Identification and characterisation of vacuolar proton-pumping pyrophosphatase genes in bread wheat

Abstract

Vacuolar proton-pumping pyrophosphatase (H+-PPase) genes encode membrane bound proteins responsible for hydrolysing cytosolic pyrophosphate (PPi) and generating an electrochemical potential difference for protons (H+) across cellular membranes. Constitutive expression of H+-PPase genes, in particular the Arabidopsis thaliana AVP1 gene, significantly improves the growth of several plant species, under control and stress conditions. Despite considerable research, little is known about these genes in bread wheat (Triticum aestivum) and whether they can be utilised to improve wheat growth and/or stress tolerance. The first focus of this research project was to further investigate the role of AVP1 in Arabidopsis. Metabolomics analysis of AVP1 mutant and AVP1 over-expressing Arabidopsis lines showed the concentrations of multiple metabolites were reduced in the mutants compared to wild-type, while concentrations of many metabolites were increased in the AVP1 over-expressing lines compared to wild-type. This analysis suggests that expression of AVP1 could potentially influence pathways involved in the biosynthesis of ascorbic acid, tryptophan and sucrose. The second focus of this project was to investigate the role of AVP1 in bread wheat. To address this, transgenic wheat lines (cv. Bob White), containing constitutive (ubi:AVP1) and stress-inducible (rab17:AVP1) AVP1 expression, were characterised under control and saline conditions. When grown in control and saline hydroponics conditions, however, no phenotypic differences were observed between the ubi:AVP1 or rab17:AVP1 transgenic lines and the null segregants. These findings suggest that, either AVP1 expression does not have a beneficial impact on growth and salt tolerance in bread wheat, or that the promoters and/or experimental methodologies used to characterise these lines were not suitable. The third focus of this project was to identify native wheat H+-PPase (TaVP) genes, and to investigate the role of these genes in vitro and in planta. Using the NRGene wheat reference genome assembly, 15 TaVP genes were identified and were shown to vary in gene sequence and expression. Expression patterns differed greatly between genes, tissue types, developmental stages and wheat varieties, which will be useful for the development of genetic markers for breeding purposes. To characterise the role of these genes, TaVP homologs from the B genome were expressed in a salt-sensitive mutant yeast strain. Analysis of TaVP expressing yeast suggested that TaVP2-B and TaVP4-B may be more beneficial for improving salt tolerance. Transgenic wheat lines (cv. Fielder) constitutively expressing the TaVP1-B and TaVP2-B genes were generated and phenotyped in soil under control and saline conditions. The transgenic wheat lines had significantly reduced plant biomass at maturity, decreased time to flowering, and increased leaf Na+ and Cl- accumulation. These results indicate that TaVP1-B and TaVP2-B influence ion accumulation, plant development and floral transition when constitutively expressed in bread wheat. Overall, the findings of this research project suggest that wheat TaVP genes are likely to have diverse roles in regulating plant growth and salt tolerance, and variation amongst these genes could potentially be utilised to enhance the growth and stress tolerance of bread wheat through selective breeding and gene editing in the future