植物木质素合成调控与生物质能源利用

植物木质素生物合成调控研究已在造纸树种与饲草品质的改良中取得了许多进展。随着对木质纤维原料乙醇发酵研究的兴起,植物木质素合成调控再次成为研究热点。该文总结了目前生物质能源利用的现状,同时针对木质素在木质纤维乙醇发酵中的限制作用,综述了近年来植物木质素合成调控的研究进展,提出了今后的研究方向和内容,并展望了木质素合成调控在木质纤维乙醇发酵中的应用。     

植物木质素合成调控与生物质能源利用

植物木质素生物合成调控研究已在造纸树种与饲草品质的改良中取得了许多进展。随着对木质纤维原料乙醇发酵研究的兴起, 植物木质素合成调控再次成为研究热点。该文总结了目前生物质能源利用的现状, 同时针对木质素在木质纤维乙醇发酵中的限制作用, 综述了近年来植物木质素合成调控的研究进展, 提出了今后的研究方向和内容, 并展望了木质素合成调控在木质纤维乙醇发酵中的应用。     

植物4香豆酸:辅酶A连接酶研究进展

4香豆酸:辅酶A连接酶(4-coumarate:coenzyme A ligase EC6.2.1.12,4CL)是木质素生物合成途径中的一个关键酶,催化肉桂酸及其衍生物生成相应的硫酯。同时也是苯丙烷类代谢途径中的第三个步骤,连接木质素前体和各个分支途径的纽带,在木质素合成过程中发挥了重要的调控作用。近年来利用基因工程手段调控木质素生物合成,降低木质素含量,减少制浆造纸过程中的污染已成为研究热点。结合国内外关于4CL的研究成果,对植物4CL基因家族、酶学性质、晶体结构以及4CL在调控木质素生物合成中的作用等方面进行综述,并对4CL的研究方向提出展望,旨为对该基因的研究提供参考。     

木质素合成研究进展

木质素是植物体内一类重要的大分子有机物,具有支持、保护和运输等生物学功能。然而,木质素的存在对植物资源的利用有一些不利影响,主要体现在导致造纸污染严重及影响牲畜对饲草的消化和吸收等。通过生物工程手段调控木质素的合成具有重要的环境保护价值和经济价值,已经成为国际上生命科学研究的热点之一。近年来,这一领域发展十分迅速。本文介绍了木质素生物合成途径,重点综述了调控木质素合成的手段及限速酶研究方面的最新进展,并提出存在的问题及展望。     

植物次生细胞壁生物合成的转录调控网络

植物次生细胞壁包含纤维素、半纤维素和木质素, 赋予细胞壁机械强度及疏水性, 这种特性对植物直立生长、水分和营养物质运输以及抵御生物和非生物胁迫十分重要。该文总结了调控次生细胞壁生物合成的转录因子及其调控机制, 包括NAC转录因子调控次生壁合成的一级开关作用, AtMYB46/AtMYB83及其下游调控因子的二级开关作用, 以及其它转录因子对次生壁生物合成的调控作用, 并对未来研究内容和方法进行了展望, 以期为深入系统理解次生细胞壁生物合成的转录调控网络提供参考。     

植物次生细胞壁生物合成的转录调控网络

植物次生细胞壁包含纤维素、半纤维素和木质素,赋予细胞壁机械强度及疏水性,这种特性对植物直立生长、水分和营养物质运输以及抵御生物和非生物胁迫十分重要。该文总结了调控次生细胞壁生物合成的转录因子及其调控机制,包括NAC转录因子调控次生壁合成的一级开关作用,AtMYB46/AtMYB83及其下游调控因子的二级开关作用,以及其它转录因子对次生壁生物合成的调控作用,并对未来研究内容和方法进行了展望,以期为深入系统理解次生细胞壁生物合成的转录调控网络提供参考。     

植物咖啡酸-O-甲基转移酶的研究进展

单木质素醇(H型、G型和S型)是构成植物木质素和木脂素的基本单元,其组成的不同直接决定木质素和木脂素的化学多样性和生物活性差异。咖啡酸-O-甲基转移酶(caffeic acid O-methyltransferase, COMT)可催化苯丙素类化合物羟基上氧原子的甲基化,在不同类型单木质素醇的构成中起决定作用,是木质素和木脂素生物合成途径的关键酶。2010年的相关综述主要对COMT的基因特征和在木质素生物合成中的调控作用作了介绍,文中聚焦了近十多年来COMT的最新研究进展,从基因特征、表达特征、结构特征和调控作用几个方面进行全面综述,并对COMT的研究和应用前景进行展望。     

基于转录组测序的慈竹笋木质素基因筛选及其表达分析

慈竹（Bambusa emeiensis）纤维素含量丰富,是较好的造纸原料,但竹茎中木质素影响着制浆生产及纸浆质量。目前,对慈竹木质素生物合成机制所知甚少,这限制了遗传调控竹木质素的研究。本文以拟南芥、水稻等植物的已知木质素基因作为查询序列,通过BLASTp和系统进化分析,从10、50、100和150 cm慈竹笋转录组数据中筛选到351个木质素生物合成相关Unigenes,包括51个LAC,37个4CL、26个PAL、34个CCR和25个CAD相关转录子,其数量高于其他已报道的竹类植物。转录丰度和定量基因表达分析发现16个木质素基因,包括2个PAL、5个CCR、3个4CL、2个CADH2和4个LAC,随着笋发育而表达上调,表明其可能与发育性木质素积累相关。     

木质素的生物合成及其调控研究进展

木质素是植物体中仅次于纤维素的一种重要大分子有机物质,具有重要生物学功能,其3种主要单体的生物合成途径已经基本清楚。从木质素生物合成及基因工程在调控木质素生物合成中的作用等方面的研究进展进行了综述,并提出了存在的问题及对策。     

烟草4CL蛋白免疫荧光定位研究

4-香豆酸辅酶A连接酶(4CL)是维管植物木质素生物合成途径的关键酶,应用原核表达系统获得了毛白杨可溶性4CL1融合蛋白,以Ni2 -Agrose亲和柱层析纯化得到的SDS-PAGE电泳纯的毛白杨4CL1融合蛋白为抗原,免疫家兔获得毛白杨4CL1多克隆抗体,Western blotting鉴定表明兔抗毛白杨4CL1多克隆抗体具有高度特异性,免疫荧光定位发现普通烟草4CL1蛋白特异性地在木质部表达.为进一步应用木质部特异表达启动子定向调控木质素生物合成奠定了理论基础.     

Syringyl lignin is unaltered by severe sinapyl alcohol dehydrogenase suppression in tobacco

The manipulation of lignin could, in principle, facilitate efficient biofuel production from plant biomass. Despite intensive study of the lignin pathway, uncertainty exists about the enzyme catalyzing the last step in syringyl (S) monolignol biosynthesis, the reduction of sinapaldehyde to sinapyl alcohol. Traditional schemes of the pathway suggested that both guaiacyl (G) and S monolignols are produced by a single substrate-versatile enzyme, cinnamyl alcohol dehydrogenase (CAD). This was challenged by the discovery of a novel sinapyl alcohol dehydrogenase (SAD) that preferentially uses sinapaldehyde as a substrate and that was claimed to regulate S lignin biosynthesis in angiosperms. Consequently, most pathway schemes now show SAD (or SAD and CAD) at the sinapaldehyde reduction step, although functional evidence is lacking. We cloned SAD from tobacco (Nicotiana tabacum) and suppressed it in transgenic plants using RNA interference-inducing vectors. Characterization of lignin in the woody stems shows no change to content, composition, or structure, and S lignin is normal. By contrast, plants additionally suppressed in CAD have changes to lignin structure and S:G ratio and have increased sinapaldehyde in lignin, similar to plants suppressed in CAD alone. These data demonstrate that CAD, not SAD, is the enzyme responsible for S lignin biosynthesis in woody angiosperm xylem.     

Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii

Syringyl lignin, an important component of the secondary cell wall, has traditionally been considered to be a hallmark of angiosperms because ferns and gymnosperms in general lack lignin of this type. Interestingly, syringyl lignin was also detected in Selaginella, a genus that represents an extant lineage of the most basal of the vascular plants, the lycophytes. In angiosperms, syringyl lignin biosynthesis requires the activity of ferulate 5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase, and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). Together, these two enzymes divert metabolic flux from the biosynthesis of guaiacyl lignin, a lignin type common to all vascular plants, toward syringyl lignin. Selaginella has independently evolved an alternative lignin biosynthetic pathway in which syringyl subunits are directly derived from the precursors of p-hydroxyphenyl lignin, through the action of a dual specificity phenylpropanoid meta-hydroxylase, Sm F5H. Here, we report the characterization of an O-methyltransferase from Selaginella moellendorffii, COMT, the coding sequence of which is clustered together with F5H at the adjacent genomic locus. COMT is a bifunctional phenylpropanoid O-methyltransferase that can methylate phenylpropanoid meta-hydroxyls at both the 3- and 5-position and function in concert with F5H in syringyl lignin biosynthesis in S. moellendorffii. Phylogenetic analysis reveals that Sm COMT, like F5H, evolved independently from its angiosperm counterparts.     

Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis gene encoding ferulate 5-hydroxylase

Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H over-expression increases lignin syringyl monomer content and abolishes the tissue-specificity of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauliflower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on lignification in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a significant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H-F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H-F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H-F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.     

Lignin engineering

Lignins are aromatic polymers that are present mainly in secondarily thickened plant cell walls. Several decades of research have elucidated the main biosynthetic routes toward the monolignols and demonstrated that lignin amounts can be engineered and that plants can cope with large shifts in p-hydroxyphenyl/guaiacyl/syringyl (H/G/S) lignin compositional ratios. It has also become clear that lignins incorporate many more units than the three monolignols described in biochemistry textbooks. Together with the theory that lignin polymerization is under chemical control, observations hint at opportunities to design lignin structure to the needs of agriculture. An increasing number of examples illustrates that lignin engineering can improve the processing efficiency of plant biomass for pulping, forage digestibility and biofuels. Systems approaches, in which the plant's response to engineering of a single gene in the pathway is studied at the organismal level, are beginning to shed light on the interaction of lignin biosynthesis with other metabolic pathways and processes.     

Caffeoyl coenzyme A O-methyltransferase and lignin biosynthesis.

Lignin, a complex phenylpropanoid compound, is polymerized from the monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These three monolignols differ only by the 3- and 5-methoxyl groups. Therefore, enzymatic reactions controlling the methylations of the 3- and 5-hydroxyls of monolignol precursors are critical to determine the lignin composition. Recent biochemical and transgenic studies have indicated that the methylation pathways in monolignol biosynthesis are much more complicated than we have previously envisioned. It has been demonstrated that caffeoyl CoA O-methyltransferase plays an essential role in the synthesis of guaiacyl lignin units as well as in the supply of substrates for the synthesis of syringyl lignin units. Caffeic acid O-methyltransferase has been found to essentially control the biosynthesis of syringyl lignin units. These new findings have greatly enriched our knowledge on the methylation pathways in monolignol biosynthesis.     

Multiform biosynthetic pathway of syringyl lignin in angiosperms

To clarify the pathway for biosynthesis of sinapyl alcohol in angiosperms, tracer experiments using stable isotopes were performed on robinia ( Robinia pseudoacacia L.), oleander ( Nerium indicum Mill.), magnolia ( Magnolia kobus DC.) and Arabidopsis thaliana (L.) Heynh. Precursors used in the experiment were (13)C- and (2)H ( D)-labeled [8-(13)C, 3-OCD(3)]ferulic acid and [8-(13)C, 3,5-OCD(3)]sinapic acid. The incorporation of labeled precursor into lignin was confirmed by gas chromatography-mass spectrometry of the products of derivatization followed by reductive cleavage. Crude extracts of differentiating xylem or stems from these plants were also assayed for 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) activity using sinapic acid and ferulic acid as substrates. In robinia and oleander, 4CL activity toward sinapic acid was detected, and labeled sinapic acids were incorporated into syringyl lignin. These results indicate that robinia and oleander have a pathway that produces sinapyl alcohol from sinapic acid via sinapoyl-CoA. By contrast, in magnolia and Arabidopsis, 4CL activity toward sinapic acid could not be detected, and labeled sinapic acid was not incorporated into lignin. These results suggest that syringyl lignin biosynthesis in angiosperms operates via multiple pathways that depend on the species.     

The Last Step of Syringyl Monolignol Biosynthesis in Angiosperms Is Regulated by a Novel Gene Encoding Sinapyl Alcohol Dehydrogenase

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) has been thought to mediate the reduction of both coniferaldehyde and sinapaldehyde into guaiacyl and syringyl monolignols in angiosperms. Here, we report the isolation of a novel aspen gene (PtSAD) encoding sinapyl alcohol dehydrogenase (SAD), which is phylogenetically distinct from aspen CAD (PtCAD). Liquid chromatography-mass spectrometry-based enzyme functional analysis and substrate level-controlled enzyme kinetics consistently demonstrated that PtSAD is sinapaldehyde specific and that PtCAD is coniferaldehyde specific. The enzymatic efficiency of PtSAD for sinapaldehyde was approximately 60 times greater than that of PtCAD. These data suggest that in addition to CAD, discrete SAD function is essential to the biosynthesis of syringyl monolignol in angiosperms. In aspen stem primary tissues, PtCAD was immunolocalized exclusively to xylem elements in which only guaiacyl lignin was deposited, whereas PtSAD was abundant in syringyl lignin-enriched phloem fiber cells. In the developing secondary stem xylem, PtCAD was most conspicuous in guaiacyl lignin-enriched vessels, but PtSAD was nearly absent from these elements and was conspicuous in fiber cells. In the context of additional protein immunolocalization and lignin histochemistry, these results suggest that the distinct CAD and SAD functions are linked spatiotemporally to the differential biosynthesis of guaiacyl and syringyl lignins in different cell types. SAD is required for the biosynthesis of syringyl lignin in angiosperms.     

Conversion of guaiacyl to syringyl moieties on the cinnamyl alcohol pathway during the biosynthesis of lignin in angiosperms

Aglycons derived from 4-O-β-D-glucosides of both caffeyl and 5-hydroxyconiferyl alcohols were incorporated into guaiacyl (G) and syringyl (S) units in the lignin of newly formed xylem of several angiosperms. It is likely that these aglycons enter the cinnamyl alcohol pathway as intermediates in the introduction of methoxyl groups onto aromatic rings, and serve as precursors for the biosynthesis of lignin. The S/G ratio in this pathway was coincident with the ratio in the cell wall lignin of each tree. Our results indicate that the cinnamyl alcohol pathway involves the same mechanisms as the cinnamic acid and cinnamyl CoA pathways and they suggest that this novel pathway might be part of a metabolic grid in the biosynthesis of lignin. Received: 8 September 1999 / Accepted: 4 October 1999     

Red Xylem and Higher Lignin Extractability by Down-Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity in trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.