A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/ quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.

SummaryThe monomeric composition of tobacco lignin has been modified by genetic engineering. Sense or antiseose expression of sequences encoding O-methyltransferese (OMT), a lignin biosynthetic enzyme, was shown to modulate enzyme activity. Ten constructs harboring the entire or a partial OMT cDNA were used. Populations of 20 transgenic plants per construct were analyzed for OMT activity and compared with untransformed controls. As expected, expression of only the full-length sense construct led to an increase in OMT activity. An important reduction of activity was found in a variable number of plantlets from all other transgenic populations but the inhibition was sustained through the adult stage only in plants transformed with the complete cDNA. T-DNA genes were shown to be stably integrated into the tobacco genome and to be transmitted to the progeny. By using gene-specific probes, OMT inhibition in stems was correlated to a parallel disappearance of OMT transcripts originating from both the resident gene and the transgene. In contrast, transgene transcripts were detected in leaf tissues where the resident gene is poorly expressed, thus indicating that relative expression of the two OMT genes controls transcript turnover. In stems of inhibited plants, a marked decrease of syringyl units and the appearance of 5-hydroxy guaiacyl units were demonstrated. These two structural features are also characteristic of natural mutants of maize with an improved digestibility compared with wild lines. These data demonstrate the feasibility and the potential benefits of lignin manipulation.

Bilberry is a characteristic field layer species in the boreal forests and is an important forage plant for herbivores of the North European ecosystem. Bilberry leaves contain high levels of phenolic compounds, especially hydroxycinnamic acids, flavonols, catechins, and proanthocyanidins. We investigated the phenolic composition of bilberry leaves in two studies, one following foliar development in forest and open areas, and the other along a wide geographical gradient from south to north boreal forests in Finland. An analysis of bilberry leaves collected in open and forest areas showed that major phenolic changes appeared in the first stages of leaf development, but, most importantly, synthesis and accumulation of flavonoids was delayed in the forest compared to the high light sites. Sampling along a geographical gradient in the boreal zone indicated that leaves from higher latitudes and higher altitudes had greater soluble phenolic and flavonol levels, higher antioxidant capacity, and lower contents of chlorogenic acid derivatives. The ecological significance of the results is discussed.