Cinnamate-4-Hydroxylase Expression in Arabidopsis (Regulation in Response to Development and the Environment)

Abstract

Cinnamate-4-hydroxylase (C4H) is the first Cyt P450-dependent monooxygenase of the phenylpropanoid pathway. To study the expression of this gene in Arabidopsis thaliana, a C 4 H cDNA clone from the Arabidopsis expressed sequence tag database was identified and used to isolate its corresponding genomic clone. The entire C4H coding sequence plus 2.9 kb of its promoter were isolated on a 5.4-kb Hindlll fragment of this cosmid. lnspection 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 C4H was analyzed by RNA blot hybridization analysis and in transgenic Arabidopsis carrying a C4H-P-glucuronidase transcriptional fusion. C4H message accumulation was light-dependent, but was detectable even in dark-grown seedlings. Consistent with these data, C4H mRNA was accumulated to light-grown levels in etiolated detl-7 mutant seedlings. C4H is widely expressed in various Arabidopsis tissues, particularly in roots and cells undergoing lignification. The C4H-driven /3-glucuronidase expression accurately reflected the tissue-specificity and woundinducibility of the C4H promoter indicated by RNA blot hybridization analysis. A modest increase in C4H expression was observed in the tt8 mutant of Arabidopsis.The phenylpropanoid pathway gives rise to a wide array of metabolites. These compounds participate in many plantdefense responses (Nicholson and Hammerschmidt, 1992) and absorb potentially damaging UV-B radiation (Caldwell et al., 1983;Li et al., 1993;Landry et al., 1995). The pathway also generates the monomers required for lignin biosynthesis: ferulic acid and sinapic acid (Lewis and Yamamoto, 1990). The purification of the soluble enzymes of the phenylpropanoid pathway over the last 10 years has permitted the cloning of their respective genes. These include the genes encoding Phe ammonia-lyase, p-coumarate COA ligase, caffeic acidl5-hydroxyferulic acid, O-methyltransferase, chalcone synthase, and chalcone isomerase. In comparison, the genes encoding two of the Cyt P450-dependent monooxygenases (P450s) in this pathway, C4H and ferulate-5-

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism

“…At least in a qualitative manner, the MetArray expression patterns matched most previously reported data by Northern, RT-PCR, or promoterreporter analyses of single CYP genes (Bell-Lelong et al, 1997;Mizutani et al, 1998), of genes encoding UGTs and glutathione-dependent enzymes (Warnecke et al, 1997;Jackson et al, 2001;Jones et al, 2003;Loutre et al, 2003;Rodriguez Milla et al, 2003), or groups of ABC transporter genes van den Bruˆle and Smart, 2002). A compilation of examples are provided as supplementary information (Supplementary Table 2).…”

The Phenylpropanoid Pathway in Arabidopsis

“…The single C4H gene from Arabidopsis was cloned and characterized over a decade ago (Table 1; Bell-Lelong et al, 1997;Mizutani et al, 1997); however, the identification of viable C4H mutants was only reported recently. By using the UV-screening method described previously, Schilmiller and coworkers were able to isolate three allelic mutants harboring missense mutations in the Arabidopsis C4H gene.…”

Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene

“…For plant transformation, the promoter-terminator cassette from pJR19 (Abbott et al, 2002) was isolated as an EcoRI/HindIII fragment and ligated into pMOG1006 cut with the same enzymes to make pMOG1006-35S. The 35S promoter was later replaced with the Arabidopsis C4H promoter (Bell-Lelong et al, 1997) to make pMOG-C4H. GUS2A-F5H was isolated as a SalI fragment and inserted into SalI-digested pMOG1006-35S or pMOG-C4H plasmids.…”