Transcriptional regulation of a seed-specific carrot gene, DC8.

Abstract

Many late embryogenesis abundant (Lea) protein genes in plants are regulated by abscisic acid (ABA). The RNA level of a carrot gene, DC8, increases in response to ABA in developing seeds. However, DC8 cannot be induced by ABA in adult tissues. We used chimeric genes made of various DC8 promoter fragments fused to beta-glucuronidase (GUS) to analyze the transcriptional regulation of DC8. DC8:GUS expression was measured in electroporated carrot protoplasts and in stably transformed carrots. The region of the DC8 promoter from -170 to -51 contained ABA-responsive sequences that required a 5' upstream region for high levels of expression in embryogenic callus protoplasts. 505 bp of the DC8 promoter conferred GUS expression in stably transformed somatic and zygotic embryos. DC8:GUS was expressed only in tissues formed in the seed. This includes cells in the embryo, the endosperm and the germinating seedlings. Gel retardation and competition experiments were performed to analyze the embryo nuclear protein-DNA binding activities in vitro. No binding activity was detected on the putative ABA-responsive region; however the 5' upstream regions located between -505 and -301 interacted with embryo nuclear factors. An additional site of DNA-protein interaction was located between positions -32 and +178. The nuclear proteins that bind these sequences were found in the embryo nuclei only, not in the nuclei from leaves or roots.