Rerouting the plant phenylpropanoid pathway by expression of a novel bacterial enoyl-CoA hydratase/lyase enzyme function

The gene for a bacterial enoyl-CoA hydratase (crotonase) homolog (HCHL) previously shown to convert 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to the corresponding hydroxybenzaldehydes in vitro provided an opportunity to subvert the plant phenylpropanoid pathway and channel carbon flux through 4-hydroxybenzaldehyde and the important flavor compound 4-hydroxy-3-methoxybenzaldehyde (vanillin), Expression of the Pseudomonas fluorescens AN103 HCHL gene in two generations of tobacco plants caused the development of phenotypic abnormalities, including stunting, interveinal chlorosis and senescence, curled leaf margins, low pollen production, and male sterility. In second generation progeny, the phenotype segregated with the transgene and transgenic siblings exhibited orange/red coloration of the vascular ring, distorted cells in the xylem and phloem bundles, and lignin modification/reduction. There was depletion of the principal phenolics concomitant with massive accumulation of novel metabolites, including the glucosides and glucose esters of 4-hydroxybenzoic acid and vanillic acid and the glucosides of 4-hydroxybenzyl alcohol and vanillyl alcohol, HCHL plants exhibited increased accumulation of transcripts for phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate:CoA ligase, whereas beta -1,3-glucanase was suppressed, This study, exploiting the ability of a bacterial gene to divert plant secondary metabolism, provides insight into how plants modify inappropriately accumulated metabolites and reveals the consequences of depleting the major phenolic pools.