Functional characterisation of plant cytosolic thioredoxins.

Abstract

Thioredoxins are small, ubiquitous, disulfide oxidoreductase proteins characterised by a conserved dicysteine active site. Within the cell, they are believed to maintain the redox environment and participate in a broad range of biochemical processes. Plant thioredoxins are a diverse multigene family, primarily classified according to the system by which they are reduced and their subcellular localization. Thioredoxins located in the cytoplasm (type -h) are usually dependent on NADPH for reduction by NADPH-thioredoxin reductase. There are four cytosolic thioredoxins in grass species, with subclass 4 believed to be the most ancient. The highly conserved nature of thioredoxin-h4, in plant species as diverse as angiosperms and gymnosperms, implies a conservation of gene function. Discovery of thioredoxin-h4 function in barley (Hordeum vulgare) was the core focus of the research presented in this thesis. The characterisation of thioredoxin-h4 was approached from both, genetic, and protein biochemistry perspectives. To commence the research, the transcript profile of barley thioreodoxin-h4 (HvTrx-h4) was examined in barley reproductive tissues. As a direct consequence of findings, anther and stigma tissues were used in protein interaction studies employing a mono-cystenic active-site HvTrx-h4 affinity chromatography technique. HvTrx-h4 was mutated, recombinantly expressed, purified and immobilised in order to isolate and identify proteins with which it interacted. Identification of HvTrx-h4 protein targets sought to reveal the pathways in which thioredoxin-h4 is involved. To further characterise the expression of HvTrx-h4, the promoter and 5′ untranslated regions of genomic sequence were isolated and used to drive expression of green fluorescent protein in transgenically modified barley. This enabled examination of the temporal and spatial regulation of HvTrx-h4 under normal growth conditions, as well as in response to abiotic stress and plant hormone treatments. Through these studies it was discovered that HvTrx-h4 is likely to be the subject of post-transcriptional modifications. Subsequent investigations revealed HvTrx-h4 is also regulated at the post-translational level through glutathionylation. Previous studies have ascribed a role for thioredoxins in plant oxidative stress defence. The question of whether modulation of HvTrx-h4 expression could be manipulated to alter plant oxidative stress tolerance was considered. To investigate, transgenic tobacco plants (Nicotiana tabacum) containing altered amounts of thioredoxin-h4 protein were subjected to various stresses; abiotic, biotic and chemical, in nature. Tobacco constitutively over-expressing thioredoxin-h4 displayed increased tolerance to ultraviolet light B, heat and methyl viologen treatment. Knowledge acquired by this study and presented in this thesis, suggest a role for barley thioredoxin-h4 in the oxidative stress response. Furthermore, the description of both post-transcriptional and post-translational regulation of HvTrx-h4 constitutes the first report of this level of regulation for a plant cytosolic thioredoxin.