#4

Caffeoyl-CoA O-methyltransferase (CCOMT)

Since the discovery of a distinct class of O-methyltransferases catalyzing the conversion of caffeoyl coenzyme A (CoA) into feruloyl CoA (16-18), it is now well established that vascular plants contain at least two classes of O-methyltransferases involved in phenylpropanoid/monolignol pathway metabolism. Depending upon the class, these can either act on the corresponding CoA esters (CCOMTs), or are involved in the final methylation step leading to syringyl moieties (so called caffeic acid O-methyltransferases, COMTs). However, the rather broad enzyme versatility displayed by each class of the O-methyltransferases in vitro has led to much uncertainty as regards precise physiological functions (particularly for CCOMT). For example, alfalfa CCOMT is capable of converting caffeoyl and 5-hydroxyferuloyl CoA into feruloyl and sinapoyl CoA, but does not efficiently use p-coumaric, ferulic and sinapic acids as substrates (19). Thus, there is currently some uncertainty as to whether CCOMT is involved in guaiacyl, syringyl and/or guaiacyl-syringyl lignin biosynthesis (5).

Arabidopsis contains some four CCOMT homologues [CCOMT1 – CCOMT4] which, when compared to the alfalfa CCOMT (19), display between ~94 – 65% identity and ~89 – 53% similarity at the amino acid level, respectively. Relative to Arabidopsis CCOMT 1 (the closest homologue to the alfalfa CCOMT), the three other Arabidopsis homologues have 70.3, 63.6 and 67.1% similarity and 58.6, 50.2 and 54.8% identity, respectively; all are cytosolic and lack any targeting peptide sequence(s). Currently, however, no CCOMT has been functionally characterized in Arabidopsis, nor are their detailed patterns of gene expression known.

Analysis of the EST database entries indicates that of the four putative CCOMTs, only CCOMT1 is expressed in all of the tissues examined, except for the above-ground organs (1). On the other hand, CCOMT2 was only found in green siliques, whereas CCOMT4 was present in mixed tissues and CCOMT3 was not detected. It is quite unusual that no CCOMT homologue was detected in the above-ground organs, and again calls into question the stage(s) of Arabidopsis development that were selected for the EST databases, particularly if CCOMT1 has a role in lignification. Obviously, there is an urgent need for the systematic delineation of the function of each CCOMT, with both a biochemical and metabolic context being established.

There are thus three ongoing subprojects: (i) cloning of Arabidopsis CCOMT genes and characterization of the corresponding recombinant proteins; (ii) analysis of CCOMT gene “knockouts”; and (iii) identifying temporal and spatial patterns of expression of the four CCOMT promoters when fused to GUS/GFP reporter genes. Progress is shown in Tables 7 and 8.