GLUCAN PHOSPHORYLASES IN VICIA-FABA L - CLONING, STRUCTURAL-ANALYSIS AND EXPRESSION PATTERNS OF CYTOSOLIC AND PLASTIDIC FORMS IN RELATION TO STARCH

We have isolated and characterised cDNA sequences from a Vicia faba co tyledonary library encoding a plastidic isoform (VfPho1) and a cytosol ic isoform (VfPho2) of an alpha-1,4-glucan phosphorylase (EC 2.4.1.1; Commission on Plant Gene Nomenclature 1994). The Phol isoform is chara cterized by the presence of a plastidial transit peptide and an 81-res idue stretch of additional amino acids in the middle of the polypeptid e which are not found in the Pho2 isoform. We define the position of t his so-called insertion sequence differently from previous authors. Th e Pho1 transcripts were found predominantly in the early seed coat and in cotyledons, and accumulated until the late desiccation phase, wher eas Pho2 transcripts were about equally abundant in all investigated t issues. Activity patterns of both enzymes in cotyledons roughly follow ed mRNA accumulation patterns, with the exception of the late desiccat ion 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 accumu lation 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 pattern s were associated with starch formation during seed development, and c ould simply reflect a continuous accumulation of enzyme protein, ensur ing immediate participation in starch degradation during germination.