Abstract
To elucidate the genetic and biochemical regulation of elicitor-induced p-coumaraldehyde accumulation in plants, we undertook a multifaceted approach to characterize the metabolic flux through the phenylpropanoid pathway via the characterization and chemical analysis of the metabolites in the p-coumaryl, coniferyl, and sinapyl alcohol branches of this pathway. Here, we report the identification and characterization of four cinnamyl alcohol dehydrogenases (CADs) from cucumber (Cucumis sativus) with low activity toward p-coumaraldehyde yet exhibiting significant activity toward other phenylpropanoid hydroxycinnamaldehydes. As part of this analysis, we identified and characterized the activity of a hydroxycinnamoyl-coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) capable of utilizing shikimate and p-coumaroyl-coenzyme A to generate p-coumaroyl shikimate. Following pectinase treatment of cucumber, we observed the rapid accumulation of p-coumaraldehyde, likely the result of low aldehyde reductase activity (i.e. alcohol dehydrogenase in the reverse reaction) of CsCAD enzymes on p-coumaraldehyde. In parallel, we noted a concomitant reduction in the activity of CsHCT. Taken together, our findings support the hypothesis that the up-regulation of the phenylpropanoid pathway upon abiotic stress greatly enhances the overall p-coumaryl alcohol branch of the pathway. The data presented here point to a role for CsHCT (as well as, presumably, p-coumarate 3-hydroxylase) as a control point in the regulation of the coniferyl and sinapyl alcohol branches of this pathway. This mechanism represents a potentially evolutionarily conserved process to efficiently and quickly respond to biotic and abiotic stresses in cucurbit plants, resulting in the rapid lignification of affected tissues.
Highlights
To elucidate the genetic and biochemical regulation of elicitor-induced p-coumaraldehyde accumulation in plants, we undertook a multifaceted approach to characterize the metabolic flux through the phenylpropanoid pathway via the characterization and chemical analysis of the metabolites in the p-coumaryl, coniferyl, and sinapyl alcohol branches of this pathway
These results suggest that the biosynthesis of lignin precursors in pathogen-challenged cucurbits is directed toward the synthesis of p-coumaryl derivatives rather than the more highly substituted methoxylated analogs
P-coumaraldehyde is an important substrate required for the formation of p-coumaryl alcohol, a lignin precursor (Kim et al, 2003)
Summary
P-Coumaraldehyde and a Lignin-Like Material Accumulate in Cucumber Hypocotyls upon Pectinase Treatment. As monitored by HPLC (e.g. no reduction of p-coumaraldehyde) and by spectrophotometry (e.g. oxidation of p-coumaryl alcohol to its corresponding aldehyde), with any of the tested substrates (Fig. 5) Based on these results, we conclude that CsCAD5 and CsCAD6 activity is extremely low, below the limits of detection, as was observed with several members of the Arabidopsis CAD family (Kim et al, 2004). C4H is considered a key point for entering the monolignol pathway (Fig. 1), and in agreement with the induction of mRNA accumulation in pectinasetreated plants, we detected increased levels of p-coumaric acid in pectinase-treated hypocotyls of cucumber (Fig. 3) Based on this observation, we hypothesized that CsCAD1, CsCAD2, and, to a lesser extent, CsCAD4 are the primary enzymes responsible for the generation of hydroxycinnamyl alcohol formation in cucumber hypocotyls. Based on these initial observations, we eliminated substrate availability as the rate-limiting step controlling the formation of H-lignin follow-
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