The 4-Coumarate:coenzyme A ligases from Nicotiana tabacum and Arabidopsis thaliana : characterization of cDNA clones, gene families, recombinant proteins, and antisense transgenic-plants

The cDNAs encoding 4-coumarate:coenzyme A ligase (4CL), an enzyme in the general phenylpropanoid pathway, were cloned from Nicotiana tabacum and Arabidopsis thaliana. In tobacco, 4CL was encoded by a gene family and northern blot analysis demonstrated that the steady-state RNA levels were highest in stems, ovaries, and non-pigmented portions of the corolla. Two 4CL cDNAs, which were 80% identical to each other at the nucleotide level, were expressed in E. coli. The relative abilities of the recombinant-4CL proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were comparable to that of the 4CL activity found in tobacco-stem extracts. Both recombinant-4CL proteins utilized cinnamate as a substrate, an activity not observed in tobacco extracts. This activity towards cinnamate was inhibited by a modifying-component found in tobacco extracts and the evidence suggests that the substrate specificity of 4CL is, in part, determined by post-translational modification such as phosphorylation. In Arabidopsis, 4CL was shown to be encoded by a single gene. Northern blot analysis indicated that, like tobacco, 4CL steady-state RNA levels were highest in the bolting stem. The Arabidopsis-4CL cDNA was inserted in antisense orientation behind the CaMV 35S or parsley 4CL1 promoters and introduced into Arabidopsis. Transgenic plants were analyzed by western blot analysis and plants with severely suppressed-4CL protein levels were further analyzed. One transgenic-Arabidopsis line had greater than 90% decrease in 4CL enzyme activity and accumulated significantly less (50%) lignin in the bolting stem as compared to wild-type untransformed plants. Despite the decrease in 4CL mRNA, 4CL protein, and 4CL enzyme activity, anthocyanin accumulation was unaffected in the antisense-4CL Arabidopsis-lines. Mature, fully-expanded Arabidopsis-leaves were wounded and this resulted in a coordinated increase in RNA transcripts from genes encoding enzymes in the oxidative pentose phosphate pathway, the shikimic acid pathway, and the general phenylpropanoid pathway. The coordinated activation of gene expression was observed in the wild-type and antisense-4CL transgenic lines suggesting that wound-induced gene expression is not dependent on the carbon flow through the 4CLcatalyzed step.Science, Faculty ofBotany, Department ofGraduat