Identification, Cloning And Characterization Of HvEXPB7 In Root Hairs Of Tibetan Wild Barley Under Drought Stress

Drought is a major limiting factor for crop production. One of the cost-effective solutions for sustainable crop production in water-limiting areas is crop genetic improvement for higher drought tolerance; the identification of drought tolerant crop germplasm and understanding the underlying tolerance mechanisms is necessary, therefore, to improve the plant adaptation to drought-prone environments. Root hair is an important organ in uptake water and nutrients, and plays an important role in drought tolerance. Root hair development and physiological function are restricted by genetic variation as well as different environmental factors like soil moisture. Root epidermal cell specialization, root hair cell differentiation, elongation and apical growth related genes have been studied extensively in Arabidopsis. There is a very little information about the genetic and molecular basis of root hair development in monocots in response to drought. Tibetan wild barley is rich in genetic diversity and provides elite genes for crop improvement in such abiotic stress tolerance as drought. In this study, the drought-tolerant wild barley XZ5 （screened by our lab previously） was used. Based on the genotypic differences of root hair development and root hair transcriptome in response to drought stress, the root hair development-regulated gene, HvEXPB7, was isolated and cloned, and the molecular drought-tolerant mechanism in wild barley was studied. The main results were summarized as follows:1. Root hair phenotype of two contrasting Tibetan wild barley genotypes XZ5 and XZ54 of drought-tolerance and sensitive and a drought-tolerant cv. Tadmor were compared in response to polyethylene glycol-induced drought stress. Most of the root hairs were fell off under drought stress for 1 d with no significant differences among the three barley genotypes; the root hairs were regrown under drought stress for 3 d and 5 d, and the drought-tolerant wild barley XZ5 exhibits significantly more root hairs than the drought-sensitive wild barley XZ54 and the drought-tolerant cv. Tadmor; drought induced pronounced development of root hair was only observed in XZ5 with increasing drought treatment time.2. Isolation and collection sufficient amount of root hairs in liquid nitrogen. Gene expression profile in root hairs of the three barley genotypes in response to drought stress were compared via transcriptome sequencing. XZ5 has far more DEGs than XZ54 and cv. Tadmor in response to drought. Comparative transcriptome analysis revealed extensive changes with the expression of 216,19,77 genes, respectively, in root hairs of XZ5, XZ54 and Tadmor significantly affected under drought stress. Based on all of these genes, we identified 36 drought-tolerance-associated genes, being up-regulated in XZ5 but down-regulated/unaltered in XZ54, or unaltered in XZ5 but down-regulated in XZ54 （drought vs control）. These genes were mainly involved in energy metabolism, antioxidant stress, defence, cell wall modification, root hair development. Among them,16 genes including a P-expansin gene were expressed different between XZ5 and Tadmor in response to drought. The P-expansin gene expression was significantly up-regulated in XZ5 but unaltered in Tadmor by drought stress.3. The full length cDNA and DNA sequences of the β-expansin gene was cloned using RACE method. Sequence comparison indicates that the β-expansin gene carries unique DPBB₁ and Pollon_allerg₁ domains as well as some highly conserved domains of the expansin gene family. The phylogenetic tree analysis indicated this β-expansin gene belongs to the EXPB subfamily and is most closely related to OsEXPB7, being named HvEXPB7. Comparative analysis of CDS and promoter sequences of HvEXPB7 among the three barley genotypes revealed that this gene reserves the highest genetic polymorphism and RHEs （Root hair specific cis-elements） in XZ5. Tissue expression analysis revealed that HvEXPB7 is predominantly expressed in roots, subcellular localization verified that HvEXPB7 is a secretory protein located on the plasma membrane.4. The HvPDS was successfully silenced in XZ5 using BSMV-VIGS （Barley stripe mosaic virus induced gene silencing） method, constructing the BSMV-VIGS system of wild barley for the first time. BSMV-VIGS induced HvEXPB7 silencing leads to less and shorter root hairs than the wild-type plants both in control and drought conditions, significantly reduced plant root biomass and potassium （K+） uptake, whereas these effects were partially alleviated by exogenous ethylene, especially under drought stress. And function identification of HvEXPB7 demonstrate the importance of this gene in root hair development, and subsequently K+ uptake, finally, plant growth under drought stress. Our findings provide an insight into gene discovery and the molecular mechanisms related to drought tolerance in Tibetan wild barley. All of these results offer novel molecular resource for improving plant drought-tolerance.