Characterising two newly identified Arabidopsis thaliana SUMO proteases

Abstract

As food production efforts are under escalating threat particularly with abiotic and biotic stresses depleting crop yield, there is an increasing need to understand and manipulate the plant stress signalling pathways to generate stress-resilient crops. Recently, the post-translational modification (PTM) system, Small Ubiquitin-like Modifier (SUMO), has been shown to regulate a wide spectrum of plant adaptation processes. The research in this thesis explores our current knowledge of the SUMO pathway and investigates the SUMO proteases regulating deSUMOylation. Two proteases from a newly discovered class of SUMO proteases, deSUMOylating Isopeptidases (DeSis), in Arabidopsis thaliana (Arabidopsis) were extensively investigated in this study. The At4g25660 (AT60) and At4g25680 (AT80) DeSi proteases, displayed similar characteristics to one another and were both found to localise outside the nucleus towards the plasma membrane. An in vitro deSUMOylation assay displayed signs of the SUMO protease activity of the AT60 protease. Although functional redundancy was speculated between the two DeSi proteases, findings suggested unequal redundancy was more likely with AT80 being more important. Double knockout (KO) AT60-AT80 mutants using the clustered regularly interspaced short palindromic repeats (CRISPR) system and single AT60 and AT80 overexpressing transgenics were generated and subjected to stress-response assays. AT60-AT80KO mutants were hypersensitive to the presence of the stress modulator phytohormone, abscisic acid (ABA), and the pathogen response elicitor, flg22. Overexpressing lines displayed either no difference or increased tolerance to the stress elicitors relative to wild-type (WT) plants. The findings provided evidence that the AT60 DeSi protease was implicated in negatively regulating ABA signalling and plant immune responses. The AT80 protease was found to play a regulatory role in ABA and immune signalling responses, as well as showing potential implications in pathogen-induced guard cell responses. This study provides evidence the two DeSi proteases play a significant role in regulating the stress-induced growth and defence responses in Arabidopsis.