Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota 2: Confirming biosynthetic steps through feeding of inhibitors and precursors

Biosynthesis of hydroxybenzoates even at enzymatic level is poorly understood. In this report, effect of feeding of putative biosynthetic precursors and pathway-specific enzyme inhibitors of early phenylpropanoid pathway on p-hydroxybenzoic acid accumulation in chitosan-elicited hairy roots of Daucus carota was studied. Three selective metabolic inhibitors of plant phenylpropanoid pathway, namely, aminooxyacetic acid (AOAA), piperonylic acid (PIP) and 3,4-methylenedioxycinnamic acid (MDCA), which are known to inhibit phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL) respectively, the three early enzymes of phenylpropanoid metabolism, were chosen with the anticipation that selective inhibition of these enzymes in vivo may provide information on the metabolic route to p-hydroxybenzoic acid formation. Supplementation of AOAA (0.2–1.0 mM) and PIP (0.2–1.0 mM) resulted in the reduced accumulation of p-hydroxybenzoic acid in the wall-bound fraction. However, addition of MDCA (0.2–1.25 mM), did not suppress p-hydroxybenzoic acid accumulation but suppressed lignin and total flavonoid accumulation, suggesting that 4CL enzyme activity is not required for p-hydroxybenzoic acid formation. Feeding of elicited hairy roots with phenylalanine, coumaric acid and p-hydroxybenzaldehyde had a stimulatory effect on p-hydroxybenzoic acid accumulation; however, maximum stimulatory effect was shown by p-hydroxybenzaldehyde. This suggests that p-hydroxybenzaldehyde might be the immediate precursor in p-hydroxybenzoic acid biosynthesis. Finally, in vitro conversion of p-coumaric acid to p-hydroxybenzoic acid with p-hydroxybenzaldehyde as intermediate using cell-free extract provided an unequivocal support for CoA-independent and non-β-oxidative route of p-hydroxybenzoic acid biosynthesis in Daucus carota.