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 13 C-1 H 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.lignin ͉ HCT ͉ tracheary elements T he global trend toward a biomaterials-based economy makes plant cell walls increasingly important as renewable resources for the production of biofuels and biocomposites. Lignin is the second most abundant terrestrial biopolymer after cellulose and a major structural component of cell walls in woodforming tissues (1). The content, composition, and structure of lignin all have considerable impact on the utilization of plantderived materials and have therefore been the subject of intensive research (1, 2). Lignins are heterogeneous cell wall polymers derived primarily from hydroxycinnamyl alcohols via combinatorial radical coupling reactions (3). Typically, they make up 20-30% of the cell wall material in woody tissue of both angiosperm and gymnosperm species. Lignin in coniferous gymnosperms such as Pinus radiata does not contain syringyl (S) components, which makes it different from lignin of many other vascular plants including angiosperms (4).We have developed a P. radiata callus culture system to better assign function to genes associated with cell wall-related processes such as lignification in conifers. These callus cultures can be transformed and subsequently induced to differentiate into tracheary elements (TEs), the main cellular components of wood in conifer species (5). The biochemical composition of cell wall polymers in differentiated TEs is similar to those produced in P. radiata wood...

Mass Spectrometry-Based Sequencing of Lignin Oligomers

“…Furthermore, p-hydroxyphenylglycerol end units were observed in a dehydrogenation polymer (that was never subjected to ball milling) obtained using p-coumaryl alcohol as monomer . Finally, arylglycerol end units were shown to be increased in lignins rich in p-hydroxyphenyl units and in lignins that were obtained by down-regulating p-coumarate 3-hydroxylase in alfalfa (Medicago sativa; Ralph et al, 2006) or hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata lignifying cell cultures (Wagner et al, 2007). These results indicate that arylglycerol end units are a feature of natural lignins, as suggested previously (Higuchi et al, 1974).…”

Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects

“…RNAi has recently been established for white spruce (A. Séguin, unpublished data) and has not yet been reported to be successful in any other conifer except for radiata pine (Pinus radiata; Wagner et al, 2007Wagner et al, , 2009). Since Sitka spruce and white spruce are closely related species (Yeh and Arnott, 1986), we used white spruce as a suitable system for analysis of the effect of targeted RNAi on CYP720B transcript levels and diterpenoid metabolites.…”

Formation of plant tracheary elements in vitro – a review

“…These include: a thioglycolic acid lignin assay (Möller et al 2003); Klason lignin analysis (Möller et al 2005b); thioacidolysis (Möller et al 2005a(Möller et al , 2005bWagner et al 2013); two dimensional nuclear magnetic resonance (Wagner et al 2007); an acetyl bromide lignin assay (Wagner et al 2007); alkalinenitrobenzene oxidation (Sato et al 2011); pyrolysis-gas chromatography coupled mass spectroscopy (Pillai et al 2011); and Fourier-transform infrared (FTIR) spectroscopy (Pillai et al 2011). In some cases, similar methods are applicable to the analysis of cell-wall polysaccharides, e.g.…”

“…Möller et al (2005b) reported the silencing of cinnamyl alcohol dehydrogenase, an enzyme associated with the formation of cell wall polymers such as lignin, using RNA interference. Suppression of genes associated with the monolignol pathway in pine, such as 4CL and HCT (encoding 4-coumarate CoA ligase and p-hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase), resulted in substantial lignin reductions of up to 50-60% (Wagner et al 2007;Wagner et al 2009). Suppression of a putative Pinus radiata clone (PrCCoAOMT) encoding the lignin-related enzyme caffeoyl CoA 3-O-methyltransferase (CCoAOMT) caused a reduction in lignin content of 5-20% in Pinus radiata TE cultures.…”

“…Xylogen, a proteoglycan-like factor accumulates in vascular tissues and mediates cell-cell interactions required for xylem differentiation in Zinnia cells cultured in vitro (Motose et al 2004). The cell-wall lignin content and composition in in vitro differentiated TEs is similar to those produced in Pinus radiata wood, demonstrating that the in vitro P. radiata TE system is well suited to study the formation of lignified secondary cell walls in gymnosperms (Möller et al 2005a;Wagner et al 2007). In vitro cultured Zinnia elegans mesophyll cells are a single cell-based system so cannot be used to study cellto-cell communication that is occurring during xylogenesis in plants.…”