scissorsProteases play crucial roles in stress acclimation and cell death in plants. Degradomics – mapping the substrates and cleavage sites of proteases – was only recently applied to plants, but will be crucial to understand the impact of proteases during stress acclimation and cell death.

Protease function has been implicated in a variety of biological processes, such as meiosis, embryogenesis, seed coat formation, stomata development, chloroplast biogenesis, pathogen defense responses and cell death (van der Hoorn, 2008). Moreover, there has been a shift in our perception of proteases from being mere protein-degrading enzymes to being key signaling molecules (Turk et al, 2012). They exert this signaling role through the processing of substrate proteins, which in plants is a largely uncharted domain (Tsiatsiani et al, 2012). Degradomics – mapping the substrates and cleavage sites of proteases – is a novel omic-approach that is only recently introduced to plants and will be crucial to understand the impact of proteases during stress acclimation and cell death.

The COFRADIC (Combined Fractional Diagonal Chromatography) technology enables the discovery of protease substrate proteins on proteomic scale. This technology was founded by the group of Kris Gevaert at VIB-Ghent (Staes et al, 2011) and in close collaboration, COFRADIC was adapted to plant systems by our laboratory, resulting in the first sets of protease substrates – or degradomes (Tsiatsiani et al, 2013).

Our lab has a longstanding interest in a group of proteases called metacaspases and their function and regulation during cell death in Arabidopsis thaliana (Coll et al, 2010; Tsiatsiani et al, 2011; Vercammen et al, 2004). Metacaspases are a family of distant caspase homologs in plants, fungi and protozoa. Caspases are cysteine-dependent aspartate-specific proteases that play a prominent role as the “death executioners” in apoptotic cell death in mammalian systems. Caspases cut a wide range of apoptotic substrates, finally resulting in cell death. In plants, programmed cell death shares morphological features with mammals, although the molecular mechanisms underlying this process are mostly unknown (van Doorn et al, 2011).

The Arabidopsis thaliana genome contains nine metacaspase genes divided into two categories: type I (AtMC1 to AtMC3, with an N-terminal extension or prodomain) and type II (AtMC4 to AtMC9, without the prodomain). Importantly, we demonstrated that type II metacaspases – in contrast to mammalian caspases – are arginine/lysine-specific cysteine dependent proteases (Vercammen et al, 2004). The type II metacaspase AtMC9 has been thoroughly characterized in our lab during the last ten years. It has rather different characteristics than the other investigated metacaspases, having an acidic pH optimum and no requirement for calcium (whereas other metacaspases typically have neutral pH optima and a strict calcium requirement for activity). A screening of a combinatorial tetrapeptide library of approximately 130,321 substrates with AtMC9, indicated the tetrapeptide Val-Arg-Pro-Arg (VRPR) as the optimized substrate. AtMC9 activity is regulated by a serine protease inhibitor, AtSerpin1, by covalently binding to AtMC9 and S-nitrosylation of the active site cysteine (Belenghi et al, 2007; Vercammen et al, 2006). Degradome studies by the COFRADIC technology have revealed a potential developmental function of AtMC9 during early seedling development, where the activity of phosphoenolpyruvate carboxykinase 1 (PEPCK1), a key enzyme in gluconeogenesis, is enhanced upon MC9-dependent proteolysis (Tsiatsiani et al, 2013). These and other roles of type II metacaspases are being investigated with the help of degradome studies.

The type I metacaspases AtMC1 and AtMC2 control hypersensitive-type cell death upon pathogen attack (Coll et al, 2010). In a collaborative effort with the group of Jeff Dangl (University of North Carolina at Chapel Hill, USA) we demonstrated that AtMC1 positively drives cell death in the highly localized patches of cell death that are typical to the rapid response to pathogens, called the hypersensitive response. Interestingly, AtMC2 antagonizes this action and therefore is a negative regulator of cell death. We further investigate the intriguing spatiotemporal regulation of AtMC1 and AtMC2 during hypersensitive response, as well as upstream and downstream actors (degradome) of the pathway.

Further reading

Stael S., Kmiecik P., Willems P., Van Der Kelen K., Coll N.S., Teige M. and Van Breusegem F. (2015). Plant innate immunity – sunny side up? Trends Plant Sci. 20, 3-11.

Wrzaczek M., Vainonen J.P., Stael S., Tsiatsiani L., Help-Rinta-Rahko H., Gauthier A., Kaufholdt D., Bollhöner B., Lamminmäki A., Staes A., Gevaert K., Tuominen H., Van Breusegem F., Helariutta Y. and Kangasjärvi J. (2015). GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis. EMBO J. 34, 55-66.

Bollhöner B., Zhang B., Stael S., Denancé N., Overmyer K., Goffner D., Van Breusegem F. and Tuominen H. (2013). Post mortem function of AtMC9 in xylem vessel elements. New Phytol. 200, 498-510.

Tsiatsiani L., Timmerman E., De Bock P.-J., Vercammen D., Stael S., van de Cotte B., Staes A., Goethals M., Beunens T., Van Damme P., Gevaert K. and Van Breusegem F. (2013). The Arabidopsis METACASPASE9 degradome. Plant Cell 25, 2831-2847.

Tsiatsiani L., Gevaert K. and Van Breusegem F. (2012). Natural substrates of plant proteases: how can protease degradomics extend our knowledge? Physiol. plant. 145, 28-40.

Turk B., Turk D. and Turk V. (2012). Protease signalling: the cutting edge. EMBO J. 31, 1630-1643.

Staes A., Impens F., Van Damme P., Ruttens B., Goethals M., Demol H., Timmerman E., Vandekerckhove J. and Gevaert K. (2011). Selecting protein N-terminal peptides by combined fractional diagonal chromatography. Nat. Prot. 6, 1130-1141.

Tsiatsiani L, Van Breusegem F, Gallois P, Zavialov A, Lam E, Bozhkov PV (2011). Metacaspases. Cell Death Differ. 18, 1279-1288.

van Doorn W.G., Beers E.P., Dangl J.L., Franklin-Tong V.E., Gallois P., Hara-Nishimura I., Jones A.M., Kawai-Yamada M., Lam E., Mundy J., Mur L.A., Petersen M., Smertenko A., Taliansky M., Van Breusegem F., Wolpert T., Woltering E., Zhivotovsky B. and Bozhkov P.V. (2011) Morphological classification of plant cell deaths. Cell Death Differ. 18, 1241-124.

Coll N.S., Vercammen D., Smidler A., Clover C., Van Breusegem F., Dangl J.L. and Epple P. (2010). Arabidopsis type I metacaspases control cell death. Science 330, 1393-1397.

van der Hoorn R.A. (2008). Plant proteases: from phenotypes to molecular mechanisms. Annu. Rev. Plant Biol. 59, 191-223.

Belenghi B., Romero-Puertas M.C., Vercammen D., Brackenier A., Inzé D., Delledonne M. and Van Breusegem F. (2007) Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue. J. Biol. Chem. 282, 1352-1358.

Vercammen D., Belenghi B., van de Cotte B., Beunens T., Gavigan J.A., De Rycke R., Brackenier A., Inzé D., Harris J.L. and Van Breusegem F. (2006). Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9. J. Mol. Biol. 364, 625-636.

Vercammen D., van de Cotte B., De Jaeger G., Eeckhout D., Casteels P., Vandepoele K., Vandenberghe I., Van Beeumen J., Inzé D. and Van Breusegem F. (2004). Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine. J. Biol. Chem. 279, 45329-45336.