Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis

Abstract

The phenylpropanoid pathway provides precursors for the biosynthesis of soluble secondary metabolites and lignin in plants. Ferulate-5-hydroxylase (F5H) catalyzes an irreversible hydroxylation step in this pathway that diverts ferulic acid away from guaiacyl lignin biosynthesis and toward sinapic acid and syringyl lignin. This fact led us to postulate that F5H was a potential regulatory step in the determination of lignin monomer composition. To test this hypothesis, we have used Arabidopsis to examine the impact of F5H overexpression. Arabidopsis is a useful model system in which to study lignification because in wild-type plants, guaiacyl and syringyl lignins are deposited in a tissue-specific fashion, while the F5H-deficient fah1 mutant accumulates only guaiacyl lignin. Here we show that ectopic overexpression of F5H in Arabidopsis abolishes tissue-specific lignin monomer accumulation. Surprisingly, overexpression of F5H under the control of the lignification-associated cinnamate-4-hydroxylase promoter, but not the commonly employed cauliflower mosaic virus 35S promoter, generates a lignin that is almost entirely comprised of syringylpropane units. These experiments demonstrate that modification of F5H expression may enable engineering of lignin monomer composition in agronomically important plant species.

Figure 1

Developmental changes in lignin monomer composition during development of the Arabidopsis rachis in wild type and 35S-F5H transgenics. Lignin monomer composition was determined by nitrobenzene oxidation of lignocellulosic material derived from 4-cm rachis segments from 5-week-old Arabidopsis plants.

Figure 2

Histochemical staining for lignin monomer composition in Arabidopsis stem cross-sections. Lower rachis segments were hand sectioned, stained with the Mäule reagent and observed by light microscopy using cross-polarizing optics. Red staining indicates the presence of syringyl residues in the plant secondary cell wall.

Figure 3

RNA blot analysis of F5H and C4H expression in Arabidopsis rachis internodes. RNA was isolated from the uppermost 12 internodes of 5-week-old Arabidopsis plants beginning near the top of the inflorescence with those internodes ≥2 mm in length. Blots were probed with cDNAs corresponding to the Arabidopsis C4H and F5H genes. Equal loading of lanes was verified by ethidium bromide staining of the 28S ribosomal RNA band.

Figure 4

Impact of promoter choice on F5H overexpression in transgenic Arabidopsis. Stem tissue from 5-week-old plants of the wild type, the fah1–2 mutant, and nine independent fah1–2 lines homozygous for the 35S-F5H transgene (A) was harvested and used for RNA isolation. Blots were probed with the F5H cDNA and were exposed to film for 24 hr to visualize the level of F5H expression in the wild type and the fah1–2 mutant (Left), and for 2 hr to evaluate F5H expression in the 35S-F5H transgenics (Right). Identical analyses were carried out on 5-week-old stem tissue from the wild type, the fah1–2 mutant, and nine fah1–2 lines homozygous for the C4H-F5H transgene (B). Blots were probed with the F5H cDNA and were exposed to film for 12 hr to visualize the level of F5H expression.

Figure 5

GC analysis of DFRC lignin degradation products from representative lines described in Fig. 4 and Table 2. Retention times of trans-coniferyl alcohol diacetate (G), trans-sinapyl alcohol diacetate (S), and the tetracosane internal standard (IS) are indicated.