CIS-ELEMENTS AND TRANS-ACTING FACTORS AFFECTING REGULATION OF A NONPHOTOSYNTHETIC LIGHT-REGULATED GENE FOR CHLOROPLAST GLUTAMINE-SYNTHETASE

The glutamine synthetase (GS) gene family in pea (Pisum sativum) consists of four nuclear genes encoding distinct isoenzymes. Molecular studies have shown that the GS2 gene encoding chloroplast-localized GS is expressed in specific cell types and is regulated by diverse factors such as light and photorespiration. Here, we present the nucleotide sequence of the pea GS2 gene promoter. To identify the elements involved in regulation of GS2 expression, GS2 promoter-deletion analyses were performed using GS2-GUS fusions in tobacco (Nicotiana tabacum). This analysis revealed that the GS2 transit peptide is not required for mesophyll cell-specific expression of beta-glucuronidase (GUS). GUS activity was induced 2- to 4-fold in light-grown versus etiolated T-1 seedlings. However, high levels of GUS activity were observed in etiolated seedlings. This observation demonstrated that regulation of expression of GS2, a nonphotosynthetic light-regulated gene, involves additional factors. A 323-bp GS2 promoter sequence is sufficient to confer light regulation to the GUS reporter gene in leaves of mature transgenic tobacco. Light-regulated expression of this pea gene promoter is observed in both tobacco and Arabidopsis, suggesting that the regulatory elements are conserved. Gel-shift analysis detected DNA-protein complexes formed with potential transcription elements within this short, light-responsive GS2 promoter fragment.