Cloning and characterization of the genomic sequence and promoter region of pea chloroplastic fructose-1,6-bisphosphatase

Abstract

Chloroplastic fructose-1,6-bisphosphatase (FBPase) is an essential enzyme for carbohydrate synthesis during photosynthesis. One of its most interesting feature is its activation during the illumination through a light modulated reduction of a -S-S- bridge located in the enzyme (1). The cytosolic version of plant FBPase is involved in the sucrose synthesis and is inhibited by AMP and fructose-2,6-bisphosphate. The philogenetic tree built up with FBPases from different species arrange plant cytosolic and chloroplastic FBPases in separate groups. However, the alignment of the amino acid sequences of these enzymes shows highly conserved regions suggesting a gene duplication and a common origin for both genes. The main difference is the primary structure of both enzymes, an extra region of the chloroplastic FBPase, which supports two cisteines involved in the formation of the -S-S- bridge. Using PCR amplification and the earlier known cDNA sequence (2), a 1.6 kb genomic DNA sequence of a pea chloroplastic FBPase has been obtained. This genomic DNA contains 4 open reading frames (ORFs), and 3 introns at the same positions that those found for the genomic DNA sequence of wheat (3), suggesting the possibility of a common ancestor for chloroplastic FBPases. The promoter region of the chloroplastic FBPase has been isolated by the PCR walking method. The computer analysis of this region shows several interesting regulatory elements. GATA, GT1 and I boxes, which have been already described as Light Responsive Elements (LRE) in different light-dependent genes (4). This strengthens the previous assumption of a light regulated expression of the pea chloroplastic FBPase. (1) Cséke et al. (1986).Biochem.Biophys.Acta 853: 43-63 (2) Carrasco et al. (1994). Planta 193: 494-501 (3) Lloyd et al. (1991). Mol Gen Genet 225: 209-216 (4) Arguello et al. (1998). Ann.Rev.Plant Physiol.Plant Mol.Biol. 49:525-555