237Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_15, © Springer Science+Business Media New York 2014
Chapter 15
Plant Metacaspase Activation and Activity
Elena A. Minina , Simon Stael , Frank Van Breusegem ,
and Peter V. Bozhkov
Abstract
Metacaspases are essential for cell death regulation in plants. Further understanding of biochemistry of
metacaspases and their molecular function in plant biology requires a set of robust methods for detection
of metacaspase activation and quantitative analysis of corresponding proteolytic activity. Here we describe
methods for purifi cation of recombinant metacaspases, measurement of enzymatic activity of recombinant
and endogenous metacaspases in vitro and in cell lysates, respectively, and fi nally detection of metacaspase
activation in vivo. Additionally, an in vitro metacaspase protein substrate cleavage assay based on the cell-
free production of substrate protein followed by proteolysis with recombinant metacaspase is presented.
These methods have been originally developed for type II metacaspases from Arabidopsis and Norway
spruce ( Picea abies ), but they can be used as templates for type I metacaspases, as well as for type II meta-
caspases from other species.
Key words Cell lysate , Metacaspase , Natural substrate , Programmed cell death , Proteolytic activity ,
Synthetic substrate1 Introduction
Most plants have two types of structurally distinct metacaspases
[ 1 – 3 ]. Type I metacaspases have a variable sized N-terminal exten-
sion upstream of the p20 caspase-like domain and a short (<40
aa-long) linker between the p20 and p10 domains. Such structure
is not unique to plant metacaspases and is shared with metacas-
pases from fungal and protozoan lineages [ 3 ]. Type II metacas-
pases lack a prodomain, while possessing a longer (>100 aa-long)
interdomain linker. This type of metacaspases is plant-specifi c,
potentially derived from primary endosymbiosis [ 3 ], and therefore
cannot be found in other organisms. The number of metacaspase
genes in the genomes of different plant species varies considerably,
from one or two in some of green algae to several homologues of
each type (I and II) in angiosperms [ 2 ].