植物中松脂醇-落叶松脂素还原酶催化特征研究进展

松脂醇-落叶松脂素还原酶(PLR)是植物中木脂素生物合成的关键酶,能够催化松脂醇转化为落叶松脂素,并进一步催化落叶松脂素生成开环异落叶松脂素,且存在底物立体选择性,是一种NADPH依赖型还原酶。PLR的催化产物位于不同类型8-8′木脂素的源头,其底物选择性直接决定木脂素的骨架类型,如呋喃、二苄基丁烷、二苄基丁内酯和芳基四氢萘木脂素。因此, PLR的催化特性和表达特征在植物木脂素组成及其生物活性多样性中发挥重要作用。该文综述了PLR在植物木脂素生物合成中的作用、对映异构体选择性及其催化机制,以期为进一步研究PLR基因的生物学功能以及催化机制奠定基础,并为不同类型木脂素的精确生物合成指明方向。     

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。     

木质素合成关键酶——肉桂醇脱氢酶的研究进展

肉桂醇脱氢酶(CAD)是木质素合成途径的关键酶之一,它作用于木质素单体生物合成的最后一步。重点综述了肉桂醇脱氢酶(CAD)的在基因家族方面,基因调控方面以及蛋白结晶方面的研究进展,讨论了存在的问题并提出了相关策略。     

过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。     

尾叶桉COMT和CCoAOMT基因定向调控木质素单体合成的烟草转化研究

目的:利用烟草遗传转化体系,研究尾叶桉(Eucalyptus urophylla)咖啡酸氧甲基转移酶基因(Eu COMT)和咖啡酰Co A氧甲基转移酶基因(Eu CCo AOMT)对木质素单体合成的定向调控效果。方法:分别利用Eu COMT的正义片段、Eu CCo AOMT的全长RNAi片段进行单基因和二价基因的烟草转化研究,并对转基因烟草植株中目标基因表达水平、木质素和纤维素的含量、茎部解剖结构及木质素单体含量进行检测。结果:分别获得了转基因植株C-S(转Eu COMT正义片段)、CR(转Eu CCo AOMT全长RNAi片段)、C-CR(Eu COM和Eu CCo AOMT二价基因转化)。烟草中转入的正义Eu COMT的片段能够正常表达,而Eu CCo AOMT的全长RNAi片段对烟草CCo AOMT基因引发了强烈的抑制。转基因烟草的生长形态、木质素、纤维含量及解剖结构与野生型无显著差异。转基因植株C-S中G木质素含量升高17.72%,S/G值降低17.99%;CR中S/G值升高61.62%,CCR中G木质素降幅达到57.38%,S/G比值升幅达到114.94%。结论:抑制CCo AOMT对G木质素合成具有显著的抑制效果,Eu COMT和Eu CCo AOMT二价基因转化对S/G比值的定向调控效果最为理想。     

植物肉桂醇脱氢酶及其基因研究进展

肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)作为植物次生代谢特别是木质素合成的关键酶,与植物生长发育和抵御病原菌入侵关系密切,研究CAD基因表达调控及其与组织木质化的关系具有重要的植物生理学意义.该文综述了植物CAD的蛋白特征、酶学性质、基因分布和分类、基因结构和表达调控以及CAD表达与木质素合成的关系,为研究CAD在植物生长发育和抗病中的作用提供理论指导.     

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。     

木质素生物合成及其基因工程研究进展

木质素是维管植物的一种主要组成成分,是植物适应陆地环境的重要特征之一.然而,它的存在严重影响植物材料在造纸工业与畜牧业生产中的应用,因此其生物合成调控的研究引起人们极大关注.随着各种分析技术和手段的提高,该领域研究取得了突破性的进展.该文重点阐述这些新进展,同时较系统地介绍利用基因工程技术调控木质素生物合成的研究成果,并提出一些关于更有效地利用生物技术手段改良造纸资源植物品质的建议.     

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。     

A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acid O-methyltransferase activity

Lignin is a polymeric constituent of the cell wall that needs to be removed during the paper making process. Bi-specific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) catalyses the O-methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively. These compounds are intermediates in the biosynthesis of the lignin precursors. Therefore, COMTs are potential target enzymes for reducing the amount, or modifying the composition, of lignin in plants. Different antisense and sense constructs have been expressed of a gene encoding a COMT from poplar (Populus trichocarpa x P. deltoides) in a P. tremula x P. alba clone under the control of the cauliflower mosaic virus 35S promoter. From all analysed transformants, four lines transformed with an antisense construct had a reduced COMT activity. Two showed a 50% reduction of COMT activity, which altered only slightly the monomeric composition. In the two other transformants, the COMT activity was reduced by 95%. In the latter case, the syringyl/ guaiacyl ratio (S/G) was reduced by sixfold (due to a decrease of S and an increase of G), as analysed by thioacidolysis. A new component of lignin, the 5-hydroxyguaiacyl residue, was detected among the thioacidolysis products. Moreover, in contrast to the white/yellow colour of wild-type wood, the xylem of the transgenic lines with a 95% reduction of COMT activity was pale rose. A similar phenotype was observed in brown-midrib mutants of maize and sorghum, known for their altered lignification. Although the lignin composition was consistently modified, the lignin content of the transgenic poplars was similar to that of the controls.     

Both caffeoyl Coenzyme A 3-<Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 and caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase 1 are involved in redundant functions for lignin,flavonoids and sinapoyl malate biosynthesis in Arabidopsis

Two methylation steps are necessary for the biosynthesis of monolignols, the lignin precursors. Caffeic acid O-methyltransferase (COMT) O-methylates at the C5 position of the phenolic ring. COMT is responsible for the biosynthesis of sinapyl alcohol, the precursor of syringyl lignin units. The O-methylation at the C3 position of the phenolic ring involves the Caffeoyl CoA 3-O-methyltransferase (CCoAOMT). The CCoAOMT 1 gene (At4g34050) is believed to encode the enzyme responsible for the first O-methylation in Arabidopsis thaliana. A CCoAOMT1 promoter-GUS fusion and immunolocalization experiments revealed that this gene is strongly and exclusively expressed in the vascular tissues of stems and roots. An Arabidopsis T-DNA null mutant named ccomt 1 was identified and characterised. The mutant stems are slightly smaller than wild-type stems in short-day growth conditions and has collapsed xylem elements. The lignin content of the stem is low and the S/G ratio is high mainly due to fewer G units. These results suggest that this O-methyltransferase is involved in G-unit biosynthesis but does not act alone to perform this step in monolignol biosynthesis. To determine which O-methyltransferase assists CCoAOMT 1, a comt 1 ccomt1 double mutant was generated and studied. The development of comt 1 ccomt1 is arrested at the plantlet stage in our growth conditions. Lignins of these plantlets are mainly composed of p-hydroxyphenyl units. Moreover, the double mutant does not synthesize sinapoyl malate, a soluble phenolic. These results suggest that CCoAOMT 1 and COMT 1 act together to methylate the C3 position of the phenolic ring of monolignols in Arabidopsis. In addition, they are both involved in the formation of sinapoyl malate and isorhamnetin.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Immunolocalization of two ligninO-methyltransferases in stems of alfalfa (Medicago sativa L.)

Summary Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT) catalyze parallel reactions that are believed to be involved in the biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativa L.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli, and were used for immunolocalization studies in lignifying alfalfa stem tissue. Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by light microscopy and immunocytochemistry. Electron microscopy revealed that both enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser extent, in the cytoplasm of phloem cells. There was no significant difference in the localization pattern of COMT and CCOMT, suggesting that the two enzymes may be part of a metabolic grid leading to production of lignin monomers in lignifying tissue of mature alfalfa stem internodes.     

O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.     

Molecular characterization of a brown midrib3 deletion mutation in maize

The caffeic acid O-methyltransferase (COMT) gene plays an important role in the synthesis of lignin. We have used the polymerase chain reaction in conjuction with genomic analysis to characterize deletion mutations of this gene in maize. In addition, we have analyzed and compared regions of the COMT gene from three distinct heterotic groups. Both PCR and Southern analysis indicate that the active wild-type COMT gene can be polymorphic. We suggest that the intron domain of at least one heterotic inbred can contribute to the alteration of the wild-type gene. In addition, multiple deletion mutations have occurred at this locus. We have found a previously uncharacterized deletion mutation in which segments of both the intron and exon have been deleted and replaced by other sequences. Precise knowledge of its sequence has allowed us to develop an assay by which we can follow this mutation in a breeding program.     

Structural basis for the modulation of lignin monomer methylation by caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase

Caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) from alfalfa is an S-adenosyl-L-Met-dependent O-methyltransferase involved in lignin biosynthesis. COMT methylates caffeoyl- and 5-hydroxyferuloyl-containing acids, aldehydes, and alcohols in vitro while displaying a kinetic preference for the alcohols and aldehydes over the free acids. The 2.2-A crystal structure of COMT in complex with S-adenosyl-L-homocysteine (SAH) and ferulic acid (ferulate form), as well as the 2.4-A crystal structure of COMT in complex with SAH and 5-hydroxyconiferaldehyde, provide a structural understanding of the observed substrate preferences. These crystal structures identify residues lining the active site surface that contact the substrates. Structurally guided site-directed mutagenesis of active site residues was performed with the goal of altering the kinetic preferences for physiological substrates. The kinetic parameters of the COMT mutants versus wild-type enzyme are presented, and coupled with the high-resolution crystal structures, they will serve as a starting point for the in vivo manipulation of lignin monomers in transgenic plants. Ultimately, this structurally based approach to metabolic engineering will allow the further alteration of the lignin biosynthetic pathway in agronomically important plants. This approach will lead to a better understanding of the in vivo operation of the potential metabolic grid for monolignol biosynthesis.     

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.     

RNAi‐suppression of barley caffeic acid O‐methyltransferase modifies lignin despite redundancy in the gene family

Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.