Characterising the Gene Expression, Enzymatic Activity and Subcellular Localisation of Arabidopsis thaliana Metacaspase 5 (AtMCA-IIb)

SIMPLE SUMMARY: Metacaspases are proteases present in bacteria, protists, algae, plants and fungi but not in animals. In a given organism, metacaspases are present in families with multiple members, making it difficult to determine which family member is responsible for what activity. One way to solve this issue is to search for specific cells or specific conditions where a single family member is active in the absence of other members. With this aim in mind, we report the characterisation of metacaspase number 5 from the plant Arabidopsis. It is a little-studied member of the metacaspase class that we detected only under very specific contexts, opening a window for the study of how it helps plants to cope with stress. Furthermore, we gathered important information on the property of the protein itself. The data presented provide additional insights into deciphering the function of metacaspases in plants and will support future research toward further understanding of their mode of action. This knowledge will ultimately inform the creation of climate-resilient crops to provide food security for a growing human population. ABSTRACT: Metacaspases are a class of proteases found in plants that have gained attention in recent years due to their involvement in programmed cell death (PCD) and other essential cellular processes. Although structurally homologous to caspases found in animals, metacaspases have distinct properties and functions. There are nine metacaspase genes in the Arabidopsis thaliana genome; these can be type I or type II, and working out the function of each member of the gene family is challenging. In this study, we report the characterisation of one Arabidopsis type II metacaspase, metacaspase-5 (AtMC5; AtMCA-IIb). We detected the expression of AtMC5 only under specific conditions with a strong upregulation by ER stress and oxidative stress at a narrow 6 h time point. Recombinant AtMC5 was successfully purified from E. coli, with the recombinant AtMC5 working optimally at pH 7, using an optimised reaction buffer containing 10 mM calcium chloride together with 15% sucrose. Like other metacaspases, AtMC5 cleaved after arginine residue and demonstrated a substrate preference towards VRPR. Additionally, AtMC5-RFP was shown to be localised in the cytosol and nucleus of transfected cells. We found no evidence of a strong link between AtMC5 and PCD, and the data provide additional insights into the function of metacaspases in plants and will aid in future research toward further understanding their mode of action.