Reprogramming the Phenylpropanoid Metabolism in Seeds of Oilseed Rape by Suppressing the Orthologs ofREDUCED EPIDERMAL FLUORESCENCE1   

Abstract

(C.M.) As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, and trilignols, altered accumulation patterns of kaempferol glycosides, and changes in minor conjugates of caffeate, ferulate, and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1 suppression.Phenylpropanoid metabolism provides plants with a vast array of phenolic compounds that contribute to nearly all aspects of plant life (Vogt, 2010). In species of the Brassicaceae family, soluble hydroxycinnamate conjugates, mainly sinapate esters, constitute an abundant metabolite fraction produced from a branch of the phenylpropanoid pathway (Fraser and Chapple, 2011). As major compounds, sinapoylmalate accumulates in leaves (Hause et al., 2002) and sinapoylcholine (sinapine) in seeds (Bouchereau et al., 1991; Fig. 1).In Arabidopsis (Arabidopsis thaliana), the disturbed accumulation of sinapoylmalate caused the mutant phenotype reduced epidermal fluorescence (ref; Ruegger and Chapple, 2001). Molecular characterization of the ref1 mutant led to the identification of the gene At3g24503 (REF1) encoding coniferaldehyde dehydrogenase/ sinapaldehyde dehydrogenase (CALDH/SALDH; EC 1.2.1.68; Nair et al., 2004). The bifunctional enzyme CALDH/SALDH was shown to catalyze the NADP + -dependent oxidation of coniferaldehyde and sinapaldehyde to yield the corresponding hydroxycinnamates ferulate and sinapate. As a potential branching enzyme, the enzymatic activity of the REF1-encoded CALDH/ SALDH might be crucial for the partition ratio of metabolites between lignin and hydroxycinnamate biosynthesis ( Fig. 1). Therefore, manipulation of REF1 expression coupled to a comprehensive metabolite analysis of transgenic plants appeared as an interesting strategy to gain a deeper understanding of the plant phenylpropanoid metabolic network. Moreover, in crop plants like oilseed rape (Brassica napus), where high levels of sinapate esters contribute to antinutritive features, the suppression of REF1 orthologs might cause increased quality.This work describes the isolation of REF1 o...

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