Cloning and molecular features of cytosolic fructose-1,6-bisphosphatase from pea

Abstract

A cDNA clone encoding for pea (Pisum sativum L.) cytosolic fructose-1,6-bisphosphatase (E.C. 3.1.3.11) has been isolated by reverse transcription-polymerase chain reaction of the total mRNA. The sequence analysis displayed a 341-amino acid protein of about 37300 Da molecular mass, corresponding to the subunit of this homotetrameric enzyme; it showed about 80% homology with the other ten higher plant cytosolic FBPases sequenced so far. The enzyme displayed a strong transcriptional expression in green organs (sessile and petioled leaves, stem, pod and grain), and poor expression in root and senescent basal leaves. It is noteworthy the high FBPase transcriptional expression in pod, which displays up to 4-fold higher content of FBPase-specific mRNA than that of root. The mRNA related to cytosolic FBPase was detected after 24 h continuous illumination of 24-h-dark-grown seedlings; this light-induced transcriptional expression is slower than that of chloroplast FBPase, which appears soon after 2 h light. In both cases the corresponding mRNAs disappeared when the light was turned off. The translational expression was also manifested, both as FBPase protein and activity, after 24 h illumination. This delay in the expression of cytosolic FBPase with respect to that of the plastidic enzyme can be interpreted as an indirect effect induced by a metabolite of the photosynthetic carbon pathway, rather than a direct effect of light on the DNA-expression mechanism. Pea cytosolic FBPase was not activated by dithiothreitol, with or without coupling to thioredoxins f or m. The enzyme showed a half-life of 6 h.