Exploitation Of Salt-Tolerant Germplasm And Its Mechanisms In Tibetan Wild Barley

Soil salinity is a major abiotic stress of restricting crop productivity worldwide, posing a great threat to agricultural sustainability. A thorough understanding of salt-tolerant mechanisms is necessary for alleviating salt injury to agricultural production by improving cultural practices and developing salt-tolerant varieties. Currently, it is well documented that osmotic regulation, ion homeostasis and anti-oxidation are the three aspects of salt tolerance in plants. However, the genetic and molecular mechanisms of salt tolerance have not been fully elucidated.Barley (Hordeum vulgare) is the fourth most important cereal crop in the world, characterized by its wide adaptability and high salinity tolerance. Consequently, it is frequently used as a model crop in the attempts to understand salinity tolerance in the cereal crops. Tibetan annual wild barley (H. vulgare ssp. Spontaneum and ssp. agriocrithum)(hereafter referred to as Tibetan wild barley) is considered as one of the ancestors of cultivated barley, and shows wide genetic variations in the tolerance to harsh environments. It is possible for us to explore the elite germplasm in terms of salt tolerance from the wild barley.In the present study, an association analysis was used to identify and explore salt-tolerant germplasm based on the evaluation of salt tolerance among around200Tibetan wild barley accessions, and the mechanisms of salt tolerance between wild and cultivated barleys were systematically resolved by ionomic, membrane proteomic and metabolomic methods. The main results were summarized as follows:1. Identification of salt-tolerant germplasm and genetic association analysis within Tibetan wild barleys.Hundred and eighty-eight accessions of Tibetan wild barley were exposed to salt stress of300mM NaCl in hydroponics at three-leaf stage for3weeks, to compare the difference among these accessions in salt tolerance as indicated by relative dry weights. Salt stress significantly reduced shoot and root dry weight by27.6%to73.1%and the root weight showed greater reduction than the shoot weight. There was a significant difference among the Tibetan wild barley accessions. Compared with CM72, a salt-tolerant cultivated cultivar, some accessions showed higher shoot, root or whole-plant dry weight under salt stress.The linkage disequilibrium (LD) structures of the chromosome5were evaluated using57diversity arrays technology (DArT) markers over this chromosome. The LD decay of genetic distance was8.9cM (R2<0.1) or1.5cM (R2<0.2). The genetic variation of HvCBF1, HvCBF3, HvCBF4and HvHVA1was studied by sequencing the gene coding regions. There were2,15,16and10single nucleotide polymorphisms (SNP) sites, corresponding to3,8,13and6haplotypes for the four genes, respectively, with0.3,2,2.4and1.6SNPs in each100bp of the coding sequence. Furthermore, the certain LD relationship was detected between SNPs.Association analysis was conducted between the genetic diversity and the genotypic salt tolerance. It was found that marker bpb-4891was significantly associated with salt tolerance, explaining the2.2%and2.3%of phenotypic variation for the relative shoot and whole-plant dry weight, respectively. The haplotype13of HvCBF4gene exhibited highly significant association with shoot and whole-plant relative dry weight, explaining7.7%and6.4%of the variations, respectively. Based on the distance in the barley genetic map, it may be assumed that the marker bpb-4891was closely linked with the HvCBF4gene. The results suggest that the marker bpb-4891and haplotype13of HvCBF4gene could be related to salinity tolerance. Tibetan wild barley accessions, XZ16and XZ26have haplotype13of HvCBF4genotypes, and could be considered as high salt tolerance.2. Mechanisms of ionome responding to salt stress in barleyIn order to reveal mechanisms of ionome responding to salt stress in barley, two cultivated barleys (CM72, salt-tolerant; Gairdner, salt-sensitive) and two wild barleys (XZ16, salt-tolerant; XZ169, salt-sensitive) differing in salt tolerance were used to investigate shoot biomass and ion changes in response to salt stress of150and300mM NaCl in hydroponics at0,1,2,3and5weeks after treatment. After35days treatment, there was no significant difference in shoot weight between salt stress of150mM NaCl and control for4barley genotypes, but under salt stress of300mM NaCl, the reduction of shoot weight could be found, ranging from30%to53%. Among the four genotypes, XZ16maintained the largest shoot biomass under salt stress, and it had1.7and1.4times larger shoots dry weight than CM72, respectively. In conclusion, the results show that XZ16is a fast-growing and salt-tolerant barley.In roots, Na content reached the maximum at2weeks after treatments, and then tended to decrease. The contents of P, S, Mg, K, Mn, Zn and B decreased steadily during salt treatment, while Mn and Zn contents decreased greatly under salt stress of300mM NaCl. By contrast, the contents of Ca, Cu and Fe increased under salt stress. In shoots, Na content increased and the contents of P, K, Ca, Mg, S, Cu and B decreased due to salt treatments. The contents of Mn and Zn increased under salt stress of150mM NaCl, and increased under salt stress of300mM NaCl. In contrast, Fe content decreased under salt stress of150mM NaCl, but increased under salt stress of300mM NaCl. These results suggest that maintenance of low Na content and Na/K ratio in shoots is a key important for salt tolerance in both cultivated and wild barleys. In addition, the increased contents of Ca, Cu and Fe in roots and Fe in shoots may enhance tissues tolerance to salt stress.3. Mechanisms of membrane proteins responding to salt stress in barley.In order to reveal mechanisms of membrane proteins responding to salt stress in barley, wild barley (XZ16) and cultivated barley (CM72) were used to investigate differential expression of membrane proteomes in roots and leaves under normal condition and salt stress of200mM NaCl after48hours, using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-TOF-MS) techniques.In roots, salt stress induced6proteins up-expressed, being more than1.5times than those in the control, and these proteins included ATP synthase (subunit a), aspartate aminotransferase, lactoylglutathione lyase, class III peroxidase and pathogenesis related protein No.10. On the other hand,7proteins were down-expressed under salt stress, including ATP synthase (subunit β), dihydrolipoamide dehydrogenase family protein, serine protease, cysteine proteinase, Calmodulin-like protein, NADP-malic enzyme and class Ⅲ peroxidase. In leaves, salt stress induced eight proteins down-expressed, and these proteins included ATP synthase, Bp2A protein, Cp31AHv protein, Cp31BHv protein, glutamine synthetase, germin-like protein phosphoglycerate kinase, and oxygen-evolving enhancer protein No.2. In contrast, NAD-dependent epimerase, DegP protease and harpin binding protein No.1were up-expressed under salt stress.Based on the membrane proteome analysis, it can be suggested that the main mechanisms of membrane proteins for salt tolerance in roots were protection of membrane stability, scavenging reactive oxygen species (ROS) and function of ion homeostasis, while the mechanisms of membrane proteins in leaves were protection of photosynthetic reaction activity. Furthermore, there was genotypic difference in expression levels of membrane proteins, with a wild barley, XZ16having higher expression of proteins involved in osmotic regulation, ion transport and protection of photosynthesis than CM72, which could be considered as the molecular basis of high salt tolerance for XZ16.4. Mechanisms of metabolome responding to salt stress in barley.In order to reveal adaptive metabolic pathways in barley under salt stress, CM72and XZ16were used to investigate metabolome changes in response to salt stress of300mM NaCl after three weeks using gas chromatography-mass spectrometry (GC-MS). A total of82key metabolites were identified and their concentrations were affected by salt stress. Compared with the control, salt stress caused the significant changes in the contents of53and55metabolites for CM72and XZ16, respectively. The response of metabolites in roots was enhanced TCA cycle and sugar accumulations, and inhibited glycolysis and amino acid synthesis. In contrast, Calvin cycle, glycolysis and amino acid synthesis were enhanced, while TCA cycle was inhibited in leaves exposed to salt stress.There was an obvious difference in metabolites in response to salt stress between roots and leaves. The results showed that the most important compatible solutes are proline, sugars (sucrose, raffinose and trehalose), mannitol and inositol for roots, and raffinose, proline and some amino acids for leaves. Osmotic adjustment is a basic mechanism for salt tolerance in roots. In leaves, a dramatic enhancement of proline, sugars and amino acid synthesis was found, which is obviously favorable for osmotic adjustment, and may provide sufficient carbohydrates and energy for roots. In addition, metabolites in response to salt stress differ between cultivated and wild barleys. CM72accumulated more metabolites associated with photosynthesis and TCA cycle in leaves, but less amino acids and organic acids, suggesting that the cultivated barley enhances its salt tolerance through increasing photosynthesis and energy consumption. While the contents of fructose-6-P, glucose-6-P and PEP were not affected by salt stress in the leaves of XZ16. Wild barley has higher contents of compatible solutes, more active metabolite synthesis and rapid growth than cultivated barley under salt stress.In summary, XZ16, a Tibetan wild barley accession was identified in this study, being higher salt tolerance than CM72, a standard cultivated cultivar. Salt tolerance of barley is involved in a complex molecular network, and to our understanding it is first attempt that we systematically investigated the different mechanisms of salt tolerance between wild barleys and cultivated barleys using ionomic, membrane proteomic and metabolomic methods. The results surely enrich the knowledge of crop salt tolerance and can provide a theoretical reference and practical guidance for exploring genetic resources and breeding crop cultivars with salt stress.