Confirmation of the barley vacuolar pyrophosphatase HvHVP10 as a candidate gene for salinity tolerance

Abstract

Salinity is one of the most significant environmental issues affecting agricultural production in Australia and around the world. One main salinity tolerance mechanism among plants is the exclusion of sodium ions from the aerial parts of the plants. Conventional breeding techniques and genetic engineering approaches have been employed in the effort to develop varieties with lower levels of shoot sodium accumulation under saline conditions. A gene identification approach in the salt laboratory at the Australian Centre for Plant Functional Genomics (ACPFG) identified a major quantitative trait locus (QTL) for sodium exclusion (HvNax3) in the barley mapping population Barque-73 (Hordeum vulgare; a high yielding South Australian cultivar) × CPI-71284-48-48, (a wild barley; Hordeum spontaneum). Syntenic mapping revealed a number of candidate genes underlying the HvNax3 QTL, the most promising of which encodes a vacuolar pyrophosphatase proton pump (V-PPase, HVP10 gene in barley) responsible for establishing an electrochemical gradient across the tonoplast that drives the secondary transport of ions such as sodium into the vacuole. The compartmentalisation of sodium in the vacuole reduces the toxic effect of high sodium concentration in the cytoplasm, whilst at the same time facilitating water uptake into the plant. Orthologues of this gene have been shown to confer salinity and drought tolerance in a variety of plant species. In order to confirm that V-PPase is the gene responsible for the HvNax3 phenotype, analysis of the accumulation of sodium ions and HvHVP10 gene expression in root and shoot tissue was conducted over 10 days of salinity stress. Results of this work suggested that the accumulation of sodium in the roots of lines carrying the CPI-71284-48 HvNax3 allele may be attributed to a peak in HvHVP10 expression at day 3 of salinity stress. This finding is further supported by analysis of pyrophosphatase activity within root tonoplast enriched vesicles, results of which suggest that pyrophosphatase activity was higher in the line with the CPI-71284-48 HvNax3 allele in response to salinity stress. Sequence analysis of the HvHVP10 CDS, gene and promoter region suggests that this difference in expression and enzyme activity may be due to the presence of a DREB transcription binding site just upstream of the HvHVP10 start codon in CPI-71824-48. Future analysis is required to validate this finding and warrants further investigation, possibly by employing the newly emerging CRISPR technology. A significant component of this research project was the development of HvHVP10 overexpression (OEX) lines. The salinity tolerance of these lines was characterised in the T1 and T2 generation using a supported hydroponics system and destructive analysis. Results from this study indicated that is some cases the constitutive OEX of HvHVP10 lead to increased biomass of transgenic plants under both salinity stress and control conditions. Several transgenic lines also displayed altered patterns of sodium accumulation in the root and shoot tissue under 150 mM and 200 mM NaCl. Further analyses of these transgenic lines at later generations is required to confirm this phenotype and warrants future investigation.