The origin and metabolic fate of 4-hydroxybenzoate in Arabidopsis

The contribution of p-coumarate β-oxidation and kaempferol cleavage to the pools of glycosylated, free and cell wall-bound 4-hydroxybenzoate is organ-dependent in Arabidopsis. 4-Hydroxybenzoate (4-HB) is a vital precursor for a number of plant primary and specialized metabolites, as well as for the assembly of the plant cell wall. In Arabidopsis, it is known that 4-HB is derived independently from phenylalanine and tyrosine, and that the metabolism of phenylalanine into 4-HB proceeds via at least two biosynthetic routes: the β-oxidation of p-coumarate and the peroxidative cleavage of kaempferol. The precise contribution of these precursors and branches to 4-HB production, however, is not known. Here, we combined isotopic feeding assays, reverse genetics, and quantification of soluble (i.e., free and glycosylated) and cell wall-bound 4-HB to determine the respective contributions of phenylalanine, tyrosine, β-oxidation of p-coumarate, and peroxidative cleavage of kaempferol to 4-HB biosynthesis in Arabidopsis tissues. Over 90% of 4-HB was found to originate from phenylalanine in both leaves and roots. Soluble 4-HB level varied significantly between organs, while the proportion of cell wall-bound 4-HB was relatively constant. In leaves and flowers, glycosylated and cell wall-bound 4-HB were the most and least abundant forms, respectively. Flowers displayed the highest specific content of 4-HB, while free 4-HB was not detected in roots. Although p-coumarate β-oxidation and kaempferol catabolism were found to both contribute to the supply of 4-HB in all tissues, the proportion of kaempferol-derived 4-HB was higher in roots than in leaves and flowers. Within the β-oxidative branch, p-coumaroyl-CoA ligase 4-CL8 (At5g38120) bore a preponderant role in the production of soluble and cell wall-bound 4-HB in leaves, while p-coumaroyl-CoA ligase At4g19010 appeared to control the biosynthesis of soluble 4-HB in flowers. Furthermore, analysis of a series of Arabidopsis T-DNA mutants corresponding to the three major UDP-glucosyltransferases known to act on 4-HB in vitro (UGT75B1, UGT89B1, and UGT71B1) showed that none of these enzymes appeared in fact to have a significant role in the glycosylation of 4-HB in vivo.