In vivo proteolytic profiling of the type I and type II metacaspases in Chlamydomonas reinhardtii exposed to salt stress.

Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using mass spectrometry-based degradomics approach, during control conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated the relevance of the predicted metacaspase substrates to the physiology of C. reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in the catabolism of carboxylic acids, while CrMCA-II plays an important role in photosynthesis and translation. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.

Detection and Quantification of the Hypersensitive Response Cell Death in Arabidopsis thaliana.

In plants, the hypersensitive response (HR) is a programmed cell death modality that occurs upon recognition of harmful non-self. It occurs at the site of pathogen infection, thus preventing pathogens to live off plant tissue and proliferate. Shedding light on the molecular constituents underlying this process requires robust and quantitative methods that can determine whether plants lacking functional genes are defective in HR execution compared to wild-type controls. In this chapter, we provide two quantitative protocols in which we measure cell death from Arabidopsis thaliana leaves infected with avirulent HR-causing bacterial strains. Firstly, we use trypan blue staining to quantify the stained area of leaves upon bacterial infection using a personalized macro in the Image J (Fiji) software. Alternately, we incorporate an electrolyte leakage protocol in order to measure HR caused by different avirulent bacterial strains at different bacterial titers. We encourage users to perform a combination of both methods when assessing HR in different plant genotypes.

AtSERPIN1 is an inhibitor of the metacaspase AtMC1-mediated cell death and autocatalytic processing in planta.

The hypersensitive response (HR) is a localized programmed cell death phenomenon that occurs in response to pathogen recognition at the site of attempted invasion. Despite more than a century of research on HR, little is known about how it is so tightly regulated and how it can be contained spatially to a few cells. AtMC1 is an Arabidopsis thaliana plant metacaspase that positively regulates the HR. Here, we used an unbiased approach to identify new AtMC1 regulators. Immunoaffinity purification of AtMC1-containing complexes led us to the identification of the protease inhibitor AtSerpin1. Our data clearly showed that coimmunoprecipitation between AtMC1 and AtSerpin1 and formation of a complex between them was lost upon mutation of the AtMC1 catalytic site, and that the AtMC1 prodomain was not required for the interaction. AtSerpin1 blocked AtMC1 self-processing and inhibited AtMC1-mediated cell death. Our results constitute an in vivo example of a Serpin acting as a suicide inhibitor in plants, reminiscent of the activity of animal or viral serpins on immune/cell death regulators, including caspase-1. These results indicate a conserved function of a protease inhibitor on cell death regulators from different kingdoms with unrelated modes of action (i.e. caspases vs metacaspases).