#3

Coumarate CoA ligase (4CL)

Generically, this ATP-requiring class of enzymes converts p-coumaric acid and potentially other substituted cinnamic acids into the corresponding coenzyme A (CoA) esters, respectively. Initially, in Arabidopsis (13), 4CL was reported to exist as a single gene (4CL1), this subsequently being extended to a three membered gene family (4CL1 – 4CL3) by 1999 (14). However, following completion of the Arabidopsis genome, some 14 genes were annotated as putative 4CLs, with identities ranging from ~83 to ~40% relative to the originally isolated 4CL1 (1). It should be noted, though, that the precise biochemical and physiological functions of each are largely unknown, although all contain an AMP binding domain.

The available EST database entries provide some preliminary insight into possible patterns of putative 4CL expression in developing Arabidopsis (1). In the various tissues examined, only eight of the putative 4CLs were detected, namely 4CL1 – 4CL4, 4CL6, 4CL8, 4CL9 and 4CL13, with 4CL8 being the most abundant, i.e. 4CL1, 2, 8 and 13 were noted in developing seeds, 4CL2, 3, 8 and 9 were present in roots, 4CL2, 3, 4 and 8 were in green siliques, whereas 4CL4, 8 and 9 were in above-ground organs. Additionally, 4CL2, 3 and 8 were found in flower buds and 4CL6, 8 and 13 were present in mixed tissues. The remaining six isoforms were not detected, this perhaps being due to low copy number, transient expression and/or only being inducible under certain conditions. As to be expected, these data provisionally suggest that different 4CL isoforms are specifically targeted to distinct metabolic pathways, e.g. to lignins, lignans, various hydroxycinnamate derivatives, as well for formation of, for example, the phenolic components of suberin, sporopollenin and cutin.

Our studies are currently directed towards (i) determining catalytic properties of recombinant proteins including the synthesis of the various possible phenylpropanoid CoA enzymatic products and the corresponding substrates, (ii) defining the patterns of expression of each of the 4CL promoters (fused to GUS and/or GFP), and (iii) analysis of the 4CL gene “Knockouts”. Progress is shown in Tables 4 – 6.(15).