Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation. Developmental regulation of the gene, and expression in yeast

Abstract

The general phenylpropanoid pathways generate a wide array of aromatic secondary metabolites that range from monolignols, which are ubiquitous in all plants, to sinapine, which is confined to crucifer seeds. The biosynthesis of these compounds involves hydroxylated and methoxylated cinnamyl acid, aldehyde, or alcohol intermediates. Of the three enzymes originally proposed to hydroxylate the 4-, 3-, and 5-positions of the aromatic ring, cinnamate 4-hydroxylase (C4H), which converts trans-cinnamic acid to p-coumaric acid, is the best characterized and is also the archetypal plant P450 monooxygenase. Ferulic acid 5-hydroxylase (F5H), a P450 that catalyzes 5-hydroxylation, has also been studied, but the presumptive 3-hydroxylase converting p-coumarate to caffeate has been elusive. We have found that Arabidopsis CYP98A3, also a P450, could hydroxylate p-coumaric acid to caffeic acid in vivo when expressed in yeast (Saccharomyces cerevisiae) cells, albeit very slowly. CYP98A3 transcript was found in Arabidopsis stem and silique, resembling both C4H and F5H in this respect. CYP98A3 showed further resemblance to C4H in being highly active in root, but differed from F5H in this regard. In transgenic Arabidopsis, the promoters of CYP98A3 and C4H showed wound inducibility and a comparable developmental regulation throughout the life cycle, except in seeds, where the CYP98A3 promoter construct was inactive while remaining active in silique walls. Within stem and root tissue, the gene product and the promoter activity of CYP98A3 were most abundant in lignifying cells. Collectively, these studies show involvement of CYP98A3 in the general phenylpropanoid metabolism, and suggest a downstream function for CYP98A3 relative to the broader and upstream role of C4H.

Figure 1

Biochemical transformations of the ring and side chain in cinnamic acid. It is the first phenolic acid in the general phenylpropanoid metabolism arising from deamination of l-Phe by PAL. 4-Hydroxylation of cinnamic acid by C4H generates p-coumaric acid, but further ring transformations can potentially occur when the γ-carbon is an acid, aldehyde, or ester. In the current depiction of the pathways, 3-hydroxylation occurs on p-coumaric acid or p-coumaroyl CoA, and the later 5-hydroxylation by ferulic acid 5-hydroxylase (F5H) occurs when the γ-carbon is in aldehyde or alcohol form. A fully methoxylated acid, i.e. 4-hydroxy,3,5-dimethoxycinnamic acid (sinapic acid) is an intermediate in sinapine and other sinapate ester synthesis. The gene encoding 3-hydroxylation has been elusive. Further details appear in the text.

Figure 2

Expression patterns of some Arabidopsis CYP genes. PCR-amplified segments of the CYPs (http://drnelson.utmem.edu/CytochromeP450.html) were used for probing 15 μg of RNA. After initial analyses, C4H, F5H, and CYP98A3 expression was determined again for quantitation using a phosphor imager. A, The stem RNA signal was set as the reference (100%) for each probe. Expression of C4H, F5H, and CYP98A3 were analyzed successively in the indicated order after removal of the previous probe. B, Collectively, three membranes with identical RNA loading were used; a representative for rRNA loading is shown.

Figure 3

Hydroxylation of p-coumaric acid by yeast WAT21 (pRAM51) cells that produce Arabidopsis CYP98A3. Inset, Immunoblot of 15 μg of protein from the control strain containing the vector (pYeDP60) and the recombinant strain fractionated on SDS-PAGE. The new peak appearing in the CYP98A3⁺ strain matched an authentic caffeic acid standard. Negative ion electrospray tandem mass spectrometry identified a predominant daughter ion (m/z 135) from the parent ion (m/z 179).

Figure 4

CYP98A3-catalyzed production of caffeic acid in yeast WAT 21 cells. The cells were grown in YPLA medium (Pompon et al., 1996) supplemented with 5 mm p-coumaric acid. Samples withdrawn at indicated times were analyzed for caffeic acid content by HPLC as described in “Materials and Methods.” Inset, Caffeic acid standard plot constructed with authentic sample.

Figure 5

Developmental regulation of CYP98A3 and C4H. Transgenic plants with promoter::GUS fusion constructs were analyzed for 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc) staining. a, g, c, i, p, and t, Compound microscope; the remainder are from a dissecting microscope. p and t, Differential interference contrast microscopy. The material in a through l and u and v were from soil-grown plants, and the rest from seedlings germinated on Murashige and Skoog agar. The deliberately wounded parts of excised leaves are marked in boxes. Hy, Hypocotyl; Pr, primary root; Lr, lateral root. Cross sections: s, stele; e, endodermis; c, cortex. Bars: 100 μm for p and t; 400 μm for a through l, o, s, u, and v; 1 mm for n and r; and 4 mm for m and q.

Figure 6

CYP98A3 localization, and lignification. Immunochemistry with polyclonal antiserum raised against a truncated CYP98A3 produced in E. coli (inset). Paraffin sections of stem (a–d) and root (e) were probed with pre-immune serum (a) or antiserum (b–e). Hand sections of stem (f–h) or root (i) were stained with phloroglucinol for lignin. Bar = 100 μm in a through e and 400 μm in f through i. Arrows indicate tissues that are lignified. vb, Vascular bundles; if, interfascicular fiber; st, stele.