Metacaspase-dependent programmed cell death is essential for plant embryogenesis

In plants, as in animals, programmed cell death (PCD) is a key process responsible for the elimination of unneeded structures and for overall shape remodeling during development [1]; however, the molecular mechanisms remain poorly understood. Despite the absence of canonical caspases in plants, dying plant cells show an increased proteolytic caspase-like activity [2]. Moreover, the cell death can be suppressed using synthetic [2] or natural [3] caspase inhibitors. This raises the question of whether plants have specific cysteine proteases with a role similar to metazoan caspases in the execution of PCD. Metacaspases are the best candidates to perform this role, because they contain a caspase-specific catalytic diad of histidine and cysteine as well as conserved caspase-like secondary structure [4,5]. Here we show the first experimental evidence for metacaspase function in the activation and/or execution of PCD in plants, and also demonstrate the fundamental requirement of plant metacaspase for embryogenesis. We explored the role of plant metacaspases in PCD using a model system of somatic embryogenesis of Norway spruce (Picea abies), where the pathway of embryo development (Figure 1A) resembles zygotic embryogeny, even though the embryo origin is different in each case (i.e., somatic cells in proembryogenic mass (PEM) versus zygote) [6]. In this developmental pathway autophagic PCD ablates PEMs at the time of their differentiation to embryos and then eliminates terminally differentiated embryo suspensor as the embryos enter late embryogeny [6,7] (Figure 1A). We have isolated a 1687 bp cDNA sequence from the embryogenic cell cultures (EMBL database accession number AJ534970). The encoded protein shows a significant degree of conservation with metacaspases and falls into the type II plant metacaspase subfamily (Figure S1A). The protein was named mcII-Pa. The predicted secondary structure of mcII-Pa contains conserved domains and motifs present in all members of the caspase/metacaspase/paracaspase superfamily [5] (Figure S1B). The putative mcII-Pa catalytic diad of cysteine and histidine is placed in the α α/β β fold characteristic for the caspase-hemoglobinase fold (CHF)-containing proteins [5]. The predicted mcII-Pa protein lacks both the death-effector domain and the caspase-activating recruitment domain found in classical initiator caspases, but has a p20-like domain including the active-site pentapeptide DXCHS (where X is A or S) shared by all metacaspases [5] (Figure S1B). This domain is fused to the 268 amino acid carboxy-terminal region consisting of a large insert of approximately 180 amino acids and a p10-like domain. In situ hybridization analysis has revealed restricted accumulation …