TRANSCRIPTOMIC ANALYSIS IDENTIFIES POTENTIAL REGULATORS INVOLVED IN PROGRAMMED CELL DEATH AND REMODELLING OF LACE PLANT LEAVES (APONOGETON MADAGASCARIENSIS)

Abstract

Programmed cell death (PCD) is a highly controlled, regulated process of deleting cells. Plant PCD is either developmentally regulated or environmentally induced due to biotic or abiotic stress. Leaves of the lace plant Aponogeton madagascariensis undergo developmentally regulated PCD to form perforations in areoles framed between longitudinal and transverse veins throughout the leaf lamina. The lace plant is an emerging model system to study PCD due to its spatiotemporal predictability of PCD on thin, almost translucent leaves, making them ideal for live cell imaging and the existence of protocols for sterile propagation and experimentation. The molecular mechanisms that control animal PCD are well defined but less understood in plant PCD. The objectives of this thesis were to identify potential regulators of lace plant PCD using RNA sequencing analysis and test the role of selected genes using pharmacological experiments, western blotting, qPCR, anthocyanin quantification, reactive oxygen species (ROS) detection and observing changes in leaf perforations. By taking advantage of the visible cell death gradient, cells destined to die (PCD) and cells destined to survive (NPCD) were separated using laser capture microdissection. Transcriptomes of different stages of lace plant leaf development, NPCD and PCD cells were assembled to profile differentially expressed genes during PCD and leaf remodelling. Genes encoding heat shock protein 70 (Hsp70) and autophagy-related protein 16 (Atg16) were highly expressed in early leaf development when PCD is active. Treatment with PCD regulator ROS increased Hsp70 levels 2-fold and caspase-like activity but no effect on perforations formed. Antioxidants yielded opposite effects, inhibiting the number of perforations. Treatment with an Hsp70 inhibitor increased Hsp70 levels 4-fold and yielded similar results to antioxidants, while inhibiting anthocyanin accumulation. Autophagy promotion by rapamycin increased Atg16 levels and inhibited anthocyanin and perforation formation, while autophagy inhibitors had the opposite effect. Results from this work generated a workflow for future lace plant omics studies and improved the understanding of Hsp’s and autophagy’s (through Atg16) role in PCD during lace plant leaf remodelling. The ultimate goal of lace plant research is to improve our understanding of PCD to manipulate the process for future applications in medicine and agriculture.