Wagner Armin, Ralph John, Akiyama Takuya, Flint Heather, Phillips Lorelle, Torr Kirk, Nanayakkara Bernadette, Te Kiri Lana
Cellwall Biotechnology Centre, Scion (New Zealand Forest Research Institute), Private Bag 3020, Rotorua, New Zealand.
Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11856-61. doi: 10.1073/pnas.0701428104. Epub 2007 Jul 3.
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.
羟基肉桂酰辅酶A：莽草酸羟基肉桂酰转移酶（HCT）参与甲氧基化单木质醇的生成，这些甲氧基化单木质醇是被子植物中愈创木基和紫丁香基木质素的前体。我们从辐射松（一种不产生紫丁香基木质素的针叶裸子植物）中鉴定并克隆了一个假定的HCT基因。该基因在辐射松细胞培养物的管状分子（TE）形成过程中上调，并且与先前分离的烟草HCT的氨基酸序列具有72.6%的同一性。RNA干扰介导的假定HCT基因沉默对木质素含量、单木质醇组成和单元间连接分布有强烈影响。乙酰溴（AcBr）分析显示TE中木质素含量降低了42%。热解-气相色谱/质谱（Pyrolysis-GC/MS）、硫代酸解和核磁共振（NMR）检测到木质素组成有显著变化。最值得注意的是硫代酸解释放的对羟基苯基单元的增加，从野生型对照中的痕量增加到转基因植物中的高达31%。二维13C-1H相关核磁共振证实了转基因植物中对羟基苯基单元的增加，并揭示了结构差异，包括树脂醇的增加、二苯并二恶英的减少以及甘油端基的存在。在沉默的转基因植物中观察到的修饰证实了该靶向基因与辐射松中的木质素生物合成相关，因此可能编码HCT。因此，这种酶代表了被子植物和针叶裸子植物（如辐射松）中甲氧基化苯丙烷类生物合成的代谢起始点。
