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.

Expression of DC8 is associated with, but not dependent on embryogenesis

“…Transgenic carrots were constructed as described by Goupil et al [14]. Arabidopsis plants were transformed with the DC8(505):GUS construct according to procedures described by Valvekens etal.…”

“…Total RNA was electrophoresed in a 10J~o agarose/3YSo formaldehyde gel, blotted onto nylon membranes (GeneScreen, New England Nuclear, Boston, MA), and hybridized at high stringency [ 37 ]. Plasmid p87P 1.7 [14], a transcription vector containing coding sequences from the carrot DC8 gene, was linearized with BamHI and single stranded RNA probe was synthesized using T3 polymerase and 32p-dCTP according to the manufacturer's instructions (Promega, Madison, WI),…”

“…Cis-elements and transcription factors mediating ABA-inducible LEA gene expression have been identified [ 16,29,38]. Some ABA-inducible LEA genes are inducible only in cells formed in the seed [ 14,32], while others appear to lack seed specificity [30,38]. Carrot LEA genes that encode hydrophilic proteins, such as DC8, are expressed in carrot zygotic embryo and endosperm tissues, in somatic embryos, and in callus [2,4,9,46].…”

“…Carrot LEA genes that encode hydrophilic proteins, such as DC8, are expressed in carrot zygotic embryo and endosperm tissues, in somatic embryos, and in callus [2,4,9,46]. The ABA treatment causes large increases in levels of mRNA and protein in cultured carrot cells, but ABA cannot induce DC8 expression in mature leaf or root tissues [14,18]. Other carrot LEA genes, such as DC24, and the carrot oleosin protein gene, DC59, have similar expression patterns [17, 191. Because LEA mRNA and protein abundance was correlated with late embryonic development, LEA gene expression was thought to be regulated by the embryonic developmental program and LEA gene activities have been used as stagespecific markers to characterize embryo-lethal mutants [43,44].…”

“…Other carrot LEA genes, such as DC24, and the carrot oleosin protein gene, DC59, have similar expression patterns [17, 191. Because LEA mRNA and protein abundance was correlated with late embryonic development, LEA gene expression was thought to be regulated by the embryonic developmental program and LEA gene activities have been used as stagespecific markers to characterize embryo-lethal mutants [43,44]. Therefore, it was puzzling to find DC8 expression in early embryogenesis and in non-embryo tissues such as callus and endosperm [ 14]. On the other hand, since both the embryo and the endosperm desiccate during seed maturation, we might expect the expression of genes involved in desiccation protection in both of these tissues.…”