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Title: An investigation into halotolerance mechanisms in Arabidopsis thaliana
Author: Price, Jillian
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2005
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Soil salinity is one of the most significant abiotic stresses that can reduce agricultural productivity. Consequently there is much interest in the mechanisms halophytes employ to tolerate high salt environments. The model organism Arabidopsis is salt sensitive questioning the suitability of this plant for salt tolerance studies. A salt tolerant Arabidopsis IffiS (Habituated to High Salt) cell line was, however, established. Wild type Arabidopsis cell cultures cannot tolerate 70mM NaCI, but the IffiS cell line was adapted from the wild type cell lines by sub-culturing into progressively higher levels of NaCI, The generation of the HHS cell line suggests that although Arabidopsis is generally considered to be salt sensitive, it does have the genetic potential to be salt tolerant. These cell cultures are useful for salt tolerance studies as to survive in high salinity, unlike the intact root system of a plant, each cell in culture must be expressing the required traits for salt tolerance. Measurements of total ion content of the cell lines indicate that the HHS Arabidopsis cells do not accumulate Na+; instead mechanisms for either Na exclusion, or Na efflux must function. Arabidopsis activation tagged lines were used in large scale gain-of-function screens to identify single sequences that allow seedlings to survive better under NaCI stress. 23 000 lines were screened on MS media with reduced r (200JlM) and Ca2+ (530J.1M) concentrations, supplemented with 0.75% sucrose and 80mM NaCI. It is well established that the toxic effects ofNa+ can be ameliorated by high external rand Ca2+ concentrations. Under these conditions wild type seedling growth was impaired. Therefore, any mutants that were more efficient at K+ uptake, preventing Na+ uptake, Na+ efflux, or show an increased sensitivity to the Ca2+ response flourished and were easily identified in the screen. Primary screening isolated 631 putative salt, tolerant mutants. Secondary screening under identical conditions isolated 50 mutants displaying a strong phenotype, 36 a weak phenotype and 109 were found to be false positive. When compared with wild type, the activation tagged mutant JP5 was more salt tolerant. The IP5 mutant contained a single insertion of the pSKI015 activation tag vector located downstream from the AtMYB64 gene. Expression·of AtMYB64 was up regulated in the JP5 mutant compared with wild type. Homozygous lines reported to carry a T-DNA insertion in the first intron of AtMYB64 (a putative AtMYB64 knockout) was hypersensitive to salt. Overexpression of AtMYB64 conferred salt tolerance to transgenic Arabidopsis lines. These results suggest AtMYB64 is a transcription factor that activates salt tolerance mechanisms in Arabidopsis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available