Differential Production of meta Hydroxylated Phenylpropanoids in Sweet Basil Peltate Glandular Trichomes and Leaves Is Controlled by the Activities of Specific Acyltransferases and Hydroxylases

Abstract

Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid.Basil (Ocimum basilicum) plants synthesize significant quantities of phenylpropanoid derivatives that contain an hydroxyl group at the meta position on the aromatic ring. Several of these compounds, such as eugenol, rosmarinic acid, and caffeic acid (Fig. 1), are found in high concentrations in specialized structures that are located on the surface of the aerial parts of the plant and are known as peltate glandular trichomes (glands; Gang et al., 2001). These specialized metabolites have been found in other plant species as well, but the nature of the enzymes that catalyze the meta hydroxylation has so far remained poorly understood.One of the difficulties in identifying the enzymes catalyzing the 3-hydroxylations in the phenylpropanoid pathways is that they appear to be found at very low abundance in the tissues examined (Petersen, 1997). We have recently shown (Gang et al., 2001) that, as in peppermint (Mentha ϫ piperita; Gershenzon et al., 1992;McCaskill and Croteau, 1995), the basil gland cells can be removed from the plant and studied in isolation from the rest of the plant, greatly facilitating biochemical and molecular investigations of a single, fully differentiated cell type (Gershenzon et al., 1992;McCaskill et al., 1992;McCaskill and Croteau, 1995;Lange et al., 2000;Gang et al., 2001). Furthermore, because these gland cells are so highly specialized for production of plantspecialized metabolites, the enzymes in these metabolic pathways are highly express...

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…A mechanistically similar hydroxylation was suggested for the biosynthetic route to the phenylpropanoid specialized metabolite rosmarinic acid, which proceeds via 3-hydroxylation of the 4 0 -phenyllactic ester of 4-coumarate. Two CYP98A enzymes from species accumulating rosmarinic acid, one from Ocimum basilicum (sweet basil) of the Lamiaceae family, and the other from Lithospermum erythrorhizon of the Boraginaceae family, were suggested to catalyse the required 3-hydroxylation, albeit at a lower rate than with the shikimate ester (O. basilicum) or without comparison to the shikimate ester (L. erythrorhizon) [19,20]. The proof for a truly neofunctionalized CYP98A in rosmarinic acid biosynthesis came in 2009, when Eberle et al [21] published their study on Coleus blumei (Lamiaceae) CYP98A14.…”

A coumaroyl-ester-3-hydroxylase Insertion Mutant Reveals the Existence of Nonredundant meta-Hydroxylation Pathways and Essential Roles for Phenolic Precursors in Cell Expansion and Plant Growth

“…Interestingly enough, the ectopic CYP98A3 overexpression for complementation of the null mutant does not promote any ectopic lignification. It is worth mentioning that the analysis of precursor conversion by peltate glands isolated from two sweet basil (Ocimum basilicum) lines that differ in their ability to produce eugenol also led Gang et al (2002) to propose the existence of an alternative meta-hydroxylation pathway. The line that produced methyl chavicol instead of eugenol had a strongly decreased ability to meta-hydroxylate p-coumaroyl esters, but an unaltered capability to convert p-coumaric acid into caffeic acid.…”

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

“…Unfortunately, the mechanisms involved in biochemical differentiation and global gene expression of epidermal cells remain largely unknown, partly due to perceived difficulties associated with selective isolation of leaf epidermal cells that are intimately associated with adjacent cells within the tissue. However, several protocols have been developed for the isolation of other leaf surface cells, like glandular trichomes that protrude on the leaf surface (Lange et al, 2000;Gang et al, 2002;Wagner et al, 2004).…”