Two Divergent Members of a Tobacco 4-Coumarate:Coenzyme A Ligase (4CL) Gene Family (cDNA Structure, Gene Inheritance and Expression, and Properties of Recombinant Proteins)

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Abstract
Several cDNA clones encoding 4-coumarate:coenzyme A ligase (4CL) were isolated from a tobacco (Nicotiana tabacum) cDNA library and grouped into two classes. Sequencing of one cDNA from each class showed that the clones were similar to other 4CL genes and about 80% identical with each other. Genomic Southern blots using DNA from Nicotiana sylvestris, Nicotiana tomentosiformis, and N. tabacum demonstrated the presence of both classes of 4CL sequences (4CL1 and 4CL2) in the progenitor species and in tobacco. Northern blots indicated that 4CL mRNA transcripts are highest in old stems and higher in the unpigmented corolla tubes than in the pigmented limbs of tobacco flowers. The 4CL genes are developmentally regulated and are wound and methyl jasmonate inducible. The relative abilities of recombinant 4CL1 and 4CL2 proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were similar and comparable with that of 4CL in tobacco stem extracts. Surprisingly, both recombinant 4CL proteins utilized cinnamate as a substrate, an activity not observed in stem extracts. This activity was inhibited by a heat-labile, high-molecular-weight factor found in tobacco stem extracts, suggesting that the substrate specificity of 4CL is, in part, determined by the activity of proteinaceous cellular components.