Molecular Physiology And Evolution Of Ion Transport And Drought Tolerance In Stomata Of Wild Barley

Drought is one of the major abiotic stresses affecting sustainable agricultural development worldwide.Understanding the physiological and molecular mechanisms of drought tolerance is essential for breeding the drought-tolerant crops to cope with global climate change.Stomata act as the gate of gas and water exchange between the interior of leaf and the atmosphere,which tightly regulate photosynthesis and water use efficiency.Wild barley?Hordeum spontaneum?is highly tolerance to virous hostile environmental conditions and owns a wide genetic diversity,which make it an ideal model for revealing the crop abiotic stresses.In the present study,a series of comprehensive physiological,comparative genomic,transcriptomic,molecular,cell biological,and evolutionary biological dataset demonstrated the distinct difference among wild barley genotypes and populations,their evolutionary adaption to drought,and the functional diversity of key stomatal regulating anion channels.The results will provide useful information for the breeding of resilient barley for sustainable agriculture that is threatened by the changing global warming climate with higher temperature and more frequent drought events.The key findings of this research are:1.Evolutionary conservation of ABA signaling for stomatal closure in land plantsThe origin and evolution of mechanism of abscisic acid?ABA?-driven stomatal regulation in land plants is still under debate.36 species including algae and evolutionary representative land plants were selected in this study,and comparative genetic methods were used to analyze 23families of proteins related to ABA signaling pathway.The aquatic ferns Azolla filiculoides and Salvinia cucullata have representatives of 23 families of proteins orthologous to those of Arabidopsis?Arabidopsis thaliana?and all other land plant species studied.Moreover,comparative transcriptomic analysis has identified a suite of ABA responsive genes that differentially expressed in a terrestrial fern species,Polystichum proliferum,which fit the general ABA signaling pathway constructed from Arabidopsis and barley.Furthermore,stomatal assays have shown the primary evidence for ABA-induced closure of stomata in two terrestrial fern species P.proliferum and Nephrolepis exaltata.Phylogenetic analysis of the key ABA signaling proteins such as slow anion channels?SLACs?and open stomata1?OST1s?indicates an evolutionarily conserved stomatal response to ABA.In summary,new molecular and physiological evidences was found for the presence of active stomatal control in ferns.2.The regulatory gene network of wild barley leaf epidermis response to drought stressTibetan annual wild barley?Hordeum spontaneum?genotype XZ5 showed superior drought tolerance than XZ54.Under drought stress,X5 performed higher photosynthesis and biomass,as well as reasonable stomata aperture width to maintain the photosynthesis than XZ54.A total of6,627 differentially expressed genes?DEGs?identified in the transcriptomic analysis,including838 DEGs related to ABA signaling pathway,many positive regulation genes such as SLAC/SLAHs and OST1s were abundant in XZ5 after drought stress,but unchanged or down-regulated in XZ54.The results revealed that multiple signaling pathways are activated at the front line of plant,leaf epidermis,to perceive and transduce the signal to adapt to drought stress.Furthermore,cross-talk among different signaling pathways co-regulated stomatal movement to fine-tune the drought tolerance mechanisms in barley.3.Evolutionary genomics and transcriptomics of drought adaptive stomata of wild barleyIt was reported that the hot-dry savannoid tropical African Slope?AS?at Evolution Canyon I?EC I?,Mount Carmel,Israel,contrasting with the abutting cool humid temperate European Slope?ES?,has shaped the distinct stomatal and photosynthetic traits of H.spontaneum.These physiological traits have strong genomic and transcriptomic basis for stomatal regulation and drought tolerance.In this study,it was found that the wild barley genotypes from AS of ECI showed significantly higher values net CO₂ assimilation,stomatal conductance and transpiration rate,and lower values of SI?stomatal index?and GCV?guard cell volume?,which are likely to be significantly linked to drought tolerance.The Chromosomes 3H and 4H contained the highest number of SNPs and InDels in the wild barley genotypes of AS.Moreover,Tajima's D test indicated a balancing selection of wild barley,showing adaptive evolution to drought tolerance.Wild barley genotypes of AS exhibited higher genetic diversity,while one region at Chromosome5H of AS genotypes showed negative Tajima's D,indicating a purifying selection or as a signature of a recent population expansion.In total,161 genes related to drought adaption were found in wild barley genotypes through selective sweep and transcriptomic analysis,while the homologue gene of SLAC1/SLAHs was only found in AS,which showed higher drought tolerance,but not in ES.4.Functional analysis of barley slow anion channel HvSLAC1/HvSLAH3 and their interacting protein HvOST1sElectrophysiological investigations using Xenopus laevis oocytes showed that barley HvSLAC1 and HvSLAH3 are activated by HvOST1.1 and HvOST1.5,respectively,and AtOST1was able to induce anion currents of both channels.I transformed Arabidopsis slac1-3 knockout mutant with barley HvSLAC1 and HvSLAH3 and conducted detailed physiological measurement of the functional complementation lines.HvSLAC1,HvOST1.1,and HvOST1.5 were all highly expressed in epidermal peels,while HvSLAH3 is also expressed epidermis.Moreover,in comparison to the ABA insensitivity of slac1-3,the transgenic Arabidopsis lines all showed ABA-induced stomatal closure and increase of cytosolic Ca²⁺signal.However,the HvSLAC1 and HvSLAH3 transgenic lines failed to exhibit faster stomatal closure in Arabidopsis as compared to the wild type.It was thus concluded that it is most likely the genes related to grass stomatal structure and development that govern the rapid ABA-induced stomatal closure in barley rather than the“master switch”SLAC/SLAH anion channels.