Metacaspases (MCs), the caspase orthologs in plants are emerging as major modulators of Programmed Cell Death (PCD) in response to developmental and environmental cues. Tomato MC1 and MC5 were differentially regulated upon 3 biotic stresses- Alternaria solani, PstDC3000 and ToLCNDV and therefore was selected for dissecting the functional dynamics and biological significance of metacaspases in orchestrating PCD in plants. We found that the protein isolates of leaves infected with the virulent A.solani exhibited significantly higher metacaspase activity compared to the avirulent fungi in assays with fluorogenic oligopeptide substrates. MC1 transcript was augmented not only upon infection with the virulent pathogens, but also when inoculated with avirulent A. solani. Inline, MC1 promoter activity was upregulated upon A. solani stress. On the other hand, MC5 transcript expression patterns varied depending upon the pathogen (virulent vs. avirulent) and host (tolerant vs. susceptible). Biochemical investigation of purified MC5 protein demonstrated that it is most active at an acidic pH of 5-6, its proteolytic activity is independent of Ca2+ and drastically inhibited by cysteine protease inhibitors. Moreover, the transient overexpression of MC1 and MC5 in detached tomato leaves resembled pathogen stress as indicated by increased H2O2 accumulation and enhanced levels of ROS generating enzyme transcripts. Also, several autophagy-related gene (ATG) transcripts were observed to be differentially regulated under such conditions, hinting at crosstalk between MCs and ATGs in the regulation of the autophagic pathway during development, which was further reaffirmed by chemical induction of autophagy in tomato plants. Tissue-specific transcript expression analysis revealed MC1 level gradually increases, whereas that of MC5 declines during the course of development. Taken together, our studies propose that MC1 and MC5 are involved in two distinct arms of plant defense against biotic stress and also provides a novel insight into their role in regulating developmental PCD. Supported by CSIR-Ad-hoc fellowship
