Identification and Characterization of Genes that Control the Phenolic

Many of the crucial branch points in phenolic biosynthesis occur at the level of the coenzyme A (CoA) esters of the hydroxycinnamic acids, and formation of the CoA esters is a critical step in the biosynthesis of virtually all classes of phenolics in grape. Research on hydroxycinnamate CoA ligases in grape has been very limited. Purification of the enzyme has not been accomplished, and research has been hampered because cDNA probes are not available to study expression of the genes. In the first year of this project we were able to extract and stabilize cinnamoyl CoA ligase (4CL) from grape tissues. We adapted a procedure for enzyme extraction and assay that was described for 4CL from aspen (Populus tremuloidies) so that it can be used with grape tissues and we studied the substrate specificity of the enzyme from leaf and green shoot tissue. In leaves we found that the enzyme showed much more activity with caffeate than with 4-coumaric acid. Activity was also observed with ferulate and sinapate in these extracts. This result is notable because the enzyme from other plants typically shows more activity with 4-coumaric acid than with caffeate. The total enzyme activity extracted from developing shoots was much lower than from leaves and showed substrate specificity more typical of enzymes from other sources, preferring 4-coumaric acid over caffeic acid. Also, with shoot extracts no activity was observed when sinapic acid was used as substrate. These results are significant because they might suggest that there are different isoenzymes present with different substrate specificities in different tissues. Thus, extraction and assay of the enzyme has been successful and we have found that best source of the enzyme from grapevine appears to be developing leaf tissue. We obtained two cDNA clones of 4CL from poplar from a laboratory in British Colombia. These cDNAs were labeled with 32P and used to screen a grape cDNA phage library prepared from mRNA obtained from grape berries at the beginning of ripening. We isolated six strongly positive plaques. The DNA from the positive phage were amplified and have been sent for DNA sequencing. We will know very soon whether or not we have obtained clones of 4CL from grape berries. This would be important because we could then use the grape 4CL clones to study expression of the respective genes in grapevine.