Regulation and expression of cinnamate-4 hydroxylase, a cytochrome P450 monooxygenase, in Arabidopsis thaliana

Cinnamate-4-hydroxylase is the first cytochrome P450-dependent monooxygenase of the phenylpropanoid pathway. In order to study the expression of this gene in Arabidopsis thaliana, a cinnamate-4-hydroxylase cDNA clone from the Arabidopsis expressed sequence tag database was identified, and was used to isolate its corresponding genomic clone. The entire cinnamate-4-hydroxylase coding sequence plus 2.9 kb of its promoter was isolated on a 5.4 kb HindIII fragment of this cosmid. Inspection of the promoter sequence revealed the presence of a number of putative regulatory motifs previously identified in the promoters of other phenylpropanoid pathway genes. The expression of cinnamate-4-hydroxylase was analyzed by RNA blot hybridization analysis, and in transgenic Arabidopsis carrying cinnamate-4-hydroxylase-GUS transcriptional fusions. C4H RNA was present in dark grown seedlings but its level increased after exposure of seedlings to light. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated det1-1 mutant seedlings. Although C4H message was found to be light inducible, its expression did not follow a circadian pattern. Cinnamate-4-hydroxylase RNA is widely expressed in various Arabidopsis tissues, particularly in roots and tissues undergoing lignification. The cinnamate-4-hydroxylase promoter-driven GUS expression accurately reflected the tissue specificity and wound-inducibility of the cinnamate-4-hydroxylase promoter indicated by RNA blot hybridization analysis. The analysis of stem cross sections indicates intense staining in lignifying tissues such as the sclerified parenchyma, xylem and hydathodes. In roots, C4H is initially restricted to the vascular tissue and gradually increases in the cortex. Several putative regulatory sequences were found in the C4H promoter that are similar to regulatory sequences found in promoters of other phenylpropanoid genes in Arabidopsis and other species. To analyze the role of putative cis-acting elements in C4H tissue-specificity and environmental-inducibility, eight promoter deletion constructs were generated to drive GUS expression in transgenic Arabidopsis. 5′ deletion of the C4H promoter resulted in a gradual decrease in GUS expression in all organs and resulted in a decrease in the apparent wound inducibility of the promoter in mature leaves