Genetics - Theses

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    Investigating the mechanisms of zinc homeostasis in Arabidopsis thaliana
    Sinclair, Scott Aleksander. (University of Melbourne, 2010)
    Zn is an element essential for all forms of life, and either Zn-deficiency or excess Zn can have serious biological consequences. In plants, Zn levels are controlled by a number of physiological and cellular mechanisms to maintain appropriate physiological concentrations of Zn. This study further characterises the HMA2 and HMA4 transporters by developing a novel technique using the Zn fluorophore Zinpyr-1. A procedure for the use of Zinpyr-1 in Arabidopsis roots was developed and validated which allowed the visualisation and relative quantification of Zn. This technique showed that both hma4 and hma2/hma4 mutants differed in the amount of Zn and its distribution compared to wildtype or hma2. This correlates to previous data showing that hma4 and hma2/hma4 are Zn-deficient in shoots but accumulate Zn in roots. Thus we can say that this Zn- deficiency in shoot occurs due to an inability to load Zn into the xylem for transport to the shoot. To investigate the regulation of Zn-homeostasis, Zn-deficient and hma2/hma4 plants were compared to wildtype in micro-array experiments to test whether plants respond systemically to Zn- deficiency. hma2/hma4 plants are Zn-deficient in shoots but over-accumulate Zn in roots, so genes co-regulated in hma2/hma4 roots and shoots, and genes co-regulated in hma2/hma4 roots and Zn- deficient wildtype roots were identified. 52 and 27 genes were identified in these comparisons respectively as regulated >2-fold in the same direction. These were considered robust as their co-regulation was observed in four measurements, two technical replicates of two biological replicates. promoter
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    Selection and characterisation of Arabidopsis mutants resistant to a glutathione biosynthesis inhibitor
    Maughan, Spencer C. ( 2003)
    This thesis describes the investigation of GSH regulation and signaling in Arabidopsis thaliana by a genetic approach. A procedure is described to select for mutants resistant to a chemical inhibitor of glutathione (GSH) biosynthesis, L-buthionine-SR-sulfoximine (BSO). Fifteen mutants were selected and preliminary genetic examinations indicated that BSO-resistance phenotypes are often highly variable. Consequently five mutants were chosen for further genetic, molecular and biochemical characterisation. All five of these mutants were shown to have altered GSH levels. Moreover, root growth among these mutants varied indicating that different mechanisms may be conferring BSO-resistance. Molecular mapping techniques were used to assign the BSO-resistance loci of four of the mutants to chromosome regions. Two mutants (a T-DNA insertion mutant and an EMS-derived mutant) were found to map to the same position between the markers CHS1 and R89998 on chromosome 5. Subsequently, complementation tests confirmed that these mutants define a single locus. The mutated locus in the T-DNA insertion mutant was determined using molecular techniques. The gene that contained the insert encodes a putative transport protein and was designated the BSO Resistance Transporter1 (BRT1). No transcript was detected in the T-DNA insertion mutant (brt1-1) which suggests that brt1-1 is likely to be a null allele. The EMS-derived mutant (brt1-2) contained an intron splice-site consensus sequence mutation which resulted in transcripts that included an unspliced intron. The function of the BRT1 gene was investigated by phenotype testing and despite the brt1 mutants having 2-fold GSH levels they were not resistant to known oxidising agents. Northern analysis of GCS and the use of a GCS-GUS reporter line indicated that the increase in GSH levels in these mutants was not accompanied by increases in GCS transcription and translation. This suggests that BRT1 is a novel component influencing GSH metabolism. BRT1 defines a novel family of three genes in Arabidopsis. Preliminary experiments indicate that these three genes are expressed in the same tissues providing for possible a level of redundancy between them. Supporting this is a newly identified insertion mutant in a second BRT gene (brt2-1) which is also resistant to BS0. Moreover, the double mutant, brt1-1;brt2-1, exhibits increased BSO-resistance suggesting an additive effect of the mutations. Database searches revealed that there were homologues in other organisms including one from the malaria parasite (PfCRT). Mutations in PfCRT confer resistance to a widely used antimalarial, chloroquine (CQ). However the function of PfCRT is not known. The BRT1 mutant was shown to have a level of functional conservation with a malaria parasite homologue, PfCRT, and therefore further characterisation of these proteins may provide insights into GSH metabolism and perhaps CQ-resistance in the malaria parasite.