Glucan phosphorylases in Vicia faba L.: cloning, structural analysis and expression patterns of cytosolic and plastidic forms in relation to starch

Abstract

We have isolated and characterised cDNA sequences from a Vicia faba cotyledonary library encoding a plastidic isoform (VfPho1) and a cytosolic isoform (VfPho2) of an alpha-1,4-glucan phosphorylase (EC 2.4.1.1; Commission on Plant Gene Nomenclature 1994). The Pho1 isoform is characterized by the presence of a plastidial transit peptide and an 81-residue stretch of additional amino acids in the middle of the polypeptide which are not found in the Pho2 isoform. We define the position of this so-called insertion sequence differently from previous authors. The Pho1 transcripts were found predominantly in the early seed coat and in cotyledons, and accumulated until the late desiccation phase, whereas Pho2 transcripts were about equally abundant in all investigated tissues. Activity patterns of both enzymes in cotyledons roughly followed mRNA accumulation patterns, with the exception of the late desiccation phases when mRNAs were degraded but enzyme activities remained at high level, even in long-stored seeds. The distinct Pho1 and Pho2 gene expression patterns in seed coats coincided with the transient accumulation pattern of starch. Similarly, in-situ hybridisation revealed a relationship between Pho1 gene expression and starch granule formation in developing cotyledons. Expression data and enzyme activity patterns were associated with starch formation during seed development, and could simply reflect a continuous accumulation of enzyme protein, ensuring immediate participation in starch degradation during germination.