INVESTIGATING THE ROLES OF REACTIVE OXYGEN AND NITROGEN SPECIES IN PLANT PROGRAMMED CELL DEATH, CYTOSKELETAL AND MITOCHONDRIAL DYNAMICS

Abstract

Mitochondria are usually considered simply as the “powerhouses of the cell”, however in recent years it has become apparent that mitochondria are also of fundamental importance in programmed cell death (PCD), which refers to cell death resulting from a controlled, genetically defined pathway. In Arabidopsis, PCD induced by either heat shock or treatment with strong oxidants is found to be correlated with an early and irreversible change in mitochondrial morphology which manifests as an increase in the size of individual mitochondria. In addition, PCD causes a clustering of mitochondria and loss of motility. In this study, I have used two arginase negative mutant Arabidopsis lines (argah1-1 and argah2-1) which have elevated cellular NO concentrations to examine the effect of nitrosative stress on mitochondria undergoing PCD. Another three different Arabidopsis lines (mito-GFP/mTalin-mCherry, mito-GFP/MAP4-mCherry, mito- mCherry/EB1b-GFP) were used to visualize cytoskeletal elements alongside mitochondria to examine the mechanisms responsible for the mitochondrial morphology transition, clustering and motility inhibition. Results indicate that the elevated concentration of NO found in arginase negative mutants is not sufficient to induce PCD. There was no significant mitochondrial morphology or dynamic change detected between arginase negative mutants and wild type plants, with or without a heat shock. Disruption of either actin or microtubule (MT) cytoskeletal elements leads to the formation of mitochondrial clusters, although they showed different cluster morphology and sizes. Mitochondrial clusters were observed to be moving along the remaining actin cables after a mild heat treatment or cytoskeletal depolymerizing drug treatment. Intact microtubules or MT plus ends visualized with EB1b did not show any interaction with mitochondria under normal conditions. However, after a mild heat stress, EB1b appeared to be associated with clusters of enlarged, possibly swollen mitochondria.