Structure and expression of a developmentally regulated cDNA encoding a cysteine protease (pseudotzain) from Douglas fir

Abstract

We report the complete sequence and expression of a cDNA clone (Pm3-3) encoding a cysteine protease (CysP) from Pseudotsuga menziesii [Mirb] (Pm) Franco (Douglas fir). The sequence consists of a 5' untranslated region (UTR) of 153-bp followed by an open reading frame (ORF) of 1362 bp encoding a putative mature CysP flanked by N- and C-terminal propeptides. A 364-bp 3' UTR contains multiple putative AU-rich elements (ARE) that may be involved in the destabilization of transcripts. The deduced primary structure of the Pm CysP (designated pseudotzain) contains the same invariant amino acid (aa) residues that are involved in the catalytic reaction and make up the catalytic center of CysP from plants and animals. Northern blot analysis showed that cysP transcripts were most abundant in the megagametophyte (MGP) after germination and not detected in the MGP or embryo during embryogenesis. Various osmotic stresses slightly enhanced cysP transcript levels during early seedling development, whereas abscisic acid (ABA), gibberellin (GA) and other plant growth regulators and environmental conditions had little or no effect. The cysP transcripts were present in different amounts in the cotyledons, root and seed coat of 10-day-old seedlings, but were most abundant in the MGP, suggesting a role for this protease in storage protein mobilization. Phylogenetic analysis of mature CysP groups pseudotzain with other angiosperm CysP having both N- and C-terminal propeptides, suggesting a conserved function and/or targeting of this subgroup of enzymes.