Regulation of ferulate-5-hydroxylase expression in Arabidopsis in the context of sinapate ester biosynthesis

Abstract

Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here we show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3' of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapoylcholine accumulation in embryos.

Figure 1

The phenylpropanoid pathway and the pathways leading to sinapate esters in Arabidopsis. CCoA OMT, Caffeoyl CoA O-methyltransferase; C3H, p-coumarate-3-hydroxylase; pCCoA 3H, p-coumaroyl CoA-3-hydroxylase; SCE, sinapoylcholinesterase; SCT, sinapoylglucose:choline sinapoyltransferase; SGT, sinapic acid:UDPG sinapoyltransferase; SMT, sinapoylglucose:malate sinapoyltransferase. The enzyme catalyzing the step from sinapic acid to sinapoyl CoA is shown as “4CL?” to reflect the uncertainty surrounding the identity of the protein involved.

Figure 2

F5H transcript accumulation in tissues of wild-type Arabidopsis. Total RNA was extracted from the tissues indicated and probed with the F5H cDNA in RNA-blot analysis. The bottom panel illustrates ethidium-bromide staining of rRNA as a loading control.

Figure 3

Transcript accumulation of phenylpropanoid genes during seedling development. Arabidopsis seedlings were germinated and grown under aseptic conditions on modified Murashige-Skoog agar plates (Lorenzen et al., 1996) in light (L; 16-h light/8-h dark photoperiod), in darkness (D), or in darkness for 4 d before being shifted to light conditions (D→L). Total RNA was extracted on the days indicated and RNA analysis was performed using probes from cDNAs of the indicated genes. A, Total RNA from wild-type seedlings probed with the F5H cDNA. The lower panel illustrates ethidium-bromide staining of rRNA as a loading control. B, Total RNA from wild-type seedlings probed with cDNAs corresponding to the phenylpropanoid genes indicated. C, Total RNA from a fah1-2: F5H(HX) transgenic line probed with the F5H cDNA. All blots were exposed to film for 24 h except PAL2, which was exposed for 48 h.

Figure 4

Accumulation of sinapate esters in wild-type and transgenic fah1 Arabidopsis seedlings. Wild-type seedlings and transgenic fah1-2 seedlings were grown on modified Murashige-Skoog agar plates as described for Figure 3. Sinapate esters were fractionated by HPLC, detected at 335 nm, and quantitated using the extinction coefficient of sinapic acid. Each point represents the average of three replicates of 10 seedlings each ± se. Top row, Seedlings grown in the light (open symbols). Bottom row, Seedlings grown in the dark (solid lines, solid symbols) or shifted from dark to light on d 4 (dotted lines, open symbols). Circles, Sinapoylcholine; triangles, sinapoylglucose; squares, sinapoylmalate.

Figure 5

Diagrammatic representation of the F5H gene and F5H transgenes used in this study. Exons are represented by open rectangles. A, F5H genomic region. An inverted triangle indicates the location of a T-DNA insertion in the fah1-9 mutant. B, Constructs used for F5H expression analysis in the fah1-2 mutant background. 35S, CaMV 35S promoter; E, EcoRI; H, HindIII; K, KpnI; S, SacI; X, XhoI.

Figure 6

Leaves of wild type, fah1-2, and transgenic lines as viewed under visible and UV light. Top panel, Adaxial leaf surfaces illuminated by visible light; middle panel, adaxial leaf surfaces illuminated by UV light; bottom panel, abaxial leaf surfaces illuminated by UV light (peak wavelength = 302 nm).

Figure 7

Accumulation of F5H transcript in developing siliques. Siliques were collected in pairs from 5-week-old plants of the lines indicated, beginning with the first expanding silique. Total RNA was extracted and probed with the F5H cDNA in RNA analysis. Blots were exposed to film for 24 h (fah1-2:35S-F5H and fah1-2:C4H-F5H) or 48 h. The bottom panel illustrates ethidium-bromide staining of rRNA as a loading control. Col, Arabidopsis ecotype Columbia.

Figure 8

Accumulation of sinapoylcholine in developing siliques. Siliques were collected in pairs as described for Figure 7 and extracted in acidic 50% methanol. Sinapoylcholine was quantified by HPLC as described for Figure 4. Each point represents the average of three replicates of three silique pairs ± se. Col, Arabidopsis ecotype Columbia.

Figure 9

Sinapate ester accumulation in seeds of the fah1-9 mutant. Seed extracts were separated by TLC and photographed under UV light. Col, Arabidopsis ecotype Columbia; WS, Arabidopsis ecotype Wassilewskija.