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.

Figure 1

Proposed biosynthetic pathways to meta hydroxylated phenylpropanoids in sweet basil. Single arrows indicate verified transformations; double arrows indicate potential transformations. Enzymes are as follows: PAL, Phe ammonia lyase; C4H, cinnamate 4-hydroxylase; C3H, p-coumaric acid 3-hydroxylase; CPL3′H, p-coumaroyl hydroxyphenyllactate 3′-hydroxylase; CS3′H, p-coumaroyl shikimate 3′-hydroxylase; EOMT, eugenol O-methyltransferase.

Figure 2

Liquid chromatography (LC)/mass spectrometry (MS) analysis of acyltransferase assays with whole-leaf protein extracts from basil line SW. A, Assay with [8-¹³C]-p-coumaroyl-CoA and shikimic acid as substrates. Elution trace is the selected ion chromatogram at 320.3 mass-to-charge ratio (m/z). Compound 1, [8′-¹³C]-p-coumaroyl-4-O-shikimate. Inset, Electrospray ionization negative mode mass spectrum of compound 2 ([8′-¹³C]-p-coumaroyl-5-O-shikimate). B, Assay with [8-¹³C]-p-coumaroyl-CoA and 4-hydroxyphenyllactic acid as substrates. Elution trace is the selected ion chromatogram at 328.1 m/z. Inset, Electrospray ionization negative mode mass spectrum of compound 3 ([8′-¹³C]-p-coumaroyl-4-hydroxyphenyllactate).

Figure 3

Partial purification, properties and tissue specific activity of basil acyltransferases. A, Elution of basil acyltransferases CPLT (white circles) and CST (black squares) from a strong anion-exchange analytical FLPC column (MonoQ). Arrows indicate fractions for each enzyme activity that are essentially free from the opposing acyltransferase activity. Relative activity at maximum (100%) corresponds to 196 and 108 pkat mg⁻¹, respectively, for CPLT and CST. B, Substrate specificity of acyltransferases partially purified from basil line SW. Bars are relative activities for each respective enzyme preparation with the substrates p-coumaroyl-CoA (black), caffeoyl-CoA (dark gray), and feruloyl-CoA (light gray). Error bars are se. C, Specific enzyme activities for CST (black bars) and CPLT (light-gray bars) in crude protein extracts from selected tissues from basil lines SW and EMX-1. Error bars are se.

Figure 4

Comparison of 3′- and 3-hydroxylase activities in glands isolated from young leaves on mature plants from basil lines SW (dotted lines) and EMX-1 (solid lines). A, Representative LC/MS-selected ion chromatograms (336 m/z) for products of glands incubated with [8′-¹³C]-p-coumaroyl-5-O-shikimate. B, Representative LC/MS-selected ion chromatograms (344 m/z) for products of glands incubated with [8′-¹³C]-p-coumaroyl 4-hydroxyphenyllactate. C, Representative LC/MS-selected ion chromatograms (180 m/z) for products of glands incubated with [8-¹³C]-p-coumaric acid. The same amount of glands, as measured by total protein concentration and the same amount of substrate (0.6 mm), was added in each incubation for both basil lines. All assays were initiated and terminated at the same time, when approximately 5% conversion was observed for the assays in C. The peaks numbered were identified as follows: 1, [8′-¹³C]-caffeoyl-4-O-shikimate; 2, [8′-¹³C]-caffeoyl-5-O-shikimate; 5, [8′-¹³C]-caffeoyl 4-hydroxyphenyllactate; and 7, [8-¹³C]-caffeic acid. Relative activity levels are for each type of reaction, but scales are not comparable between panels.

Figure 5

Enzymatic activity of basil CYP98A13 in purified yeast microsomes. A, C, and E, Assays with recombinant CYP98A13v1. B, D, and F, Assays with WAT11 control. A and B, Assays with [8′-¹³C]-p-coumaroyl shikimate as substrate. C and D, Assays with [8′-¹³C]-p-coumaroyl 4-hydroxyphenyllactate as substrate. E and F, Assays with [8-¹³C]-p-coumaric acid as substrate. Solid lines, Selected ion chromatograms for major ion (m/z) of eluting product peak. Dotted lines, Selected ion chromatograms for major ion (m/z) of eluting substrate peak. Insets, Electrospray ionization negative mode mass spectra for selected peaks. Peaks were identified as follows: 1, [8′-¹³C]-caffeoyl-4-O-shikimate; 2, [8′-¹³C]-caffeoyl-5-O-shikimate; 3, [8′-¹³C]-p-coumaroyl-4-O-shikimate; 4, [8′-¹³C]-p-coumaroyl-5-O-shikimate; 5, [8′-¹³C]-caffeoyl 4-hydroxyphenyllactate; 6, [8′-¹³C]-p-coumaroyl 4-hydroxyphenyllactate; 7, [8-¹³C]-caffeic acid; and 8, [8-¹³C]-p-coumaric acid.