Functional Study of Grapevine (Vitis vinifera L.) Membrane Ion Transporters Related to Salt Tolerance

Abstract

Grapevines are economically and socially important for the production of wine grapes, table grapes, and dried fruit, and are considered to be sensitive to salt. During salt stress grapevines can suffer from osmotic stress and NaCl toxicity, which can lead to growth reduction, poor fruit set and low yields. Salt tolerant Vitis species rootstocks which exclude salts from the grafted Vitis vinifera scions can be used to achieve healthier vine growth and low salt wines in the salt affected regions. In this thesis several candidate genes for grapevine salt exclusion are characterised and their potential importance for breeding salt tolerant grapevines is discussed. Currently the rootstock trait of Cl¯ exclusion is considered to be the combined effect of multiple genes rather than a single gene. Microarray gene expression analysis has previously compared the gene expression profiles between the roots of the two contrasting grapevine rootstocks – the good Cl¯ excluder 140 Ruggeri and the poor Cl¯ excluder K51-40. Four putative Cl¯ and/or NO₃¯ transporters which were more highly expressed in 140 Ruggeri were selected for characterisation in this study. The first two proteins, VviALMT2 and VviALMT8 belong to the Aluminium-activated Malate Transporter family. Here, it was found that both transport multiple substrates including NO₃¯, Cl¯ and several organic acids using the Xenopus laevis oocyte system. The plasma membrane localised VviALMT2 was more highly expressed in the grapevine root stelar fraction than in the cortical fraction, with gene expression also up-regulated by high [NO₃¯]. Cell-type specific expression of VviALMT2 in Arabidopsis roots reduced shoot [Cl¯] under salt stress; therefore, it was proposed that VviALMT2 is beneficial to plants under salt stress by limiting shoot Cl¯ accumulation. The other two candidates examined were VviNPF2.1 and VviNPF2.2, which are in the NRT1/PTR family (nitrate/peptide transporter family). Here, it is shown that both VviNPF2.1 and VviNPF2.2 are plasma membrane localised; however, their Cl¯ and NO₃¯ conductance could not be determined using Xenopus laevis oocytes, and their expression in grapevine roots was down-regulated by high [NO₃¯]. Salt stress was applied to Arabidopsis plants that exhibited root epidermis and cortex specific VviNPF2.2 expression, and the shoot [Cl¯] of VviNPF2.2 expressing plants was lower than that of the non-VviNPF2.2-expressing plants. Again the conclusion was made that VviNPF2.2 may benefit plants by limiting shoot [Cl⁻] under salt stress. For grapevine shoot Na⁺ exclusion, a quantitative trait locus named NaE was identified, which contains six HKT genes. The root expressing plasma membrane localised VisHKT1;1 proteins were found to be strong Na⁺ transporters; their allelic variation resulted in different Na⁺ conductance and rectification properties, which were linked to the magnitude of shoot [Na⁺]. Two key amino acid residues of VisHKT1;1 were identified important for determining Na⁺ conductance and rectification properties. In this study, by mutating an equivalent residue in bread wheat TaHKT1;5-D, the Na⁺ conductance and rectification tested using electrophysiology were successfully altered, which suggested the potential importance of the residue to HKT function. All other grapevine HKTs in the NaE QTL were also characterised. The VviHKT1;6 and VviHKT1;7 allelic variants were all plasma membrane localised strong Na+ transporters, while the VviHKT1;8 allelic variants were endosomal compartment localised weak inwardly rectifying Na+ transporters. Through RT-qPCR and RNA-seq data analysis, all these HKTs were found to be lowly expressed or not expressed in all the tissue types tested. It is suggested that these HKTs could play minor roles in grapevine Na⁺ homeostasis compared to the VisHKT1;1. During this study, the two electrode voltage clamping technique was intensively used to test the substrate and transport activities of the candidate proteins. Conventional IV data analysis using the proprietary software is time consuming and found to be inefficient. Therefore, an R-based web app “Smart-IV” and an R package “abftools” were developed for electrophysiology data reading, automated current, capacitance, conductance and specific current/conductance processing and plotting in R. Two-electrode voltage clamp (TEVC) data analysed using the conventional method were re-analysed using Smart-IV, and the IV output from the Smart-IV was equivalent to the outputs of the proprietary software. The IV analysis process can be reduced to 15 seconds using Smart-IV with the default automatic interval search settings. To conclude, the findings of this thesis have improved our understanding of: how grapevine ALMT2 and NPF2.2 may be involved in reducing shoot [Cl¯] under salt stress; how some functional and structural functional features of the grapevine and plant HKT1 result in altered shoot Na⁺ accumulation; and, how an open-sourced tool for electrophysiological data processing can automate and stream data analysis.