Abstract
M.Sc.
Since plants are stationary, their immune systems have adapted to their environments to enable them to overcome or respond appropriately to various environmental, physical and physiological stresses that they may encounter by developing complicated defense mechanisms. The plant defense response activates complex biochemical and structural changes in plant cells. Heat stress per se, appears to be a priority stress response in plants, and increased disease susceptibility may be a result of this response. In this study, altered gene expression levels mediated by a heat treatment in Arabidopsis thaliana seedlings were analyzed.
Seedlings were exposed to a heat stress of 42C for 30 minutes, followed by a 2.5 hour recovery period at 25ºC. RNA that was isolated from the heat stress treated plants as well as control plants (untreated) was used to perform suppression subtractive hybridization (SSH) in order to obtain a forward and a reverse DNA library. The forward SSH library represented the genes that were up-regulated due to the heat shock and the reverse SSH library represented the down-regulated genes. Sequenced clones from these libraries were BLAST against the A. thaliana genome using the Genbank database and the Accession numbers retrieved were then used for Bioinformatics analysis to obtain functionality of the genes found. The bioinformatics tools used were TAIR tools, DAG graphs and FatiGO and genes were categorized into the biological processes, molecular functions and cellular components. The TAIR tools and FatiGO were then used to analyze microarray data obtained for a similar study, in order to compare the genes identified with SSH.
The genes coding for photosystem IID, serine-type peptidase, phospholipase D α, a S-locus protein kinase, regulator of chromosome condensation (RCC1) and Glucose-6-phosphate translocator are prominently up-regulated whilst other genes encoding photosystem I, plastocyanin-like mavicyanin, carbohydrate trans-membrane transporter MSS1, zinc finger C3HC4 ring family protein, ubiquitin conjugating enzyme 35 (UBC35) and integral membrane family proteins are significantly down-regulated. The FatiGO results helped to assign functionality to the genes that were found. For the SSH forward library, the cellular protein metabolic pathway was the most highly expressed term (19.21%), whereas in the microarray data, the term „positive regulation of response to stimulus‟ and membrane disassembly had a 100% expression. The reverse SSH data (down-regulation) found phosphate metabolic process as the most highly expressed term with an expression of 44.36%
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and the microarray data (negative fold-change) found the term photorespiration to be the most highly expressed with 93.54% expression. These high levels of negative expression indicate the down-regulation of these processes in the cell during heat shock.
From these results it can be assumed that at the onset of a heat stress, the plant‟s immediate response is to activate pathways of regulation as a response to the stimulus as a self-protection mechanism, and repress other pathways such as photorespiration in order to preserve its energy such as ATP.
These findings suggest that the plant is well equipped to overcome stress in its environment by activation/repression of specific organelles and pathways in the system, in order to maintain its equilibrium. Studies such as these can prove to be helpful to solve the interesting question of how a plant overcomes various environmental stresses in order to prevent disease susceptibility.