Author: Chevaun Anne Smith
Smith, Chevaun Anne, 2010 ALTERED EXPRESSION OF THE NON-PHOSPHORYLATION ELECTRON TRANSPORT CHAIN EFFECTS GROWTH AND STRESS TOLERANCE IN ARABIDOPSIS THALIANA, Flinders University, School of Biological Sciences
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The branched respiratory pathway of plants contains a cyanide-insensitive alternative pathway. The alternative pathway is of particular interest due to the apparent wasteful activity of the non-phosphorylating NAD(P)H dehydrogenases. Seven putative alternative NAD(P)H dehydrogenase genes have been identified from the Arabidopsis thaliana genome however, there have only been five activities characterised experimentally. Thus it is not known which gene encodes which activity in A. thaliana mitochondria. The three main aims of this study were to generate a transgenic A. thaliana line with complete suppression of the ndb4 gene to confirm that it encodes an external NAD(P)H dehydrogenase and its calcium dependence. It was also hoped to elucidate the role of these alternative NAD(P)H dehydrogenases by reducing or eliminating expression of all four external NAD(P)H dehydrogenases in one plant line. The third aim was to look at the response of the alternative pathway including Alternative Oxidase (AOX) to stress growth conditions, specifically salinity stress. RNAi technology was used to generate transgenic A. thaliana plants. The ndb4 lines showed a range of suppression which lead to the up-regulation of Atnbd2 in some lines and Aox1a in all lines, which was confirmed at the protein level. It was determined that ndb4 encodes a calcium independent NADH dehydrogenase, where ndb2 is likely to have calcium dependent NADH activity. Silencing lines of an Atndb region indicated the plant was not viable without any of the external dehydrogenases present. Microarray data indicated an altered expression profile in ndb4 knock-down lines which significantly impacted several anti-oxidant pathways, both in and outside the mitochondria indicating the altered ndb4 resulted in a change in the global expression pattern of the cell. The involvement of this alternative pathway in a stress response may be linked to its capacity to uncouple carbon metabolism from adenylate control and/or the minimisation of the formation of destructive reactive oxygen species (ROS). Salinity stress is a widespread, adverse environmental stress, which leads to an ionic imbalance, hyperosmotic stress, and oxidative stress, the latter being the result of reactive oxygen species formation. We have shown that salinity stress of A. thaliana plants resulted in the formation of ROS, increased levels of Na+ in both the shoot and the root and an increase in transcription of Aox1a, ndb2 and ndb4 genes, indicating the formation of a bridged non-phosphorylating electron transport chain in response to salinity stress. Furthermore, plants constitutively over-expressing Aox1a, with increased AOX capacity, showed lower ROS formation, 30-40% improved growth rates and lower shoot Na+ content compared to controls, when grown under salinity stress conditions, the ndb4 knock-down plants responded to salinity stress with the same trend. This was even further supported when plants had both an external dehydrogenase and AOX up regulated. Thus, more active alternative pathway in roots and shoots can improve the salt tolerance of A. thaliana as defined by its ability to grow more effectively in the presence of NaCl, and maintain lower shoot Na+ content. AOX does have an important role in stress adaptation in plants, and these results provide some validation of the hypothesis that AOX can play a critical role in cell re-programming under salinity stress.
Keywords: AOX,A.thaliana,NAD(P)H dehydrogenase,external dehydrogenase,salinity stress
Subject: Biological sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Assoc/Prof Kathleen Soole