Domain swap between two type-II metacaspases defines key elements for their biochemical properties.

Type-II metacaspases are conserved cysteine proteases found in eukaryotes with oxygenic photosynthesis, including green plants and some algae, such as Chlamydomonas and Volvox. Genetic and biochemical studies showed that some members in this protease family could be involved in oxidative stress-induced cell death in higher plants, but their regulatory mechanisms remain unclear. Biochemically, two distinct classes of type-II metacaspases are exemplified by AtMC4 and AtMC9 from Arabidopsis, with AtMC4 activation dependent on calcium under neutral pH, whereas AtMC9 is active only under mildly acidic pH, regardless of the availability of calcium. Here, we constructed all six possible combinations between the p20, linker, and p10 domains from AtMC4 and AtMC9. Our results show that calcium stimulation of AtMC4 activity is associated with essential amino acids located in its p20 domain. In contrast, the acidic pH optimum trait is lost from AtMC9 if one or two of its domains are replaced by that from AtMC4, suggesting that multiple interactions between domains in AtMC9 may be responsible for this property. Consistent with this, we found conserved 'signature' residues in each of the three domains that distinguish AtMC4- and AtMC9-like classes of metacaspases. Tracing the origin of the AtMC9 class, we found evidence for its appearance between lycophytes and gymnosperms, coincident with the evolution of more complex root archetypes in terrestrial plants. Our work suggests that the distinctive properties of the AtMC9-like protease could be associated with special cellular physiology in the roots of gymnosperms and angiosperms that are distinct from lycophytes.