The influence of peroxisomes on reactive oxygen species regulation and regeneration induction in plants

Research Doctorate - Doctor of Philosophy (PhD)Plants being sessile organisms cannot escape abiotic and biotic stresses and therefore have to adapt to the environment in order to enhance survival. In certain plant species, initiation of somatic embryogenesis is a form of morphogenic adaptation in response to stress. In somatic embryogenesis, somatic cells acquire totipotency after dedifferentiation and entry into the cell cycle. The highly regenerable genotype of <i>Medicago truncatula</i> (Jemalong 2HA; 2HA), provides an invaluable experimental system for investigating the processes involved in somatic embryogenesis. Somatic embryogenesis <i>in vitro</i> requires hormones and a stress signal. As a first step in the induction of somatic embryogenesis in the Medicago system, excision of 2HA leaf explants notably causes an oxidative burst induced from the mechanical wounding, and the extracellular reactive oxygen species (ROS) produced is a necessary trigger for regeneration. However, ROS are highly reactive and toxic, excessive amounts can cause damage to biomolecules and eventually leading to premature cell death. Successful regeneration therefore entails the ability of a plant cell to also maintain an optimal subcellular redox environment. Plants as part of their normal aerobic metabolism can produce ROS, but have evolved sophisticated mechanisms to scavenge ROS. In this context, peroxisomes are one of the major sources of ROS produced within a plant cell as part of the photorespiration pathway through glycolate oxidase; but are also involved in various metabolic processes including β-oxidation of fatty acids and biosynthesis of several plant hormones. Additionally, when first discovered, peroxisomes were noted to contain an arsenal of redox antioxidant machinery including site specific catalase. This prompted a consideration of the regulation of peroxisome dynamics during culture of Arabidopsis protoplasts, in the context of their antioxidant capacity; and how these dynamics during dedifferentiation and cell division induction compared to mitochondria and plastids previously investigated in this laboratory. In this thesis, the role of peroxisome dynamics in achieving redox homeostasis, whilst facilitating the acquisition of plant cellular totipotency through dedifferentiation and re-entry into the cell cycle was examined. This encompasses the early stages of somatic embryogenesis initiation. It was demonstrated that explant wounding in Medicago, induces a multiphasic oxidative response predominantly from the plant NADPH oxidase (RBOH <i>protein</i>) during <i>in vitro</i> culture for somatic embryogenesis. This mechanistic wound-induced oxidative burst was also shown during Arabidopsis protoplast isolation. The culture of Arabidopsis protoplasts induces another oxidative burst, and during culture, the role of mitochondria-derived ROS in the initiation of cellular dedifferentiation and cell division has to be considered. Using peroxisome-targeted fluorescent proteins, it was shown that during Arabidopsis protoplast culture, peroxisomes undergo massive proliferation and disperse uniformly around the cell before cell division. Amplification of the peroxisome population was likely induced by the increasing ROS activity from the ROS burst, and subsequently provided the appropriate redox environment for division induction of the Arabidopsis protoplasts. Peroxisome dispersion was influenced by the cytoskeleton, ensuring unbiased segregation during division to daughter cells. The subsequent decline in ROS levels after the ROS burst, correlated with the increase in peroxisomes, suggesting that peroxisome proliferation aided restoration of ROS homeostasis. Transcriptional profiling showed up-regulation of peroxisome biogenesis genes and several peroxisome-localised antioxidant enzymes, most notably catalase. Analysis of antioxidant levels, catalase activity and catalase isoform 3 mutants (<i>cat3</i>) indicates that peroxisome-localised catalase plays a primary role in restoring ROS homeostasis. Further it was shown that the <i>PEX11a</i> mutant which decreases peroxisome proliferation, also caused increased ROS and delayed cell division. To examine the relationship between abiotic stress and peroxisomes in intact Arabidopsis plants, peroxisome dynamics in different organs were examined under salt and drought stress. Peroxisome numbers, but not mitochondria, increased dramatically under both stresses, especially in the aerial portions of the plants conducting photosynthesis. This suggested that peroxisomal proliferation in these regions increase ROS levels by enhancing glycolate oxidase activity through photorespiration aiding in ROS signalling during abiotic stress; but also increasing photorespiratory carbon recycling. In addition, the increased antioxidant capacity protects the photosynthetic apparatus in response to ROS produced in these regions, and as part of long distance signalling from abiotic stress. Peroxisomes also provide an enhanced capacity for hormone biosynthesis. In conclusion, while ROS are important as signalling molecules (<i>e.g. as emphasised here by rboha mutant data</i>) in regeneration, modulating the sudden increase of ROS during culture is critical to prevent cellular damage. This thesis demonstrates that, peroxisomes are highly dynamic and metabolically responsive organelles. During plant cell regeneration, peroxisomes are able to increase their population through proliferation, thus increasing their antioxidant capacity to ameliorate ROS perturbation, maintaining redox equilibrium. Accordingly, under abiotic stress, peroxisomes are also likely to play critical roles through ROS regulation, photorespiration and hormone biosynthesis; conferring stress tolerance and thus, improving the acclimation ability of a plant