Author: Christopher Waterman
Waterman, Christopher, 2020 The generation and analysis of transgenic Arabidopsis plants with improved capacity to recover from severe drought stress, Flinders University, College of Science and Engineering
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The alternative pathway (AP) is a non-phosphorylating respiratory pathway present in plant mitochondria as well as being found in lower eukaryotes and other organelles, such as the chloroplast. Flux through this pathway generates heat from the energy carried in, the electron carriers NAD(P)H and FADH2, rather than conserving the energy in ATP synthesis. Despite its seemingly wasteful nature, the AP plays an important role in stress tolerance and signalling. Although it clearly plays a role in general stress tolerance, how this benefit is conveyed is still unclear. Previous experimenters have highlighted the importance of alternative oxidase (AOX1a) in stress tolerance using RNAi and overexpressor (OEX) models under a range of stressors, with clear detriments and benefits being found respectively. Other research has also found this isoform is commonly co-expressed with another AP member, type II NADH dehydrogenase (NDB2) in response to many stressors, but far less is known regarding its role in stress tolerance. In this study we aimed to generate lines co-overexpressing both of these AP members and exposing them to a combined drought and light stress, determining their response using a variety of different methodologies.
Three dual OEX lines generated from individual insertion events all showed increased expression of Ndb2 and Aox1a both at the transcriptional and protein level. Activity was assessed using isolated mitochondria and is the first example to show the Arabidopsis NDB2 to be NADH specific and calcium activated in a homologous expression system. Previous single Ndb2 OEX lines generated in our lab have failed to show any changes to activity. Interestingly, the co-OEX model generated in our hands were able to show significant increases in activity and further increases in the presence of AOX activators, highlighting the important connectedness these two AP members share.
Phenotype was assessed along with a number of biochemical and gas exchange measurements. Unlike the single OEX lines, the dual OEX lines showed minimal delay in growth milestones and were similar to the wildtype line under control conditions. There were minimal differences in the dual OEX lines compared to the wildtype line under control conditions. There was a small reduction in CO2 assimilation rate in the double OEX lines compared to wildtype and single Aox1a OEX. Notably, oxidative damage marker TBARS, was reduced under control conditions in all transgenic lines tested compared to wildtype.
These lines were then tested under drought and light stress conditions to determine changes to tolerance and their ability to recover from stress. In our tests, both the single Aox1a OEX and dual OEX lines show clear advantage over both wildtype and single Ndb2 OEX lines in their ability to recover from drought. With the addition of Ndb2 OEX to the single Aox1a OEX line, the number of plants able to recover from drought significantly exceeded the single Aox1a OEX. Although gas exchange and stress marker measurements were made on these lines under stress, an answer for how the dual OEX is conveying the improvements to recovery was not clear.
A global transcriptomic approach was applied to investigate the potential pathways that could have contributed to the improved recovery. A clear shift in signalling was demonstrated with a number of carbon and nitrogen metabolism pathways altered possibly linked also to a strong change in hormone signalling. Both single and dual OEX lines also showed upregulation of stress responsive pathways under control conditions suggesting these plants were primed for a general incoming stress. The large changes caused by altering expression of the AP highlighted the importance these genes have on controlling regulatory signals and their homeostatic role.
Keywords: Alternative Oxidase, Type II NAD(PH Dehydrogenase, Arabidopsis, Drought, Abiotic Stress, Alternative Pathway, Mitochondria, Transgenic, Recovery, AOX, NDB2
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Science and Engineering
Supervisor: Kathleen Soole