Ionic Mechanisms And Functional Characterization Of HvABCB25 And HvEXPA1 In Aluminium Tolerance In Tibetan Wild Barley

Al toxicity is considered the major factor limiting crop production in acid soils.Nowadays,the most important strategies to maintain plant production in acid soil are to neutralize the acidity with lime and the use of Al-tolerant genotype.However,the conventional liming practice is not always economical to correct the acid soil problem.Thus,the breeding of Al-tolerant genotype is thought to be the most cost-effective strategy to reduce Al toxicity in the agricultural regions with acid soil.Although genetic engineering provides an opportunity to improve the A1 tolerance in Al-sensitive genotypes by introducing foreign genes or by overexpressing endogenous genes,the knowledge about genes identified to link to Al tolerance is limited.Owing to its genetic diversities,Tibetan wild barley germplasm is a precious treasure of available genes and provides rich sources of genetic variation for cereal improvement.In our study,two contrasting Tibetan wild barley genotypes(Al-tolerant XZ16 and Al-sensitive XZ61)and Al-tolerant cv.Dayton was used to study the unique detoxification mechanisms of A1 toxicity in Tibetan wild barley.Microelectrode ion flux estimation(MIFE)technology was performed to investigate the common and specific ionic mechanisms of AI and low-pH tolerances.Beside,we functionally characterized two different expression genes,HvABCB25 and HvEXPA1,through barley stripe mosaic virus induced gene silencing(BSMV-VIGS)and homologous overexpression.The main result was followed:1.Genotypic differences in effects of acid and AI stresses on root tip ion flux in Tibetan wild barleyChanges in root tip H+,K+,Ca2+ fluxes and rhizosphere pH were investigated,using microelectrode ion flux estimation(MIFE)technology in Tibetan wild barley XZ16 and XZ61 of Al-tolerant and sensitive genotypes,and Al-tolerant cv Dayton.Our results showed that Al-induced ion flux changes were mainly occurrred at the elongation zone.The higher low-pH tolerance could be attributed to the higher H+ influx and rhizosphere alkalinize abilities.The Al-induced decrease in H+ influx and K+ efflux were higher in the Al-sensitive XZ61 than XZ16 and Dayton.The higher Ca2+ efflux under Al stress than low-pH treatment suggest that Ca2+ could play vital roles in Al stress tolerance of barley.2.Cloning and functional identification of Al-tolerant related gene HvABCB25 in Tibetan wild barleyWe identified a novel ABC transporter gene HvABCB25 from the result of genechip,which was up-regulated in XZ16 but down-regulated/unchanged in XZ61 and Dayton under AI stress.The sequence comparison indicated that HvABCB25 carried both transmembrane and AAA domains.HvABCB25 is predominantly expressed in shoots but induced by AI only in root tip in XZI6.Subcellular localization verified that HvABCB25 is located in the tonoplast.Barley stripe mosaic virus based virus induced gene silencing(BSMV-VIGS)of HvABCB25 led to severely suppress in Al tolerance under Al stress,and significantly induced Al uptake and accumulation in the cytosol.Conversely,homologous overexpression of HvABCB25 significantly improved A1 tolerance with markedly reduction in cytosol A1 accumulation,but no effect on total root cell sap Al accumulation.These results indicated that HvABCB25 was a tonoplast Al transporter and responsible for internal Al detoxification via reduced cytosol Al in roots of Tibetan wild barley,which provide us novel insight into genetic basis for Al-tolerance by internal detoxification in barley.3.Cloning and functional identification of HvEXPA1 in Tibetan wild barleyWe functionally characterized an Al inducible expansin gene,HvEXPA1,using a range of molecular and physiological approaches.There was a significant genotypic difference in Al-induced gene expression of HvEXPA1,where both Al-tolerant genotypes of Tibetan wild barley XZ16 and cv.Dayton showed significant upregulation but not in Al-sensitive wild barley XZ61.In XZ16,significant upregulation of HvEXPA1 is exclusively induced by A1 in low pH(4.3)at root tips,but not by normal apoplastic pH(5.8)or other metals ions such as Cr or La.Subcellular localization analysis indicated that HvEXPA1 is located in the plasma membrane.Bioinformatic analysis indicated that HvEXPA1 carried 3 domains and conserved 3D structure among 7 genuses.Barley stripe mosaic virus-induced gene silencing(BSMV-VIGS)of HvEXPA1 led to significantly decrease in root length and root dry weight under both of control and 200 pM A1 treatment,but the inhibition in root cell length only recorded in control condition.Interestingly,reduced A1 concentration via silencing of HvEXPA1 was recorded only in root cell wall but not in cell sap.Our results indicate that HvEXPA1 is an Al-inducible expansin gene,which participates in the root cell elongation and probably influences the A1 content through regulating root cell wall loosening in barley.