Email: A.Abbas@westernsydney.edu.au, j.he4@westernsydney.edu.au, yuanyuan.wang@westernsydney.edu.au, a.naznin@westernsydney.edu.au, ,d.dauda@westernsydney.edu.au, j.bose@westernsydney.edu.au, m.r.jones@westernsydney.edu.au, z.chen@westernsydney.edu.au.

Tonoplast destined chloride channel genes are characterized in model plant to perform multiple functions ranging from nutrient uptake assimilation and stomatal regulation by efficiently transporting NO3- /H+ and Cl- in response to biotic and abiotic stress conditions. We have done homology-based genome wide analysis and identified 23 chloride channel genes in wheat and 12 chloride channels genes in barley, based on maximum likelihood grouped into four clades.

Tissue specific gene expression analysis for Chloride Channel B gene in tolerant and susceptible wheat varieties showed its higher expression in aerial tissues, in response to heat and drought stress. There is a high confidence that Chloride Channels in wheat and barley are responsible for influx and efflux of the anions in and out of the vacuoles in the result of phosphorylation and dephosphorylation effectively regulating the opening and closing of stomata like in other species as orthologs shares high sequence similarities.

With the more genomes are now being published, enabling comparative genomic analysis for stomatal evolution and regulation under drought and heat stress conditions focusing Chloride Channels. Wild relatives of wheat and barley have evolved over time and are rich genetic resources for improving tolerance in commercially available cultivars.

Chloride channel B is our prime gene for functional studies using knockout mutation by utilising CRISPR Cas9 system and over expression mutants. To find suitable commercial candidate variety we have screened more than 300 wheat genotypes in greenhouse and field settings for their tolerance to heat and drought conditions using low-cost screening methodology which includes physiological measurements, stomatal imaging, stomatal gas exchange, biochemical analysis, and tissue specific gene expression analysis.

Chloride Channels are involved in complex plant tolerance mechanism, whereas in plants studies investigating Chloride Channels were limited to the model plants we have designed this study to elucidate the roles of Chloride Channel B in stomatal regulations of wheat and barley under drought and heat stress along with molecular and physiological networks involved in tolerance to heat and drought stress.

Key Words: Chloride Channel B, Wheat, Barley, Stomatal regulation, CRISPR Cas9, Over expression, Drought, and Heat stress.