Severa1 cDNA clones encoding 4-coumarate:coenzyme A ligase (4CL) were isolated from a tobacco (Nicotiana fabacum) 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. Cenomic Southern blots using DNA from Nicofiana sylvesfris, Nicofiana fomentosiformis, and N. tabacum demonstrated the presence of both classes of 4CL sequences (4CL7 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 i n 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.The flow of carbon from primary metabolism into the biosynthesis of an array of phenylpropanoid secondary products involves a minimum of three enzymatic steps, catalyzed by the actions of PAL, cinnamate 4-hydroxylase, and 4CL, which collectively form the general phenylpropanoid pathway. The phenylpropanoid products formed by the action of specific downstream branch pathways include coumarins, flavonoids, lignin, suberin, tannins, and other phenolic compounds, which serve diverse functions as phytoalexins, UV protectants, floral and fruit pigments, structural components of cell walls, and signaling molecules (Hahlbrock and Scheel, 1989).As the last enzyme in the general phenylpropanoid pathway, 4CL converts 4-coumaric acid and other hydroxy-or methoxy-derivatives of cinnamic acid, such as caffeic acid,