Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata

Abstract

The enzyme hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT) is involved in the production of methoxylated monolignols that are precursors to guaiacyl and syringyl lignin in angiosperm species. We identified and cloned a putative HCT gene from Pinus radiata, a coniferous gymnosperm that does not produce syringyl lignin. This gene was up-regulated during tracheary element (TE) formation in P. radiata cell cultures and showed 72.6% identity to the amino acid sequence of the Nicotiana tabacum HCT isolated earlier. RNAi-mediated silencing of the putative HCT gene had a strong impact on lignin content, monolignol composition, and interunit linkage distribution. AcBr assays revealed an up to 42% reduction in lignin content in TEs. Pyrolysis-GC/MS, thioacidolysis, and NMR detected substantial changes in lignin composition. Most notable was the rise of p-hydroxyphenyl units released by thioacidolysis, which increased from trace amounts in WT controls to up to 31% in transgenics. Two-dimensional 13C-1H correlative NMR confirmed the increase in p-hydroxyphenyl units in the transgenics and revealed structural differences, including an increase in resinols, a reduction in dibenzodioxocins, and the presence of glycerol end groups. The observed modifications in silenced transgenics validate the targeted gene as being associated with lignin biosynthesis in P. radiata and thus likely to encode HCT. This enzyme therefore represents the metabolic entry point leading to the biosynthesis of methoxylated phenylpropanoids in angiosperm species and coniferous gymnosperms such as P. radiata.

Fig. 1.

Enzymatic function of HCT in the phenylpropanoid pathway in tobacco (8).

Fig. 2.

Steady-state mRNA pool of the lignin-related P. radiata genes C4H, HCT, CCoAOMT, and CAD during TE formation as assessed by quantitative RT-PCR at days 0, 2, 4, and 10 after initiation of the differentiation process compared with the HCT steady-state mRNA pool in transgenics pHF5-18, pHF5-23, pHF5-31, and pHF5-40 at days 0 and 4 after initiation of the differentiation process relative to WT controls.

Fig. 3.

Partial short-range ¹³C–¹H (HSQC) correlation spectra (aromatic regions only) of acetylated enzyme lignins isolated from the WT control (A), the HCT-deficient line pHF5-18 (B), the HCT-deficient line pHF5-40 (C), and a difference spectrum revealing that the major relative difference is in enhanced H-units (D). Traces of H-units are seen in the typically G lignin in the WT tracheids, whereas H-units are strikingly more prevalent in the spectra of the transgenics.

Fig. 4.

Partial short-range ¹³C–¹H (HSQC) spectra (side-chain regions) of acetylated enzyme lignins isolated from P. radiata TEs. (A) WT control. (B) HCT-deficient line pHF5-40. HCT deficiency and the incorporation of higher levels of p-coumaryl alcohol into the lignin produce changes in the distribution of interunit linkage types (see text). Note that the contour levels used to display the two spectra were chosen to highlight the structural similarities and differences. With no internally invariant peaks, interpretation of apparent visual quantitative differences needs to be cautious. Volume integrals and semiquantitative data are given in Table 2. The analogous spectrum for line pHF5-18 is provided in SI Fig. 8. (C) Photograph shows the appearance of P. radiata TEs. (D) Interunit type designations A–D, X1, and X7 follow conventions established previously (1, 3, 15).