Integrative analysis of papain-like cysteine proteases and cystatins reveals stress-dependent regulatory modules in Arabidopsis thaliana

Plant papain-like cysteine proteases (PLCPs) and cystatins constitute a major protease-inhibitor system that contributes to plant signaling and responses to biotic and abiotic stress. Although the Arabidopsis genome encodes 31 predicted PLCPs and 7 cystatins, their coordinated regulation has not been systematically evaluated across major stress conditions. Here, we reprocessed and integrated AtGenExpress microarray datasets to analyze the expression patterns of 28 PLCPs and seven cystatins in response to bacterial infection (virulent and avirulent Pseudomonas syringae), wounding, and drought stress. In parallel, we performed in vitro enzyme activity assays to assess inhibition specificity of seven Arabidopsis cystatins against five abundantly expressed PLCPs. Finally, we used the support vector machine (SVM) package e1071 in R to integrate co-expression and inhibition data and to generate a hypothesis-generating PLCP-cystatin interaction network. Together, these analyses suggest that Arabidopsis deploys distinct PLCP and cystatin gene subsets in response to virulent versus avirulent bacterial infection, and that transcriptional modules associated with bacterial infection and wounding are largely distinct. In contrast, several drought-associated PLCP-cystatin modules overlap with those associated with basal defense, suggesting partially shared regulatory programs across abiotic and biotic stress.