Two light-responsive elements of pea chloroplastic fructose-1,6-bisphosphatase gene involved in the red-light-specific gene expression in transgenic tobaccos.

ABSTRACT

The pea chloroplast fructose-1,6-bisphosphatase (FBPase) gene was cloned from a pea genomic library and sequenced. The gene contained three introns and four exons. Both in vitro and in vivo analyses of the promoter region of the gene were carried out simultaneously to elucidate the mechanisms of light-mediated gene expression. Two light-responsive elements were identified in gel mobility shift assays a GT-1-like sequence for the binding of a GT-1-like factor (termed pea factor 1; PF1) and a binding site for a dark-specific factor (termed pea factor 2; PF2). The binding affinity of PF1 was higher in light-grown peas than in dark-grown peas and was affected by phosphorylation. The binding site was located at nucleotides (nt) -326 to -341. PF2 binding was dark-specific and the binding region was located upstream of the PF1-binding site (nt -492 to -412). In vivo experiments with transgenic tobacco plants suggested that the region between nt -411 and -272 contained a PF1-binding site that promoted light-mediated expression of the pea chloroplast FBPase. In contrast, the 81-bp region between nt -492 and -412, which is located further upstream than the PF1-binding site, negatively regulated light-mediated expression of FBPase. Moreover, activation of gene expression by the region (nt -411 to -272) contained a PF1-binding site that was sensitive to red-light irradiation, suggesting that the expression of the chloroplast FBPase was regulated by the phytochrome system. Interestingly, the binding region for the dark-specific factor (PF2; nt -492 to -412) not only repressed gene expression in the dark, but also acted as a light-dependent activating element of the chloroplast FBPase gene.