Physiological And Genetic Difference In Salt Stress Tolerance In Wild And Cultivated Barleys

Soil salinity stress is one of the major environmental constraints limiting crop production. To meet increasing global food demand in the near future, improvement of salt tolerance of crops is an important challenge for plant breeders. Intensive works in conventional breeding programs have been performed to develop elite varieties with a high level of salt tolerance. but till now this attempt has limited success. Understanding the genetic mechanisms underlying salt tolerance is of key importance to reach the desired breeding goals with respect to this trait. Salinity affects almost every feature of physiology, morphology, biochemistry and molecular events in the plants. Tibetan wild barley is considered as one of the ancestors of cultivated barley and it is characterized by wide variation of abiotic tolerance. Therefore, a comprehensive study was undertaken to compare Tibetan wild barley (Hordeum spontaneum) and cultivated barley (Hordeum vulgare) in whole-plant and cellular responses to salinity. The major aims of this study were to;(1) understand physiology of salt tolerance in barley cultivars and the difference in salt stress tolerance between wild barley and cultivated barleys;(2) determine the subcellular localization of Na+and K+and the expression of vacuolar H+-ATPase and H+PPase in wild and cultivated barley;(3) determine the expression of Na+/H+NHX antiporters genes involved in Na+homeostasis under salinity stress:(4) determine the genetic variation of HvNHX genes among Tibetan wild and cultivated barley. The main results are as follows:1. The difference in the physiological responses to salt stress between wild and cultivated barley. Differential response was observed among the examined genotypes in terms of plant biomass, Na+accumulation, Na+/K ratio in roots and shoots, chlorophyll content, photochemical efficiency (Fv/Fm), net photosynthetic rate (Pn), stomatal conductance (Gs), and intracellular CO2concentration (Ci), xylem sap osmolarity, electrolyte leakage and vacuolar H+-ATPase and H+-PPase activity. A large difference between wild and cultivated barleys was detected, with XZ16(wild barley) showing higher salt tolerance than Yerong and Gairdner (cultivated barley) in terms of all mentioned traits. 2. The physiological. cellular and molecular mechanisms of the difference in salt tolerance between wild and cultivated barleys. Root architecture study revealed that salinity induced a significantly greater decrease in total root length. surface area. average diameter and total volume in Gairdner than in CM72and XZ16. Wild barley XZ16enhanced their ROS scavenging ability by exhibiting increased levels of superoxide dismutase, peroxidase, catalase activity and proline content, along with decreased content of reactive oxygen species (O2and H2O2) as compared with cultivated barley CM72and Gairdner. The observation of transmission electron microscopy found that fundamental cell ultrastructure changes happened in both leaves and roots of all the barley genotypes under NaCl stress, with chloroplasts being most affected. Moreover, obvious difference could be detected among the three genotypes in the damage of cell ultrastructure under salt stress, with XZ16and Gairdner being least and most affected, respectively. Subcellular study showed that a primary strategy to protect the cytosol against sodium toxicity was compartmentalization of sodium ions into soluble fraction (vacuoles). Gairdner showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, than CM72and XZ16. Analysis of gene expression using quantitative RT-PCR showed that transcripts vacuolar H+-ATPase and inorganic pyrophosphatase (HvHVA/68and HvHVP1) were more abundant in leaves and roots in the order of XZ16> CM72> Gairdner.3. The genetic difference between the wild and cultivated barleys in salt stress tolerance. HvNHX isoforms gene expression was studeid. Overall, higher gene expression was induced in salt tolerant genotypes compared with sensitive one, indicating the important role of this gene in salt tolerance. In leaves and roots, expression of HvNHX1and HvNHX3in XZ16and CM72was up-regulated at all time as compared with sensitive one. The HvNHX2and HvNHX4isoforms were also induced by salt stress, although not to the same extent as HvNHX1and HvNHX3. Gene expression analysis revealed that the HvNHX1and HvNHX3are the candidate genes which could have the function of regulating ions by sequestration of Na+in the vacuole. Single nucleotide polymorphism (SNPs) was used as markers of the diversity. Evaluation of the sequencing data of38barley genotypes including Tibetan wild and cultivated barleys showed polymorphisms including SNPs and small insertion, deletion (INDELs) sites in the targeted genes HvNHX1and HvNHX3.4. Identification of the novel alleles involved in salinity tolerance. For HvNHXl gene, types of SNPs such as G/A, A/T, T/C, and C/A were identified in first fragment (753bp). For the second fragment (308bp) of the gene, SNPs such as G/T, G/A, T/C, C/A, A/T and A/G as well as with few samples having single nucleotide deletion were identified. For the third fragment (658bp) of the gene, SNPs such as G/A, C/A, C/T and T/G were found and for the fourth fragment (626bp) the SNPs such G/A, G/C, C/A, C/T, T/G and G/T were identified. For HvNHX3gene, types of SNPs such as G/T, G/C, G/A, A/T, A/C. C/T and T/G were identified in first fragment (714bp). For second fragment (947bp) of the gene HvNHX3, T/A, T/G, T/C and C/T and for the last fragment (520bp), T/A, G/T, T/G, C/T and G/A polymorphic sites were identified. Comprehensive analyses of the results revealed that Tibetan wild barley offers elite alleles of HvNHX1and HvNHX3conferring to salinity tolerance. So, evaluation of genetic variation and identification of salt tolerance mechanism in wild barley are important steps to unravel the novel alleles involved in salinity tolerance.