Vacuolar H⁺ -PPase (HVP) genes in barley: Chromosome location, sequence and gene expression relating to Na⁺exclusion and salinity tolerance

Three vacuolar H⁺-pyrophosphatase (HVP) genes have been identified in barley (HVP10, HVP1 and HVP3). The first gene, HVP10, was recently localised to chromosome 7HS in a mapping population developed from the cross Barque-73 x CPI- 71284. HVP10 showed increased gene expression after 24 hours of salt stress in roots of the variety Kashima, but not until the third day of salt stress in roots of both Barque-73 and CPI-71284. HVP10 was also more highly expressed following salt stress in shoots of one of the barley genotypes (CPI-71284). It is hypothesised that HVP10 plays tissuespecific roles in both Na⁺ exclusion (roots), and in compartmentalisation of toxic Na⁺ into vacuoles (shoots) in salinity tolerant barley genotypes. In the current study, a second gene, HVP1, was mapped to the long arm of chromosome 7H in barley. Different expression profiles for HVP1 across different barley genotypes were observed in this study in response to salt stress. HVP1 appears to be involved in two reactions of barley plants to salt stress: (1) adjustment to osmotic stress following initial NaCl application in both roots and shoots (cv. Kashima); and (2) management of toxic Na⁺ concentrations in shoots during the ionic phase of salt stress (cv. Barque-73). In this study, HVP1 gene sequence has eight exons and seven introns. The predicted amino acid sequences of HVP1 in the three cultivars used in this study were identical, and were also the same as sequences found in databases for barley cultivar Morex although there were a number of SNPs within the coding regions between genotypes. These results are similar to those previously published about HVP10. This suggests structures of both HVP10 and HVP1 are very conserved among barleys, and it supports a hypothesis that promoter regions of both genes are involved in the regulation of gene expression. A third gene, HVP3, has been previously described but it was mapped in chromosome 1H in current study, and deduced that the gene contains four exons and three introns. In our experiment HVP3 was expressed neither in roots nor in shoots of any of the barley genotypes tested, either under control conditions or in response to salt stress. Based on published EST data, it is hypothesised that HVP3 plays an important role for proton pump only during seed development and is not linked to abiotic stress responses. Nucleotide sequence analysis indicated that during evolution there may have been insertions/deletions occurring within two highly conserved domains shared by HVP1/HVP10 and HVP3 genes.