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Type: Thesis
Title: Investigating genes encoding membrane proteins in grapevine (Vitis vinifera L.) and Vitis spp. rootstocks to determine their role in chloride exclusion.
Author: Henderson, Sam William
Issue Date: 2014
School/Discipline: School of Agriculture, Food and Wine
Abstract: Plant growth and productivity is limited when sufficient salt (NaCl) accumulates in the soil solution to impose an osmotic stress, and when the concentrations of the dissociated sodium (Na⁺) and chloride (Cl⁻) ions accumulate to toxic levels in cells and tissues. One mechanism associated with plant salt tolerance is the ability to limit Na⁺ and Cl⁻ accumulation in the shoot. The aim of this thesis was to investigate the genetic basis of shoot Cl⁻ exclusion by using the Cl⁻ sensitive dicotyledonous crop grapevine (Vitis vinifera L.). Root transcriptomes of three Vitis genotypes were compared by microarray hybridisation. Genotypes were selected for their contrasting abilities to exclude Cl⁻ from shoots: 140 Ruggeri (Cl⁻ excluder), Cabernet Sauvignon (Cl⁻ intermediate) and K51-40 (Cl⁻ includer). The magnitude of transcriptional changes due to a 4 day, 50 mM Cl⁻ treatment were proportional to the amount of the Cl⁻ accumulating in leaves, with those in Cabernet Sauvignon and K51-40 indicative of secondary stresses, such as the generation of reactive oxygen species. In the Cl⁻ excluder 140 Ruggeri transcriptional changes were typical of plant primary responses to salt stress, such as the up-regulation of heat shock associated proteins and transcription factors. Transcripts encoding anion channels, that may potentially regulate Cl⁻ transport from root to shoot in grapevine, were not significantly differentially expressed in any genotype due to salt-stress. This was consistent with the observation that Cl⁻ exclusion in grapevine is not a salt-inducible trait. In contrast, a comparison of transcriptional differences between K51-40 and 140 Ruggeri in the absence of salt-stress revealed a list of gene candidates. Many of these genes were homologous to anion channels and their regulators from guard cells that facilitate stomatal closure in Arabidopsis thaliana. Genes included VvSLAH3, VvALMT1, VvSnRK2.6 and members of the NRT1 family. There are a number of genes, not regulated at the transcriptional level by salt, which have been implicated in salt tolerance in previous studies of Arabidopsis thaliana. The role of some homologous genes in grapevine was also investigated in this thesis with the aim of clarifying their putative roles in shoot Cl⁻ exclusion. To this end, the functional properties of a cation-chloride cotransporter (VvCCC) and two chloride channels (VvCLCc1, VvCLCg) were examined. VvCCC functionally complemented an A. thaliana ccc deletion mutant reversing a stunted-growth phenotype and perturbed Cl⁻ homeostasis. However, membrane localisation data indicates that VvCCC is likely to be localised to the trans-Golgi network so may not directly mediate xylem loading or retrieval of Cl⁻. Investigation into plant CLCs revealed that an Arabidopsis clcc mutant was not salt-sensitive, which contrasts with previous reports. When overexpressed, neither VvCLCc1 nor VvCLCg could improve growth of Arabidopsis or yeast on salt containing media. However both VvCLC isoforms could restore impaired stomatal functioning in the Arabidopsis clcc mutant. Together these data suggest that plant CCCs and CLCs are not crucial genes for Cl⁻ exclusion and salt tolerance despite previous speculation. Future investigation into plant Cl⁻ exclusion will benefit from the candidate genes identified in the transcriptional screen of this thesis.
Advisor: Gilliham, Matthew
Tyerman, Stephen Donald
Walker, Rob
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2014
Keywords: grapevine; salt tolerance; ion transport; viticulture; abiotic stress
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