity of several environmental stresses [ 1 ]. Salt stress is one of these stresses impacting 20% of the world's cultivable land and contributing to around 50% decrease in crop outputs [ 2 , 3 ]. Wheat is the second most globally cultivated crop and is a main source of vegetable proteins and daily calories required for human consumption [ 4 ]. Wheat in Saudi Arabia has a major role in baking industry and its production was around 3–4 million tonnes during the beginning of 1990s. However, wheat yield has decreased to 2.63 million tonnes since 1993 due to various limiting environmental factors including salt stress [ 5 ]. Soil salinity is becoming more severe in the brackish water-irrigated lands constituting a global threat for food production. High salinity represents a considerable constraint to crop production limiting the yield and quality of the crop [ 6 ]. Therefore, it is becoming a hard challenge to boost crop output and meet food security under increasing salinity conditions. High levels of sodium chloride in soil interfere with plant growth imposing various types of stresses, such as osmotic and ionic stresses. Plants have evolved several physiological and biochemical mechanisms as essential responses to these stresses. Stomatal closure has been reported for being one of the most common responses to osmotic stress. The stomatal closure results in a reduction in plant biomass as a consequence of carbon starvation; and over a period of time can lead to early senescence of mature leaves which might be followed by plant death [ 7 ]. It has been revealed that accelerated senescence is an adaptive way by which stressed plants reduce their canopy size and consume carbon and nutrients in their reproductive parts to produce seeds. Although this response is efficient for next generation survival, it leads to a yield decline in annual crops [ 8 ]. In addition, osmotic adjustment within stressed plant cells has been evidenced as a crucial contributor mechanism in acclimation to salt stress in various plant species; sugar beet [ 9 ], cotton [ 10 ], durum wheat [ 11 ] and bean [ 12 ]. Under salt stress, plants osmotically adjust to maintain cellular turgor and the structural integrity of membranes [ 13 ]. Moreover, antioxidant defence systems are another important protective mechanism that is induced under salt stress, which prevents the cellular damage caused by salt-induced ROS accumulation [ 14 ]. Wheat has been widely considered to be a moderately salt tolerant plant and its tolerance and responses to salinity stress vary among different tissues and cultivars [ 15 ]. Salt-tolerance is among the most physiologically complex traits in plants, it is controlled by a number of mechanisms some of which are specific to salt-stress and some are common to other stress types. It seems that plants vary in the set of mechanisms operated under salt-stress, depending on species and cultivar [ 16 ]. Wheat cultivars, which have been developed through selective breeding programmes and genetic selection, demonstrate diverse levels of tolerance to environmental stresses including salt stress indicating great genetic diversity within the species [ 17 ]. The exploration of variations in salt tolerance among different wheat cultivars has become a crucial objective in modern research in agriculture [ 15 ], as a strategic solution to enhance wheat production in salt-affected areas. Various cultivars of wheat have been documented to exhibit differences in their growth and yield outcomes under salinity, showing different levels of tolerance to salt stress [ 18 , 19 ]. Some wheat cultivars show remarkable resilience, exhibiting minimal decrease in growth and yield upon exposure to salinity conditions. In contrast, other cultivars are more susceptible to the stress and thus suffer significant losses. [ 20 ] previously investigated the variations in physiological and biochemical responses between two wheat cultivars under salt stress. They found that the salt-tolerant Suntop cultivar showed lower reductions in growth and photosynthetic efficiency and higher activities of antioxidant enzymes, exhibiting higher tolerance to salinity compared to the salt-sensitive cultivar (Sunmate). Hundreds of wheat cultivars have been grown in different regions of Saudi Arabia since hundreds of years. Many of these cultivars have evolved adaptations to prevailing local conditions, thus, they represent an invaluable germplasm resource that needs proper characterisation. Few studies have been conducted to evaluate the tolerance responses of different Saudi wheat cultivars to abiotic and biotic stresses. For examples, responses of agronomic performance and yield potentials to water stress [ 21 – 23 ], growth and physiological responses to heat stress [ 24 ], responses to pathogen attacks [ 25 ], and responses of morphological traits to gamma irradiations [ 26 ]. Very limited studies have attempted to investigate the different responses of typical Saudi wheat to salt stress [ 27 – 29 ]. Therefore, this investigation was conducted to characterize the differential responses to salt-stress in three Saudi wheat cultivars, Najran, Mebiah and Qiadh) in cultivation in different regions of the Kingdom to potentially reveal the underlying mechanisms for salt tolerance in wheat. The study investigated variation in the physiological and biochemical responses, as well as antioxidant scavenging capacity via phenolics accumulation among the three cultivars. The obtained knowledge constitutes an important addition towards understanding the different salt-tolerance mechanisms in wheat and potentially help to develop wheat cultivars with higher salt-tolerance. Materials and Methods Plant materials and salt stress treatment Seeds of three wheat ( Triticum aestivum) genotypes, Najran, Mebiah and Qiadh were obtained from the Ministry of Environment, Water & Agriculture, Saudi Arabia. Prior to sowing, seeds were stratified by incubation in the dark at 4 o C for 3 days to break seed dormancy and stimulate germination. Six cold-stratified seeds were sown in 2L plastic pots filled with a mixture of John Innes soil compost No. 2, vermiculite 2–5 mm and grit sand in a volume ratio of 2:1:1, respectively. Pots were irrigated with either tap water for control plant-set, 100 mM NaCl solution for yield stage plant-set or 200 mM NaCl solution for seedling stage plant-set then sealed with cling film to maintain moisture. Pots were placed in a controlled growth cabinet under a 16 h light/8 h dark and 20°C day/15°C night and constant 70% humidity. After germination, three randomized seedlings from each pot were retained and watered 3 times a week. One-month old plants were harvested at midday to conduct growth and biochemical measurements. To assess the effect of salt stress on yield output a plants set was harvested after grain filling. Growth and yield analysis Root and shoot of thirty-day old Najran, Mebiah and Qiadh wheat plants were harvested separately, and roots rinsed with tap water. Different growth parameters such as root length (RL), shoot length (SL), root fresh weight (RFW), shoot fresh weight (SFW), root dry weight (RDW) and shoot dry weight (SDW) were recorded. Roots and shoots of each cultivar were grouped into three replicates (each sample consisted of duplicate plants), then frozen in liquid nitrogen and stored at -80°C after grinding them under liquid nitrogen to a fine powder to be used in biochemical analyses. Dry weight was determined after drying plant tissues in an oven at 80°C for 2 days. To evaluate the extent to which the yield was affected by salinity, number of spikes, number of seeds per plant, seeds weight and seed germination rate were obtained. Measurement of Proline content Total free Proline content in control and salt-stressed plants of the three wheat cultivars was measured using a modified colorimetric method described by [ 30 ]. Ground root and shoot samples from each treatment (100 mg each) were transferred to a 2 ml micro centrifuge tube, then homogenized in 1 ml of 3% (w/v) sulphosalicylic acid. The homogenate was clarified by centrifugation at 10,000 g for 3 minutes at room temperature. A volume of 500 µl of each supernatant was mixed with 500 µl of glacial acetic acid and 500 µl of acidic ninhydrin reagent in a 2 ml micro centrifuge tube. To make the nihydrin reagent, 2.5 g ninhydrin was dissolved in 100 mL of a solution made of 60 mL glacial acetic acid, 30 mL diH 2 O and 10 mL 85% orthophosphoric acid. The reaction mixtures were incubated in a heat block at 98 ºC for 1 hour then cooled at room temperature. After cooling, absorbance of the red colour developed in samples was read spectrophotometry at 546 nm. The concentration of Proline in each sample was measured using a standard curve made using commercial pure L-proline and calculated on a dry weight basis (µg Proline mg − 1 DW). Measurement of soluble sugars and starch level Soluble and insoluble carbohydrates were quantified in salt-stressed and unstressed plants from all wheat cultivars using the phenol/sulphuric acid method [ 31 ] based on a colorimetric assay. From ground root and shoot samples, 100 mg plant tissue was homogenized in 1 ml of 80% methanol in an Eppendorf tube and then heated at 80°C for 40 min. The homogenate was centrifuged at 13000 rpm for 10 min at room temperature, then supernatant was transferred to a new tube to be used in soluble sugar assay and the remaining plant tissue kept for measuring starch level. To extract starch, the remaining tissue was washed several times with acetate buffer to remove any traces of glucose. After that, 1.2 ml acetate buffer and 0.2 ml enzyme cocktail were added, to digest starch molecules into glucose equivalent, and incubated overnight at 45 ºC. For enzyme cocktail, 26 mg (300 units) amyloglucosidase and 9 mg (25 units) amylase (Sigma-Aldrich, UK) were mixed in 20 ml acetate buffer. After incubation, the homogenate was centrifuged at 13000 rpm for 10 min at room temperature. Exactly 0.5 ml of each supernatant prepared for either soluble sugar or starch assays was transferred to a glass tube, then 0.5 ml diH 2 O, 0.5 ml 5% phenol and 2.5 ml sulphuric acid were added, respectively and left to cool for 15 min at room temperature. The absorbance of reaction mixtures was read using a spectrophotometer at 483 nm and then plotted against a standard curve created using commercial glucose with different known concentrations. Measurement of total organic acids The content of organic acids in the root and shoot tissues from control and salt treated plants was assessed using a basic titration method. Plant tissues (100mg) were homogenised in 1 ml 80% methanol and incubated at 80°C for 40 min. The extracts were centrifuged at 13000 rpm for 10 min and the supernatants collected. A 20 µl aliquot of plant extract was transferred to a small vial to this, 970 µl distilled water and 10 µl phenolphthalein (10 mg.ml − 1 ) as a pH indicator were added then the total acidity mixture was neutralized with 0.1 N sodium hydroxide, added from a titration burette, until a pink colour was obtained. The volume of sodium hydroxide used was obtained by reading the burette and the titration data was calculated and expressed on a dry weight basis (µmol.mg − 1 DW). Measurement of phenolics content Total phenolics in root and shoot plant materials of the three different wheat cultivars were estimated using the Folin-Ciocalteu (F-C) reagent. To 20 µl of plant extracts, prepared in previous experiment and stored at -20°C, 200 µl of 10% F-C reagent and 800 µl of 0.7 M Na 2 CO 3 were added and mixed thoroughly in a 2 ml tube. The mixture tubes were incubated at room temperature for 120 min. After incubation, tube content was transferred to cuvettes and absorbance readings taken using a spectrophotometer at 265 nm. The levels of phenolic compounds were determined from a standard curve plotted using gallic acid. Results Plant growth and development Growth performance of the three T. aestivum cultivars under salt-stress and control conditions was evaluated by measuring different parameters including RFW, SFW, RDW, SDW, RL and SL. All these growth parameters were at similar levels in the examined cultivars under unstressed conditions, however significant difference appeared under salt-stress between cultivars (Fig. 1). Fresh and dry weight under salinity treatment in both roots and shoots were significantly lower than those in control plants. The three cultivars have shown relatively similar reductions in both RFW and RDW in roots and shoots under salt-stress consisting of 8 to 9-fold and 9 to 10-fold reductions, respectively (Figs. 1A&B). In contrast, RL decreased more in Najran (39%) and Qiadh (38%) than Mebiah (23%), whereas SL was more reduced in Mebiah (40%) than in Qiadh (38%) and Najran (36%) (Figs. 1C&D) Grain yield As shown in Fig. 2A&B, there was a significant difference between the three wheat cultivars regarding their spike and seed numbers. Qiadh had the largest number of spikes in control and NaCl treated plants (3 spikes), and the largest number of seeds in control plants (54 seeds), however it had the smallest number of seeds in NaCl-treated plants (17 seeds). On the other hand, Najran had the lowest number of spikes (1 spike) and seeds (17 seeds) in control plants, while Mebiah had more seeds (43 seeds) and fewer spikes (2 spikes) in salt treated plants. In addition, salt treatment had a positive effect on spike and seed number in Najran and Mebiah, whereas Qiadh displayed a negative salt-effect on both parameters. This result reveals that Qiadh was the most affected cultivar by salt-stress as the number of seeds decreased dramatically (P 0.05), while the seeds number increased slightly and the spikes number increased significantly (P < 0.01) in Najran cultivar. Not only the number of seeds was affected under saline conditions but also the weight of seeds where all wheat cultivars exhibited a significant decline in seed weight (P < 0.001) in comparison to control plants (Fig. 2C). The produced grains of the three wheat cultivars were germinated under control (0 mM NaCl) and saline (200 mM NaCl) conditions. As depicted in Fig. 2D, Mebiah has shown the highest reduction in germination rate of seeds under salt stress (50%) followed by Qiadh (28%) and Najran (8%). Proline content Plants subjected to salt treatment displayed an increased accumulation of Proline in roots and shoots compared to control plants. As shown in Fig. 3, under un-stressed conditions the three wheat cultivars had little Proline content to be measured in their roots and shoots except Qiadh which had a tiny amount of Proline only in its shoot tissues (0.01 µg mg − 1 DW). Salt-stress induced an important increase in proline content in both roots and shoots of the three wheats (Fig. 3). However, a significant difference in free Proline content in root and shoot tissues was observed between the three wheat cultivars under salt treatment (P < 0.01). In response to salt stress, Mebiah had the highest whereas Qiadh had the lowest Proline content in root, 0.17, 0.01 µg mg − 1 DW, respectively. Moreover, Mebiah had the largest content of Proline in its shoots followed by Qiadh and Najran, at 0.86, 0.66 and 0.39 µg mg − 1 DW of the metabolite respectively. Soluble sugars and starch level There was no significant difference between levels of soluble sugars in the three wheat cultivars under unstressed conditions (Fig. 4A&B). However, soluble sugars content differed significantly among salt-treated plants of the three cultivars. It raised under salt-stress in roots, by 7.6-fold in Najran, 5.3-fold in Mebiah and 4.5-fold in Qiadh cultivar, as well as in shoots, by 1.9-fold in Najran, 4.6-fold in Mebiah and 4.9-fold in Qiadh cultivar. In contrast, there was a significant variation between wheat cultivars regarding the starch level in roots and shoots of control plants (Fig. 4C&D). Qiadh displayed almost no starch in roots while Najran exhibited 0.06 µg mg − 1 DW of starch in its roots, while 0.04 and 0.16 µg mg − 1 DW accumulated in the shoots of the two cultivars respectively. Salt stress led to a significant boost in starch accumulation where Qiadh had the most increase in starch levels in both roots and shoots whereas Mebiah and Najran had the lowest starch levels in their roots and shoots, respectively relatively to the control. Total organic acids Levels of total organic acids in the roots and shoots of control plants were significantly different between the three T. aestivum cultivars (P < 0.01). Salt-stress resulted in a big increase in total organic acids in the three wheat cultivars (P < 0.05) (Fig. 5). In the Najran cultivar which has shown higher salt tolerance, salt-stress increased content of organic acids 6.3 folds in the root whereas in Qiadh which exhibited less stress tolerance, only a 1.7-fold increase was measured under salt-stress. Salt-stress induced the highest increases in total organic acids of 35 folds and 19.3 folds in the shoots of Qiadh and Najran, respectively. Phenolics content NaCl treatment significantly enhanced the production of phenolics in root and shoot tissues of Najran, Mebiah and Qiadh wheats. As seen in Fig. 6, a pronounced increase of phenolics content was observed in the roots and shoots of salt-treated plants of the three cultivars compared to the control. Higher levels of phenolics content of 3.48 and 3.20 nmol.mg − 1 DW were recorded in Najran whereas Qiadh showed lower values of phenolic compounds of 1.83 and 1.87 nmol.mg − 1 DW in their roots and shoots, respectively. Discussion Within T. aestivum species, different cultivars respond variably to biotic and abiotic stresses [ 32 , 33 , 34 ]. Understanding these differences is essential for crop improvement. This can inform the efforts aiming at developing salt-tolerant crops including wheat and can help optimize agricultural practices in salt-affected regions such as Saudi Arabia. The variability in salt stress responses among wheat cultivars might be attributed to differences in the genetic background, these differences control salt stress perception and signalling pathways, osmotic adjustment capacity, ion transport and compartmentalization, and the activation of stress-response genes [ 35 , 36 ]. Plant growth, Salt stress reduced differentially the growth of the three wheat cultivars. Salinity alters plant growth and development, increasing NaCl concentrations in the growth medium result in adverse effects on plant growth and survival. Salinity leads to severe impact on the physiology traits of wheat plants including total biomass, however, the sensitivity of salt impact varies greatly among wheat cultivars [ 37 ]. In the current study, a distinctive variation in salt tolerance was observed between three cultivars, Najran was the most tolerant to saline cues while Qiadh was the most susceptible cultivar. Under salt stress, a dramatic decline in mean fresh and dry weights as well as lengths of root and shoot tissues was measured in all cultivars, but the reduction was significantly lower in the NaCl-tolerant cultivar Najran than in the NaCl-sensitive cultivar Qiadh. This decline in all growth parameters might be a consequence of the increased salt concentration around root area which in turn causes water deficit, nutritional imbalance and osmotic stress in the plant resulting in stomatal closure [ 38 ]. Moreover, prolonged exposure of plants to salinity leads to ion toxicity in the leaves and severely affects photosynthetic reactions, cell division and cell elongation which results in a reduction in root and shoot lengths [ 39 ]. Yield, salt-stress impacted differentially the spike and seed number and seed weight and germination. It is well known that salt stress influences negatively the expansion of plant leaves causing a reduction in photosynthetic capacity which in turn affects the quantity and quality of grain yield [ 40 ]. The results obtained in the present study confirm this, where Qiadh cultivar showed the highest reduction in fresh and dry weights of the shoot as well as in yield production compared to the other two cultivars. In contrast, salinity had a positive effect on spike and seed numbers in Najran and Mebiah whereas seed weight had been negatively affected by NaCl. These findings are in line with those of [ 18 ], who found a significant decline in yield outputs of all tested wheat cultivars except Sakha 94 and Sids 13 cultivars, suggesting that while most wheat cultivars are sensitive to salinity some cultivars are salt tolerant, our results suggest that Najran wheat is among the salt-tolerant cultivars. Interestingly, the Najran salt-tolerant cultivar seems to have invested carbon in producing more lighter seeds under salt-stress while maintaining a relatively high rate of germination (around 90%), in contrast to Mebiah which increased the number of seeds without maintaining good germination rate (less than 50%). In the salt-sensitive Qiadh cultivar not only the number of seeds produced under salinity declined but also the germination rate was reduced (around 70%). Choosing to maintain itself under salinity stress via an increase in seed number while maintaining a very high germination rate testifies for an advanced adaptation to salt-stress in Najran wheat. It is crucial to find the genetic determinants of this trait, several genes might be involved in the control of spike and seed numbers, the discovery of these genes is underway and some suspected transcription factors involved in the control of the developmental processes leading to spikes and seeds have already been identified mainly in rice [ 41 ]. Considerable effort is being put into identifying the orthologs of these genes as well as other genes potentially involved in the control of yield in wheat using mainly quantitative trait loci mapping. Osmoregulation, concomitant production of different substances for osmotic adjustment is not essentially required for salt tolerance. Prolonged high salt concentrations impair plant growth due to the resulting hyperionic and hyperosmotic stresses. Plants respond to these stresses by implementing biochemical mechanisms to facilitate water uptake and therefore maintain cell turgor and plant growth [ 42 ]. Osmotic adjustment is one of the crucial biochemical strategies in plant acclimation to salt stress. Proline, soluble sugars, starch and organic acids are of the main organic osmotica which are synthesized within plants to assist survival under salt stress. It is demonstrated in this study that different T. aestivum cultivars might employ various mechanisms to alleviate the harmful effects of saline stress. For example, Mebiah had the highest free Proline content, whereas Qiadh and Najran had the lowest Proline concentration in both root and shoot tissues. These results might be interpreted that Mebiah responded to the high level of NaCl by producing Proline which not only plays a great role in osmoregulation but also protects plants from the damage caused by ROS and toxic ions. Proline has been found to participate in lowering osmotic potential [ 43 ], storing carbon and nitrogen [ 44 ], detoxifying ROS [ 45 ], protecting the enzyme activities of photosynthesis and production of antioxidants [ 46 ] and inducing adaptive responses by acting as a stress signal [ 47 ] under unfavourable conditions. Another example, Najran wheat exhibited higher accumulation of soluble sugars in the roots and shoots while Qiadh had the lowest content of soluble sugars. This accumulation may participate in keeping the photosynthetic activity leading to maintaining plant biomass as these soluble sugars act as building blocks of macromolecules. These findings were accompanied with what we had found in growth analysis where Najran showed the highest fresh and dry weights under salt treatment compared to the other cultivars. In contrast, Qiadh displayed the highest increase in starch levels in both roots and shoots in comparison with the two other cultivars. Increasing the level of soluble sugars and decreasing starch levels might be a critical trait in salt tolerant cultivars. Similar results were reported by [ 48 ], who found a higher accumulation of soluble sugars in salt tolerant rice genotype, Pokkali, suggesting their important role in osmotic adjustment and accumulation of carbon energy reserves in plants. [ 49 ] have pointed out a decline in starch concentration in salt-treated leaves of Oryza sativa L. as a result of carbon limitation due to the poor photosynthetic activity under salt-stress, the decline in this case might be a result of the suppression of starch biosynthesis. Organic acids are ubiquitous metabolites in plants which accumulate in response to salt stress to act as compatible solutes for osmoregulation and as ROS scavenger as well as plant protectors. The accumulation of total organic acids in the salt stressed wheat cultivars was obvious in the roots, however it decreased in the shoot of Najran and Qiadh cultivars. These findings are consistent with a previous study that confirmed the increase of organic acids in root tissues and their depletion in the leaves under saline treatment, suggesting that these different levels of organic acids might be attributed to organ-specific functions [ 50 ]. In the case of salt stress, roots uptake excessive amounts of sodium cations which require anions to balance the charge. Thus, organic acids are more accumulated in the roots to enhance the cation–anion balance. Furthermore, their high level in the roots assist plants to osmotically adjust under salinity conditions. Anti-oxidant, salt stress enhanced phenolics production as an antioxidant response Salt-stressed plants respond to oxidative stress resulting from the accumulations of ROS by operating an antioxidant-defence systems that prevent damage caused by ROS and detoxify ROS molecules. Phenolics are one of the nonenzymatic antioxidants produced to mainly protect plants against various stresses and act as ROS scavengers. It would seem that the activity of the ROS- defence systems rises under extreme environmental stresses and is more pronounced in tolerant plants than sensitive ones [ 51 – 53 ]. This suggests that the defence systems perhaps work more effectively under unfavourable conditions. In the current study, prolonged saline stress has shown significant accumulation of total phenolics in all wheat cultivars and was more pronounced in Najran followed by Mebiah and Qiadh, confirming that wheat cultivars with different sensitivity to NaCl stress exhibit different levels of metabolites alteration. Previous studies [ 54 ] have pointed out possible involvement of phenolics particularly phenylpropanoids in salt-tolerance in wheat. The expression of many of the genes involved in the productions of these substances increase under salt-stress in Najran wheat [ 54 ]. It would be of interest to extend transcriptomics analysis to the Qiadh cultivar and compare the results to those previously obtained in Najran. Conclusion In the current study, three Saudi wheat cultivars; Najran, Mebiah and Qiadh varying in their salt-tolerance have been investigated for the effect of salt stress on their physiological and biochemical responses. Salt stress caused differential reduction in physiological activities and grain yield depending on wheat cultivar. Moreover, shoot and root tissues from different wheat cultivars with different sensitivity to NaCl stress exhibited different metabolic alterations and antioxidative responses. These salinity effects were less pronounced in the Najran cultivar potentially due to its high osmotic and antioxidant responses therefore it was characterized as the most tolerant cultivar to salt stress. Declarations Consent for publication The Authors consent to the publication by BMC Plant Biology of all the the data presented in the manuscript. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors have no competing interest with any party regarding the results of the study presented in this paper. Funding Funding for this work was provided by King Khaled University, Saudi Arabia. Authors' contributions TT: Conceptualization, Methodology, Validation, Resources, Writing - Review & Editing, Visualization, Supervision, Project administration. NA: Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft, Visualization, Project administration, Funding acquisition. Acknowledgements The authors would like to thank Khaled University, Saudi Arabia for providing financial support to this work. Authors' information (optional) Dr. Tahar Taybi Room 4.57, Ridley Building 2 School of Natural and environmantal Sciences Newcastle University, Newcastle upon Tyne NE1 7RU, UK e-mail: [email protected] Dr. Norah Alyahya Devonshire building School of Natural and environmantal Sciences Newcastle University, Newcastle upon Tyne NE1 7RU, UK e-mail: [email protected] References Ahsan F, Chandio AA, Fang W.. 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PLoS One. 2020 May 29;15(5):e0233878. doi: 10.1371/journal.pone.0233878. PMID: 32470094; PMCID: PMC7259642. Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59:651-81. doi: 10.1146/annurev.arplant.59.032607.092911. PMID: 18444910. Gupta B, Huang B. Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. Int J Genomics. 2014;2014:701596. doi: 10.1155/2014/701596. Epub 2014 Apr 3. PMID: 24804192; PMCID: PMC3996477. Asheaf MA, Ashraf M, Ali Q. Response of two genetically diverse wheat cultivars to salt stress at different growth stages: leaf lipid peroxidation and phenolic contents. Pak J Bot. 2010; 42 : 559-565. Chavarria G, Dos Santos HP. Plant water relations: absorption, transport and control mechanisms. Embrapa Uva e Vinho-Capítulo em livro científico (ALICE) . 2012. Munns R. Comparative physiology of salt and water stress. Plant, cell & environment . 2002;25 : 239-250. Taleisnik E, Rodríguez AA, Bustos D, Erdei L, Ortega L, Senn ME. Leaf expansion in grasses under salt stress. J Plant Physiol. 2009 Jul 15;166(11):1123-40. doi: 10.1016/j.jplph.2009.03.015. Epub 2009 May 20. PMID: 19467732. Nadolska-Orckzyk A, Rajchel IK, Orczy W, Gasparis S. Major genes determining yield-related traits in wheat and barley. Theor Appl Genet. 2017; 130(6):1081-1098. doi: 10.1007/s00122-017-2880-x. Epub 2017 Mar 17. PMID: 28314933; PMCID: PMC5440550. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ. PLANT CELLULAR AND MOLECULAR RESPONSES TO HIGH SALINITY. Annu Rev Plant Physiol Plant Mol Biol. 2000 Jun;51:463-499. doi: 10.1146/annurev.arplant.51.1.463. PMID: 15012199. Verbruggen N, Hermans C. Proline accumulation in plants: a review. Amino Acids. 2008 Nov;35(4):753-9. doi: 10.1007/s00726-008-0061-6. Epub 2008 Apr 1. PMID: 18379856. Hare P, Cress W. Metabolic implications of stress-induced proline accumulation in plants. Plant growth regulation . 1997; 21 : 79-102. Szabados L, Savouré A. Proline: a multifunctional amino acid. Trends Plant Sci. 2010 Feb;15(2):89-97. doi: 10.1016/j.tplants.2009.11.009. Epub 2009 Dec 23. PMID: 20036181. Surender Reddy P, Jogeswar G, Rasineni GK, Maheswari M, Reddy AR, Varshney RK, Kavi Kishor PB. Proline over-accumulation alleviates salt stress and protects photosynthetic and antioxidant enzyme activities in transgenic sorghum [Sorghum bicolor (L.) Moench]. Plant Physiol Biochem. 2015 Sep;94:104-13. doi: 10.1016/j.plaphy.2015.05.014. Epub 2015 May 30. PMID: 26065619. Maggio A, Miyazaki S, Veronese P, Fujita T, Ibeas JI, Damsz B, Narasimhan ML, Hasegawa PM, Joly RJ, Bressan RA. Does proline accumulation play an active role in stress-induced growth reduction? Plant J. 2002 Sep;31(6):699-712. doi: 10.1046/j.1365-313x.2002.01389.x. PMID: 12220262. Boriboonkaset T, Theerawitaya C, Yamada N, Pichakum A, Supaibulwatana K, Cha-Um S, Takabe T, Kirdmanee C. Regulation of some carbohydrate metabolism-related genes, starch and soluble sugar contents, photosynthetic activities and yield attributes of two contrasting rice genotypes subjected to salt stress. Protoplasma. 2013 Oct;250(5):1157-67. doi: 10.1007/s00709-013-0496-9. Epub 2013 Apr 5. PMID: 23558902. Zhu Y, Chen H, Fan J, Wang Y, Li Y, Chen J, Fan J, Yang S, Hu L, Leung H, Mew TW, Teng PS, Wang Z, Mundt CC. Genetic diversity and disease control in rice. Nature. 2000 Aug 17;406(6797):718-22. doi: 10.1038/35021046. PMID: 10963595.49. Zhao X, Wang W, Zhang F, Deng J, Li Z, Fu B.. Comparative metabolite profiling of two rice genotypes with contrasting salt stress tolerance at the seedling stage. PloS one . 2014;9 : e108020. Hernandez JA, Jimenez A, Mullineaux P, Sevilla F. Tolerance of pea (Pisum sativum L.) to long‐term salt stress is associated with induction of antioxidant defences. Plant, cell & environment. 2000;23 : 853-862. Sairam R, Srivastava G, Saxena D. Increased antioxidant activity under elevated temperatures: a mechanism of heat stress tolerance in wheat genotypes. Biologia Plantarum . 2000; 43 : 245-251. Zhou R, Kong L, Yu X, Ottosen CO, Zhao T, Jiang F, Wu Z. Oxidative damage and antioxidant mechanism in tomatoes responding to drought and heat stress. Acta physiologiae plantarum. 2019; 41 : 20. Alyahya N, Taybi T. Comparative transcriptomic profiling reveals differentially expressed genes and important related metabolic pathways in shoots and roots of a Saudi wheat cultivar (Najran) under salinity stress. Front Plant Sci.2023;28:14:1225541. doi: 10.3389/fpls.2023.1225541. PMID: 37588415; PMCID: PMC10425591. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4284092","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296704321,"identity":"0397ed90-241b-40e6-837b-6dda44cf90d4","order_by":0,"name":"Norah Alyahya","email":"","orcid":"","institution":"Newcastle University","correspondingAuthor":false,"prefix":"","firstName":"Norah","middleName":"","lastName":"Alyahya","suffix":""},{"id":296704323,"identity":"3054c4ed-0102-49f0-bd76-db26f4f55560","order_by":1,"name":"Tahar Taybi","email":"data:image/png;base64,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","orcid":"","institution":"Newcastle University","correspondingAuthor":true,"prefix":"","firstName":"Tahar","middleName":"","lastName":"Taybi","suffix":""}],"badges":[],"createdAt":"2024-04-17 21:52:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4284092/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4284092/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55729204,"identity":"d0b71dba-bd64-42f1-84d8-f0fc5ea99207","added_by":"auto","created_at":"2024-05-02 10:48:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":578040,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salt-stress on (A\u0026amp;B) root and shoot fresh weight, (C\u0026amp;D) root and shoot dry weight, and (E\u0026amp;F) root and shoot lengths of three wheat \u003cem\u003e(Triticum aestivum)\u003c/em\u003e cultivars, Najran, Mebiah and Qiadh, (n=6 +/- S.E). Salt-treated plants were watered with 200 mM NaCl whereas control plants were watered with 0 mM NaCl (tap water). Asterisks refer to significant differences at confidence levels of * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/b3a774e4cbcc020eaab6c0bd.png"},{"id":55729201,"identity":"12c40052-2490-4bea-9253-8c5fba19b392","added_by":"auto","created_at":"2024-05-02 10:48:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":357102,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salt-stress on (A) spikes number, (B) seeds number, (C) seeds weight and (D) germination rate of three wheat \u003cem\u003e(Triticum aestivum)\u003c/em\u003ecultivars, Najran, Mebiah and Qiadh. Salt-treated plants were watered with 200 mM NaCl whereas control plants were watered with 0 mM NaCl (tap water). Asterisks refer to significant differences at confidence levels of * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/2faae457259cb3511eb425b6.png"},{"id":55729203,"identity":"18d51648-8721-4631-8e94-67553600c55c","added_by":"auto","created_at":"2024-05-02 10:48:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":213538,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salt-stress on Proline content in (A) roots and (B) shoots of three wheat \u003cem\u003e(Triticum aestivum)\u003c/em\u003e cultivars, Najran, Mebiah, and Qiadh (n=3 +/- S.E). Salt-treated plants were watered with 200 mM NaCl whereas control plants were watered with 0 mM NaCl (tap water). Asterisks refer to significant differences at confidence levels of * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/d3374d37adfb739ba48ccf8c.png"},{"id":55729198,"identity":"f8b712bc-d558-4090-8fe3-c934e5a58ac4","added_by":"auto","created_at":"2024-05-02 10:48:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":386373,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salt-stress on (A\u0026amp;B) levels of soluble sugars and (C\u0026amp;D) starch content in roots and shoots of three wheat \u003cem\u003e(Triticum aestivum)\u003c/em\u003ecultivars, Najran, Mediah and Qiadh (n=3 +/- S.E). Salt-treated plants were watered with 200 mM NaCl whereas control plants were watered with 0 mM NaCl (tap water). Asterisks refer to significant differences at confidence levels of * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/9aaf6379dc913f41af22b908.png"},{"id":55729200,"identity":"656089f8-c7c2-4392-9084-37355471145b","added_by":"auto","created_at":"2024-05-02 10:48:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":250426,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of sat-stress on the content of total organic acids in (A) roots and (B) shoots of three wheat \u003cem\u003e(Triticum aestivum)\u003c/em\u003e cultivars, Najran, Mediah and Qiadh (n=3 +/- S.E). Salt-treated plants were watered with 200 mM NaCl whereas control plants were watered with 0 mM NaCl (tap water). Asterisks refer to significant differences at confidence levels of * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/75ce67e7708c372ba0fcd6c7.png"},{"id":55729202,"identity":"210188d0-96e2-4bea-991d-c22c37086c91","added_by":"auto","created_at":"2024-05-02 10:48:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":195391,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salt-stress on total phenolics content in (A) roots and (B) shoots of three wheat \u003cem\u003e(Triticum aestivum)\u003c/em\u003e cultivars, Najran, Mediah and Qiadh (n=3 +/- S.E). Salt-treated plants were watered with 200 mM NaCl whereas control plants were watered with 0 mM NaCl (tap water). Asterisks refer to significant differences at confidence levels of * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/a3864c038e2c0f86f301a87f.png"},{"id":56755896,"identity":"5d36b197-cd5a-4cad-bc92-ed797e67a707","added_by":"auto","created_at":"2024-05-20 05:31:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2180003,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4284092/v1/27d8b25d-d277-4fee-be9e-55413f060ee3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative analysis of physiological and biochemical responses to salt-stress reveals key mechanisms of salt-tolerance in some Saudi Wheat Cultivars","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, climate change has had a direct impact on agricultural production and quality of yields by increasing the frequency and severity of several environmental stresses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Salt stress is one of these stresses impacting 20% of the world's cultivable land and contributing to around 50% decrease in crop outputs [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Wheat is the second most globally cultivated crop and is a main source of vegetable proteins and daily calories required for human consumption [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Wheat in Saudi Arabia has a major role in baking industry and its production was around 3\u0026ndash;4\u0026nbsp;million tonnes during the beginning of 1990s. However, wheat yield has decreased to 2.63\u0026nbsp;million tonnes since 1993 due to various limiting environmental factors including salt stress [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Soil salinity is becoming more severe in the brackish water-irrigated lands constituting a global threat for food production. High salinity represents a considerable constraint to crop production limiting the yield and quality of the crop [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, it is becoming a hard challenge to boost crop output and meet food security under increasing salinity conditions.\u003c/p\u003e \u003cp\u003eHigh levels of sodium chloride in soil interfere with plant growth imposing various types of stresses, such as osmotic and ionic stresses. Plants have evolved several physiological and biochemical mechanisms as essential responses to these stresses. Stomatal closure has been reported for being one of the most common responses to osmotic stress. The stomatal closure results in a reduction in plant biomass as a consequence of carbon starvation; and over a period of time can lead to early senescence of mature leaves which might be followed by plant death [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It has been revealed that accelerated senescence is an adaptive way by which stressed plants reduce their canopy size and consume carbon and nutrients in their reproductive parts to produce seeds. Although this response is efficient for next generation survival, it leads to a yield decline in annual crops [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition, osmotic adjustment within stressed plant cells has been evidenced as a crucial contributor mechanism in acclimation to salt stress in various plant species; sugar beet [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], cotton [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], durum wheat [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and bean [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Under salt stress, plants osmotically adjust to maintain cellular turgor and the structural integrity of membranes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Moreover, antioxidant defence systems are another important protective mechanism that is induced under salt stress, which prevents the cellular damage caused by salt-induced ROS accumulation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWheat has been widely considered to be a moderately salt tolerant plant and its tolerance and responses to salinity stress vary among different tissues and cultivars [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Salt-tolerance is among the most physiologically complex traits in plants, it is controlled by a number of mechanisms some of which are specific to salt-stress and some are common to other stress types. It seems that plants vary in the set of mechanisms operated under salt-stress, depending on species and cultivar [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Wheat cultivars, which have been developed through selective breeding programmes and genetic selection, demonstrate diverse levels of tolerance to environmental stresses including salt stress indicating great genetic diversity within the species [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The exploration of variations in salt tolerance among different wheat cultivars has become a crucial objective in modern research in agriculture [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], as a strategic solution to enhance wheat production in salt-affected areas. Various cultivars of wheat have been documented to exhibit differences in their growth and yield outcomes under salinity, showing different levels of tolerance to salt stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Some wheat cultivars show remarkable resilience, exhibiting minimal decrease in growth and yield upon exposure to salinity conditions. In contrast, other cultivars are more susceptible to the stress and thus suffer significant losses. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] previously investigated the variations in physiological and biochemical responses between two wheat cultivars under salt stress. They found that the salt-tolerant Suntop cultivar showed lower reductions in growth and photosynthetic efficiency and higher activities of antioxidant enzymes, exhibiting higher tolerance to salinity compared to the salt-sensitive cultivar (Sunmate).\u003c/p\u003e \u003cp\u003eHundreds of wheat cultivars have been grown in different regions of Saudi Arabia since hundreds of years. Many of these cultivars have evolved adaptations to prevailing local conditions, thus, they represent an invaluable germplasm resource that needs proper characterisation. Few studies have been conducted to evaluate the tolerance responses of different Saudi wheat cultivars to abiotic and biotic stresses. For examples, responses of agronomic performance and yield potentials to water stress [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], growth and physiological responses to heat stress [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], responses to pathogen attacks [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and responses of morphological traits to gamma irradiations [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Very limited studies have attempted to investigate the different responses of typical Saudi wheat to salt stress [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, this investigation was conducted to characterize the differential responses to salt-stress in three Saudi wheat cultivars, Najran, Mebiah and Qiadh) in cultivation in different regions of the Kingdom to potentially reveal the underlying mechanisms for salt tolerance in wheat. The study investigated variation in the physiological and biochemical responses, as well as antioxidant scavenging capacity via phenolics accumulation among the three cultivars. The obtained knowledge constitutes an important addition towards understanding the different salt-tolerance mechanisms in wheat and potentially help to develop wheat cultivars with higher salt-tolerance.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and salt stress treatment\u003c/h2\u003e \u003cp\u003eSeeds of three wheat (\u003cem\u003eTriticum aestivum)\u003c/em\u003e genotypes, Najran, Mebiah and Qiadh were obtained from the Ministry of Environment, Water \u0026amp; Agriculture, Saudi Arabia. Prior to sowing, seeds were stratified by incubation in the dark at 4\u003csup\u003eo\u003c/sup\u003eC for 3 days to break seed dormancy and stimulate germination. Six cold-stratified seeds were sown in 2L plastic pots filled with a mixture of John Innes soil compost No. 2, vermiculite 2\u0026ndash;5 mm and grit sand in a volume ratio of 2:1:1, respectively. Pots were irrigated with either tap water for control plant-set, 100 mM NaCl solution for yield stage plant-set or 200 mM NaCl solution for seedling stage plant-set then sealed with cling film to maintain moisture. Pots were placed in a controlled growth cabinet under a 16 h light/8 h dark and 20\u0026deg;C day/15\u0026deg;C night and constant 70% humidity. After germination, three randomized seedlings from each pot were retained and watered 3 times a week. One-month old plants were harvested at midday to conduct growth and biochemical measurements. To assess the effect of salt stress on yield output a plants set was harvested after grain filling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGrowth and yield analysis\u003c/h2\u003e \u003cp\u003eRoot and shoot of thirty-day old Najran, Mebiah and Qiadh wheat plants were harvested separately, and roots rinsed with tap water. Different growth parameters such as root length (RL), shoot length (SL), root fresh weight (RFW), shoot fresh weight (SFW), root dry weight (RDW) and shoot dry weight (SDW) were recorded. Roots and shoots of each cultivar were grouped into three replicates (each sample consisted of duplicate plants), then frozen in liquid nitrogen and stored at -80\u0026deg;C after grinding them under liquid nitrogen to a fine powder to be used in biochemical analyses. Dry weight was determined after drying plant tissues in an oven at 80\u0026deg;C for 2 days. To evaluate the extent to which the yield was affected by salinity, number of spikes, number of seeds per plant, seeds weight and seed germination rate were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Proline content\u003c/h2\u003e \u003cp\u003eTotal free Proline content in control and salt-stressed plants of the three wheat cultivars was measured using a modified colorimetric method described by [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Ground root and shoot samples from each treatment (100 mg each) were transferred to a 2 ml micro centrifuge tube, then homogenized in 1 ml of 3% (w/v) sulphosalicylic acid. The homogenate was clarified by centrifugation at 10,000 g for 3 minutes at room temperature. A volume of 500 \u0026micro;l of each supernatant was mixed with 500 \u0026micro;l of glacial acetic acid and 500 \u0026micro;l of acidic ninhydrin reagent in a 2 ml micro centrifuge tube. To make the nihydrin reagent, 2.5 g ninhydrin was dissolved in 100 mL of a solution made of 60 mL glacial acetic acid, 30 mL diH\u003csub\u003e2\u003c/sub\u003eO and 10 mL 85% orthophosphoric acid. The reaction mixtures were incubated in a heat block at 98 \u0026ordm;C for 1 hour then cooled at room temperature. After cooling, absorbance of the red colour developed in samples was read spectrophotometry at 546 nm. The concentration of Proline in each sample was measured using a standard curve made using commercial pure L-proline and calculated on a dry weight basis (\u0026micro;g Proline mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of soluble sugars and starch level\u003c/h2\u003e \u003cp\u003eSoluble and insoluble carbohydrates were quantified in salt-stressed and unstressed plants from all wheat cultivars using the phenol/sulphuric acid method [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] based on a colorimetric assay. From ground root and shoot samples, 100 mg plant tissue was homogenized in 1 ml of 80% methanol in an Eppendorf tube and then heated at 80\u0026deg;C for 40 min. The homogenate was centrifuged at 13000 rpm for 10 min at room temperature, then supernatant was transferred to a new tube to be used in soluble sugar assay and the remaining plant tissue kept for measuring starch level. To extract starch, the remaining tissue was washed several times with acetate buffer to remove any traces of glucose. After that, 1.2 ml acetate buffer and 0.2 ml enzyme cocktail were added, to digest starch molecules into glucose equivalent, and incubated overnight at 45 \u0026ordm;C. For enzyme cocktail, 26 mg (300 units) amyloglucosidase and 9 mg (25 units) amylase (Sigma-Aldrich, UK) were mixed in 20 ml acetate buffer. After incubation, the homogenate was centrifuged at 13000 rpm for 10 min at room temperature. Exactly 0.5 ml of each supernatant prepared for either soluble sugar or starch assays was transferred to a glass tube, then 0.5 ml diH\u003csub\u003e2\u003c/sub\u003eO, 0.5 ml 5% phenol and 2.5 ml sulphuric acid were added, respectively and left to cool for 15 min at room temperature. The absorbance of reaction mixtures was read using a spectrophotometer at 483 nm and then plotted against a standard curve created using commercial glucose with different known concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of total organic acids\u003c/h2\u003e \u003cp\u003eThe content of organic acids in the root and shoot tissues from control and salt treated plants was assessed using a basic titration method. Plant tissues (100mg) were homogenised in 1 ml 80% methanol and incubated at 80\u0026deg;C for 40 min. The extracts were centrifuged at 13000 rpm for 10 min and the supernatants collected. A 20 \u0026micro;l aliquot of plant extract was transferred to a small vial to this, 970 \u0026micro;l distilled water and 10 \u0026micro;l phenolphthalein (10 mg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as a pH indicator were added then the total acidity mixture was neutralized with 0.1 N sodium hydroxide, added from a titration burette, until a pink colour was obtained. The volume of sodium hydroxide used was obtained by reading the burette and the titration data was calculated and expressed on a dry weight basis (\u0026micro;mol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of phenolics content\u003c/h2\u003e \u003cp\u003eTotal phenolics in root and shoot plant materials of the three different wheat cultivars were estimated using the Folin-Ciocalteu (F-C) reagent. To 20 \u0026micro;l of plant extracts, prepared in previous experiment and stored at -20\u0026deg;C, 200 \u0026micro;l of 10% F-C reagent and 800 \u0026micro;l of 0.7 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e were added and mixed thoroughly in a 2 ml tube. The mixture tubes were incubated at room temperature for 120 min. After incubation, tube content was transferred to cuvettes and absorbance readings taken using a spectrophotometer at 265 nm. The levels of phenolic compounds were determined from a standard curve plotted using gallic acid.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth and development\u003c/h2\u003e \u003cp\u003eGrowth performance of the three \u003cem\u003eT. aestivum\u003c/em\u003e cultivars under salt-stress and control conditions was evaluated by measuring different parameters including RFW, SFW, RDW, SDW, RL and SL. All these growth parameters were at similar levels in the examined cultivars under unstressed conditions, however significant difference appeared under salt-stress between cultivars (Fig.\u0026nbsp;1). Fresh and dry weight under salinity treatment in both roots and shoots were significantly lower than those in control plants. The three cultivars have shown relatively similar reductions in both RFW and RDW in roots and shoots under salt-stress consisting of 8 to 9-fold and 9 to 10-fold reductions, respectively (Figs.\u0026nbsp;1A\u0026amp;B). In contrast, RL decreased more in Najran (39%) and Qiadh (38%) than Mebiah (23%), whereas SL was more reduced in Mebiah (40%) than in Qiadh (38%) and Najran (36%) (Figs.\u0026nbsp;1C\u0026amp;D)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGrain yield\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;2A\u0026amp;B, there was a significant difference between the three wheat cultivars regarding their spike and seed numbers. Qiadh had the largest number of spikes in control and NaCl treated plants (3 spikes), and the largest number of seeds in control plants (54 seeds), however it had the smallest number of seeds in NaCl-treated plants (17 seeds). On the other hand, Najran had the lowest number of spikes (1 spike) and seeds (17 seeds) in control plants, while Mebiah had more seeds (43 seeds) and fewer spikes (2 spikes) in salt treated plants. In addition, salt treatment had a positive effect on spike and seed number in Najran and Mebiah, whereas Qiadh displayed a negative salt-effect on both parameters. This result reveals that Qiadh was the most affected cultivar by salt-stress as the number of seeds decreased dramatically (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and the number of spikes reduced slightly (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while the seeds number increased slightly and the spikes number increased significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in Najran cultivar.\u003c/p\u003e \u003cp\u003eNot only the number of seeds was affected under saline conditions but also the weight of seeds where all wheat cultivars exhibited a significant decline in seed weight (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in comparison to control plants (Fig.\u0026nbsp;2C). The produced grains of the three wheat cultivars were germinated under control (0 mM NaCl) and saline (200 mM NaCl) conditions. As depicted in Fig.\u0026nbsp;2D, Mebiah has shown the highest reduction in germination rate of seeds under salt stress (50%) followed by Qiadh (28%) and Najran (8%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProline content\u003c/h2\u003e \u003cp\u003ePlants subjected to salt treatment displayed an increased accumulation of Proline in roots and shoots compared to control plants. As shown in Fig.\u0026nbsp;3, under un-stressed conditions the three wheat cultivars had little Proline content to be measured in their roots and shoots except Qiadh which had a tiny amount of Proline only in its shoot tissues (0.01 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW). Salt-stress induced an important increase in proline content in both roots and shoots of the three wheats (Fig.\u0026nbsp;3). However, a significant difference in free Proline content in root and shoot tissues was observed between the three wheat cultivars under salt treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In response to salt stress, Mebiah had the highest whereas Qiadh had the lowest Proline content in root, 0.17, 0.01 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW, respectively. Moreover, Mebiah had the largest content of Proline in its shoots followed by Qiadh and Najran, at 0.86, 0.66 and 0.39 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW of the metabolite respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSoluble sugars and starch level\u003c/h2\u003e \u003cp\u003eThere was no significant difference between levels of soluble sugars in the three wheat cultivars under unstressed conditions (Fig.\u0026nbsp;4A\u0026amp;B). However, soluble sugars content differed significantly among salt-treated plants of the three cultivars. It raised under salt-stress in roots, by 7.6-fold in Najran, 5.3-fold in Mebiah and 4.5-fold in Qiadh cultivar, as well as in shoots, by 1.9-fold in Najran, 4.6-fold in Mebiah and 4.9-fold in Qiadh cultivar.\u003c/p\u003e \u003cp\u003eIn contrast, there was a significant variation between wheat cultivars regarding the starch level in roots and shoots of control plants (Fig.\u0026nbsp;4C\u0026amp;D). Qiadh displayed almost no starch in roots while Najran exhibited 0.06 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW of starch in its roots, while 0.04 and 0.16 \u0026micro;g mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW accumulated in the shoots of the two cultivars respectively. Salt stress led to a significant boost in starch accumulation where Qiadh had the most increase in starch levels in both roots and shoots whereas Mebiah and Najran had the lowest starch levels in their roots and shoots, respectively relatively to the control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTotal organic acids\u003c/h2\u003e \u003cp\u003eLevels of total organic acids in the roots and shoots of control plants were significantly different between the three \u003cem\u003eT. aestivum\u003c/em\u003e cultivars (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Salt-stress resulted in a big increase in total organic acids in the three wheat cultivars (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;5). In the Najran cultivar which has shown higher salt tolerance, salt-stress increased content of organic acids 6.3 folds in the root whereas in Qiadh which exhibited less stress tolerance, only a 1.7-fold increase was measured under salt-stress. Salt-stress induced the highest increases in total organic acids of 35 folds and 19.3 folds in the shoots of Qiadh and Najran, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhenolics content\u003c/h2\u003e \u003cp\u003eNaCl treatment significantly enhanced the production of phenolics in root and shoot tissues of Najran, Mebiah and Qiadh wheats. As seen in Fig.\u0026nbsp;6, a pronounced increase of phenolics content was observed in the roots and shoots of salt-treated plants of the three cultivars compared to the control. Higher levels of phenolics content of 3.48 and 3.20 nmol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW were recorded in Najran whereas Qiadh showed lower values of phenolic compounds of 1.83 and 1.87 nmol.mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DW in their roots and shoots, respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWithin \u003cem\u003eT. aestivum\u003c/em\u003e species, different cultivars respond variably to biotic and abiotic stresses [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Understanding these differences is essential for crop improvement. This can inform the efforts aiming at developing salt-tolerant crops including wheat and can help optimize agricultural practices in salt-affected regions such as Saudi Arabia. The variability in salt stress responses among wheat cultivars might be attributed to differences in the genetic background, these differences control salt stress perception and signalling pathways, osmotic adjustment capacity, ion transport and compartmentalization, and the activation of stress-response genes [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant growth, Salt stress reduced differentially the growth of the three wheat cultivars.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSalinity alters plant growth and development, increasing NaCl concentrations in the growth medium result in adverse effects on plant growth and survival. Salinity leads to severe impact on the physiology traits of wheat plants including total biomass, however, the sensitivity of salt impact varies greatly among wheat cultivars [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the current study, a distinctive variation in salt tolerance was observed between three cultivars, Najran was the most tolerant to saline cues while Qiadh was the most susceptible cultivar. Under salt stress, a dramatic decline in mean fresh and dry weights as well as lengths of root and shoot tissues was measured in all cultivars, but the reduction was significantly lower in the NaCl-tolerant cultivar Najran than in the NaCl-sensitive cultivar Qiadh. This decline in all growth parameters might be a consequence of the increased salt concentration around root area which in turn causes water deficit, nutritional imbalance and osmotic stress in the plant resulting in stomatal closure [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, prolonged exposure of plants to salinity leads to ion toxicity in the leaves and severely affects photosynthetic reactions, cell division and cell elongation which results in a reduction in root and shoot lengths [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYield, salt-stress impacted differentially the spike and seed number and seed weight and germination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is well known that salt stress influences negatively the expansion of plant leaves causing a reduction in photosynthetic capacity which in turn affects the quantity and quality of grain yield [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]. The results obtained in the present study confirm this, where Qiadh cultivar showed the highest reduction in fresh and dry weights of the shoot as well as in yield production compared to the other two cultivars. In contrast, salinity had a positive effect on spike and seed numbers in Najran and Mebiah whereas seed weight had been negatively affected by NaCl. These findings are in line with those of [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e], who found a significant decline in yield outputs of all tested wheat cultivars except Sakha 94 and Sids 13 cultivars, suggesting that while most wheat cultivars are sensitive to salinity some cultivars are salt tolerant, our results suggest that Najran wheat is among the salt-tolerant cultivars.\u003c/p\u003e\n\u003cp\u003eInterestingly, the Najran salt-tolerant cultivar seems to have invested carbon in producing more lighter seeds under salt-stress while maintaining a relatively high rate of germination (around 90%), in contrast to Mebiah which increased the number of seeds without maintaining good germination rate (less than 50%). In the salt-sensitive Qiadh cultivar not only the number of seeds produced under salinity declined but also the germination rate was reduced (around 70%). Choosing to maintain itself under salinity stress via an increase in seed number while maintaining a very high germination rate testifies for an advanced adaptation to salt-stress in Najran wheat. It is crucial to find the genetic determinants of this trait, several genes might be involved in the control of spike and seed numbers, the discovery of these genes is underway and some suspected transcription factors involved in the control of the developmental processes leading to spikes and seeds have already been identified mainly in rice [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Considerable effort is being put into identifying the orthologs of these genes as well as other genes potentially involved in the control of yield in wheat using mainly quantitative trait loci mapping.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsmoregulation, concomitant production of different substances for osmotic adjustment is not essentially required for salt tolerance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProlonged high salt concentrations impair plant growth due to the resulting hyperionic and hyperosmotic stresses. Plants respond to these stresses by implementing biochemical mechanisms to facilitate water uptake and therefore maintain cell turgor and plant growth [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Osmotic adjustment is one of the crucial biochemical strategies in plant acclimation to salt stress. Proline, soluble sugars, starch and organic acids are of the main organic osmotica which are synthesized within plants to assist survival under salt stress. It is demonstrated in this study that different \u003cem\u003eT. aestivum\u003c/em\u003e cultivars might employ various mechanisms to alleviate the harmful effects of saline stress. For example, Mebiah had the highest free Proline content, whereas Qiadh and Najran had the lowest Proline concentration in both root and shoot tissues. These results might be interpreted that Mebiah responded to the high level of NaCl by producing Proline which not only plays a great role in osmoregulation but also protects plants from the damage caused by ROS and toxic ions. Proline has been found to participate in lowering osmotic potential [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e], storing carbon and nitrogen [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e], detoxifying ROS [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e], protecting the enzyme activities of photosynthesis and production of antioxidants [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e] and inducing adaptive responses by acting as a stress signal [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e] under unfavourable conditions. Another example, Najran wheat exhibited higher accumulation of soluble sugars in the roots and shoots while Qiadh had the lowest content of soluble sugars. This accumulation may participate in keeping the photosynthetic activity leading to maintaining plant biomass as these soluble sugars act as building blocks of macromolecules. These findings were accompanied with what we had found in growth analysis where Najran showed the highest fresh and dry weights under salt treatment compared to the other cultivars. In contrast, Qiadh displayed the highest increase in starch levels in both roots and shoots in comparison with the two other cultivars. Increasing the level of soluble sugars and decreasing starch levels might be a critical trait in salt tolerant cultivars. Similar results were reported by [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e], who found a higher accumulation of soluble sugars in salt tolerant rice genotype, Pokkali, suggesting their important role in osmotic adjustment and accumulation of carbon energy reserves in plants. [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e] have pointed out a decline in starch concentration in salt-treated leaves of \u003cem\u003eOryza sativa\u003c/em\u003e L. as a result of carbon limitation due to the poor photosynthetic activity under salt-stress, the decline in this case might be a result of the suppression of starch biosynthesis.\u003c/p\u003e\n\u003cp\u003eOrganic acids are ubiquitous metabolites in plants which accumulate in response to salt stress to act as compatible solutes for osmoregulation and as ROS scavenger as well as plant protectors. The accumulation of total organic acids in the salt stressed wheat cultivars was obvious in the roots, however it decreased in the shoot of Najran and Qiadh cultivars. These findings are consistent with a previous study that confirmed the increase of organic acids in root tissues and their depletion in the leaves under saline treatment, suggesting that these different levels of organic acids might be attributed to organ-specific functions [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. In the case of salt stress, roots uptake excessive amounts of sodium cations which require anions to balance the charge. Thus, organic acids are more accumulated in the roots to enhance the cation\u0026ndash;anion balance. Furthermore, their high level in the roots assist plants to osmotically adjust under salinity conditions.\u003c/p\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAnti-oxidant, salt stress enhanced phenolics production as an antioxidant response\u003c/h2\u003e\n \u003cp\u003eSalt-stressed plants respond to oxidative stress resulting from the accumulations of ROS by operating an antioxidant-defence systems that prevent damage caused by ROS and detoxify ROS molecules. Phenolics are one of the nonenzymatic antioxidants produced to mainly protect plants against various stresses and act as ROS scavengers. It would seem that the activity of the ROS- defence systems rises under extreme environmental stresses and is more pronounced in tolerant plants than sensitive ones [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. This suggests that the defence systems perhaps work more effectively under unfavourable conditions. In the current study, prolonged saline stress has shown significant accumulation of total phenolics in all wheat cultivars and was more pronounced in Najran followed by Mebiah and Qiadh, confirming that wheat cultivars with different sensitivity to NaCl stress exhibit different levels of metabolites alteration. Previous studies [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e] have pointed out possible involvement of phenolics particularly phenylpropanoids in salt-tolerance in wheat. The expression of many of the genes involved in the productions of these substances increase under salt-stress in Najran wheat [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. It would be of interest to extend transcriptomics analysis to the Qiadh cultivar and compare the results to those previously obtained in Najran.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the current study, three Saudi wheat cultivars; Najran, Mebiah and Qiadh varying in their salt-tolerance have been investigated for the effect of salt stress on their physiological and biochemical responses. Salt stress caused differential reduction in physiological activities and grain yield depending on wheat cultivar. Moreover, shoot and root tissues from different wheat cultivars with different sensitivity to NaCl stress exhibited different metabolic alterations and antioxidative responses. These salinity effects were less pronounced in the Najran cultivar potentially due to its high osmotic and antioxidant responses therefore it was characterized as the most tolerant cultivar to salt stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors consent to the publication by BMC Plant Biology of all the the data presented in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interest with any party regarding the results of the study presented in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this work was provided by King Khaled University, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTT: Conceptualization, Methodology, Validation, Resources, Writing - Review \u0026amp; Editing, Visualization, Supervision, Project administration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNA: Methodology, Validation, Formal analysis, Investigation, Resources, Writing - Original Draft, Visualization, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Khaled University, Saudi Arabia for providing financial support to this work.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026apos; information (optional)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDr. Tahar Taybi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRoom 4.57, Ridley Building 2\u003c/p\u003e\n\u003cp\u003eSchool of Natural and environmantal Sciences\u003c/p\u003e\n\u003cp\u003eNewcastle University,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNewcastle upon Tyne NE1 7RU, UK\u003c/p\u003e\n\u003cp\u003ee-mail: [email protected]\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eDr. Norah Alyahya\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDevonshire building\u003c/p\u003e\n\u003cp\u003eSchool of Natural and environmantal Sciences\u003c/p\u003e\n\u003cp\u003eNewcastle University,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNewcastle upon Tyne NE1 7RU, UK\u003c/p\u003e\n\u003cp\u003ee-mail: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhsan F, Chandio AA, Fang W.. 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PMID: 37588415; PMCID: PMC10425591.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4284092/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4284092/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) is one of the most essential staple food crops since thousands of years with a massive economic importance worldwide. Wheat has been widely considered to be a moderately salt tolerant plant and its tolerance and responses to salinity stress vary among different tissues and cultivars. This study was conducted to investigate the impact of salt stress on growth and yield of three Saudi bread wheat cultivars, Najran, Mebiah and Qiadh, and characterize the differential responses of the roots and shoots to reveal different underlying mechanisms for salt tolerance. One-month old plants grown under control and salinity conditions were harvested to measure growth parameters (including fresh weight, dry weight and plant length), biochemical response (i.e. proline, soluble sugars, starch and organic acids contents) and antioxidant activity (phenolics content). A distinctive variation was observed between the three cultivars, Najran was the most tolerant to salt stress while Qiadh was the most susceptible cultivar. Under salt stress, a dramatic decline in growth parameters was noticed across all cultivars however, Qiadh exhibited the most conspicuous reduction in growth as well as in yield. In contrast, a pronounced increase of metabolite contents was shown in the three cultivars under salinity stress and was different not only between these cultivars but also between root and shoot tissues. The obtained results confirm that different wheat cultivars employ various mechanisms to alleviate the harmful effects of salt stress. The diversity in salt stress responses among different wheat cultivars can offer a promising avenue for enhancing crop productivity. In this study, the salt-tolerant Najran cultivar can serve as genetic source for breeding programs aimed at developing new varieties with enhanced salt tolerance.\u003c/p\u003e","manuscriptTitle":"Comparative analysis of physiological and biochemical responses to salt-stress reveals key mechanisms of salt-tolerance in some Saudi Wheat Cultivars","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 10:48:25","doi":"10.21203/rs.3.rs-4284092/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0937f78-548d-4bd1-89a4-17f33150d8d6","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-20T05:15:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 10:48:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4284092","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4284092","identity":"rs-4284092","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}