Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis

Caffeic acid 3-O-methyltransferase (COMT) catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, and is believed to occupy a pivotal position in the lignin biosynthetic pathway. A cDNA (TaCM) was identified from wheat and it was found to be expressed constitutively in stem, leaf and root tissues. The deduced amino acid sequence of TaCM showed a high degree of identity with COMT from other plants, particularly in SAM binding motif and the residues responsible for catalytic and substrate specificity. The predicted TaCM three-dimensional structure is very similar with a COMT from alfalfa (MsCOMT), and TaCM protein had high immunoreactive activity with MsCOMT antibody. Kinetic analysis indicated that the recombinant TaCM protein exhibited the highest catalyzing efficiency towards caffeoyl aldehyde and 5-hydroxyconiferaldehyde as substrates, suggesting a pathway leads to S lignin via aldehyde precursors. Authority of TaCM encoding a COMT was confirmed by the expression of antisense TaCM gene in transgenic tobacco which specifically down-regulated the COMT enzyme activity. Lignin analysis showed that the reduction in COMT activity resulted in a marginal decrease in lignin content but sharp reduction in the syringl lignin. Furthermore, the TaCM protein exhibited a strong activity towards ester precursors including caffeoyl-CoA and 5-hydroxyferuloyl-CoA. Our results demonstrate that TaCM is a typical COMT involved in lignin biosynthesis. It also supports the notion, in agreement with a structural analysis, that COMT has a broad substrate preference.     

Immunomodulation of gibberellin biosynthesis using an anti-precursor gibberellin antibody confers gibberellin-deficient phenotypes

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. We have recently succeeded in obtaining gibberellin (GA)-deficient phenotypes in Arabidopsis thaliana by using anti-bioactive GA antibodies. In this study, a single-chain antibody (scFv) against GA(24), a precursor GA, was utilized to repress the biosynthesis of bioactive gibberellins. Stable accumulation of the scFv in endoplasmic reticulum (ER) was achieved by being produced as a fusion with GFP as well as KDEL ER-retention signal. The transgenic plants showed GFP fluorescence in the reticulate cortical ER network in epidermal cells. The GFP-scFv fusion produced in plants maintained its binding activity. The transgenic plants showed GA-deficient phenotypes, including reduced rosette leaf development, delayed flower induction and reduced stem elongation of the main culm, especially in the early stage of inflorescence growth. Contrarily, stem elongation of the main culm at a later stage, or that of lateral shoots was much less affected by scFv production. These phenotypes were different from anti-bioactive GA scFv-producing lines, whose stem elongation was continuously repressed throughout the inflorescence development. The GA-deficient phenotypes were recovered by treatment with GA(24) and bioactive GA(4), the latter being more effective. The transgenic lines contained conspicuously higher endogenous GA(24) and clearly less GA(4) than wild-type plants. The expression of GA 20-oxidase and GA 3-oxidase genes, which are feedback-regulated by GA signaling, were up-regulated in those plants. These results demonstrate that the scFv trapped GA(24) in ER and inhibited metabolism of GA(24) to bioactive GA(4).     

Biochemical and phenotypical characterization of transgenic tomato plants overexpressing a basic peroxidase

Tomato plants ( Lycopersicon esculentum Mill. cv. Pera) were transformed via Agrobacterium tumefaciens with the binary vector pKYLX71 containing a tomato basic peroxidase (EC 1.11.1.7) gene, tpx1 , under the control of the cauliflower mosaic virus (CaMV35S) promoter. Transgenic plants showed a 2–5-fold increase in the activity of the peroxidase ionically bound to the cell wall, whereas soluble peroxidase activity remained similar or even lower than wild-type plants. Isoelectric focusing showed the presence of a new isoperoxidase of pI ca 9 in the ionically bound extract. Western blot also showed the presence of a new band at 41 kDa that was absent in the wild-type extract. A 40–220% increment of lignin content of the leaf was found in transgenic plants. Shoot phenotype of transgenic plants was similar to wild type, although under stress, the plants appeared wilted and the new leaves had a reduced area and were thicker than wild-type or older transgenic leaves. The root system was underdeveloped in transgenic plants, but the rooting ability of the stem was not affected by the overexpression of peroxidase. Finally, the morphogenetic response of cotyledon and hypocotyl explants from transgenic plants was evaluated. In the case of cotyledons, the percentage of explants with shoot was not different from wild-type plants. For hypocotyl, one of the transgenic lines showed a 30% reduction in the percentage of shoot organogenesis. The results are discussed in relation to the role of tpx1 in lignin synthesis.     

广谱抗真菌转基因水稻植株化学成分与结构动态变化

【背景】种植广谱抗真菌水稻可能会带来一定的环境生物安全问题,对其植株的化学成分进行实质等同性分析是转基因水稻安全性评价的重要内容之一。【方法】以表达广谱抗真菌蛋白转基因水稻转品1和转品8及其相应非转基因水稻七丝软粘的秸秆为研究材料,采用化学法和扫描电镜技术分析外源基因的导入对水稻秸秆化学成分以及组织显微结构的影响。【结果】（1）在整个生长发育过程中,广谱抗真菌转基因水稻转品1和转品8与其非转基因水稻七丝软粘叶片、叶鞘和茎的纤维素、半纤维素、木质素以及粗蛋白含量的变化趋势基本一致,且品种间化学成分的含量不存在显著差异。（2）广谱抗真菌转基因水稻叶片表皮的硅质瘤状结构以及气孔的形状和致密程度与其非转基因水稻七丝软粘相似;茎壁、厚壁组织、薄壁组织以及大小维管束的形态和分布情况未发生明显变化。【结论与意义】表达广谱抗真菌蛋白转基因水稻秸秆的化学成分和组织显微结构与非转基因水稻基本一致。这为广谱抗真菌转基因水稻的环境安全性评估提供了依据。     

NMR Characterization of C3H and HCT Down-Regulated Alfalfa Lignin

Independent down-regulation of genes encoding p-coumarate 3-hydroxylase (C3H) and hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) has been previously shown to reduce the recalcitrance of alfalfa and thereby improve the release of fermentable sugars during enzymatic hydrolysis. In this study, ball-milled lignins were isolated from wild-type control, C3H, and HCT gene down-regulated alfalfa plants. One- and two-dimensional nuclear magnetic resonance (NMR) techniques were utilized to determine structural changes in the ball-milled alfalfa lignins resulting from this genetic engineering. After C3H and HCT gene down-regulation, significant structural changes had occurred to the alfalfa ball-milled lignins compared to the wild-type control. A substantial increase in p-hydroxyphenyl units was observed in the transgenic alfalfa ball-milled lignins as well as a concomitant decrease in guaiacyl and syringyl units. Two-dimensional ¹³C–¹H heteronuclear single quantum coherence correlation NMR, one-dimensional distortionless enhancement by polarization transfer-135, and ¹³C NMR measurement showed a noteworthy decrease in methoxyl group and β-O-4 linkage contents in these transgenic alfalfa lignins. ¹³C NMR analysis estimated that C3H gene down-regulation reduced the methoxyl content by ~55–58% in the ball-milled lignin, while HCT down-regulation decreased methoxyl content by ~73%. The gene down-regulated C3H and HCT transgenic alfalfa lignin was largely a p-hydroxyphenyl (H) rich type lignin. Compared to the wild-type plant, the C3H and HCT transgenic lines had an increase in relative abundance of phenylcoumaran and resinol in the ball-milled lignins.     

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.     

Structural and compositional modifications in lignin of transgenic alfalfa down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase

Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase contained benzodioxanes resulting from the incorporation of the novel monomer, 5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble lignin fraction and the use of sensitive NMR instrumentation, unique structural features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol unit, was identified. It was possible to establish that coniferyl alcohol, sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise to extended chains of benzodioxane units. There is also evidence that 5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was structurally similar to the control lignin but the transgenic exhibited a dramatic decrease in lignin content (approximately 20%) and modest increase in cellulose (approximately 10%) reflecting a 30% increase in cellulose:lignin ratio. The compositional changes in both transgenics potentially allow enhanced utilization of alfalfa as a major forage crop by increasing the digestibility of its stem fraction.     

Metabolic diversion of the phenylpropanoid pathway causes cell wall and morphological changes in transgenic tobacco stems

Studies involving transgenic plants with modifications in the lignin pathway reported to date, have received a relatively preliminary characterisation in relation to the impact on vascular integrity, biomechanical properties of tissues and carbon allocation to phenolic pools. Therefore, in this study transgenic tobacco plants (Nicotiana tabacum cv XHFD 8) expressing various levels of a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) gene have been characterised for cell wall and related morphological changes. The HCHL enzyme converts p-coumaroyl-CoA to 4-hydroxybenzaldehyde thereby rerouting the phenylpropanoid pathway. Plants expressing high levels of HCHL activity exhibited reduced lignin deposition, impaired monolignol biosynthesis and vascular integrity. The plants also exhibited reduction in stem toughness concomitant with a massive reduction in both the cell wall esterified and soluble phenolics. A notable result of redirecting the carbon flux was the wall-bound accretion of vanillin and vanillic acid, probably due to the shunt pathway. Intracellular accumulation of novel metabolites such as hydroxybenzoic and vanillic acid derivatives also occurred in the transgenic plants. A line with intermediate levels of HCHL expression conferred correspondingly reduced lignin deposition, toughness and phenolics. This line displayed a normal morphology but distorted vasculature. Coloration of the xylem has been previously attributed to incorporation of alternative phenolics, whereas results from this study indicate that the coloration is likely to be due to the association of low molecular weight phenolics. There was no evidence of increased growth or enhanced cellulose biosynthesis as a result of HCHL expression. Hence, rerouting the phenylpropanoid biosynthetic pathway quantitatively and qualitatively modifies cell wall-bound phenolics and vascular structure.     

Plant growth, biomass partitioning and soil carbon formation in response to altered lignin biosynthesis in Populus tremuloides

We conducted a glasshouse mesocosm study that combined (13)C isotope techniques with wild-type and transgenic aspen (Populus tremuloides) in order to examine how altered lignin biosynthesis affects plant production and soil carbon formation. Our transgenic aspen lines expressed low stem lignin concentration but normal cellulose concentration, low lignin stem concentration with high cellulose concentration or an increased stem syringyl to guaiacyl lignin ratio. Large differences in stem lignin concentration observed across lines were not observed in leaves or fine roots. Nonetheless, low lignin lines accumulated 15-17% less root C and 33-43% less new soil C than the control line. Compared with the control line, transformed aspen expressing high syringyl lignin accumulated 30% less total plant C - a result of greatly reduced total leaf area - and 70% less new soil C. These findings suggest that altered stem lignin biosynthesis in Populus may have little effect on the chemistry of fine roots or leaves, but can still have large effects on plant growth, biomass partitioning and soil C formation.     

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.     

Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth.

We present evidence that overproduction of endogenous cytokinins (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco (Nicotiana tabacum L.) grown in vitro. It was demonstrated by overaccumulation of phenolic compounds, synthesis of pathogenesis related proteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sections proved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescence of phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco compared with controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhibited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids confirmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting of transgenic tobacco is discussed.     

Lignin modification improves fermentable sugar yields for biofuel production

Recalcitrance to saccharification is a major limitation for conversion of lignocellulosic biomass to ethanol. In stems of transgenic alfalfa lines independently downregulated in each of six lignin biosynthetic enzymes, recalcitrance to both acid pretreatment and enzymatic digestion is directly proportional to lignin content. Some transgenics yield nearly twice as much sugar from cell walls as wild-type plants. Lignin modification could bypass the need for acid pretreatment and thereby facilitate bioprocess consolidation.     

Over-expressing GsGST14 from Glycine soja enhances alkaline tolerance of transgenic Medicago sativa

Glutathione-S-transferases (GSTs) are ubiquitous enzymes that play a key role in stress tolerance and cellular detoxification. The GST gene GsGST14 selected from the gene expression profiles of Glycine soja under alkaline stress was transformed into alfalfa (Medicago sativa L.). Transgenic alfalfa plants showed 1.73–1.99 times higher GST activity than wild-type plants. Transgenic alfalfa grew well in the presence of 100 mM NaHCO₃, while wild-type plants exhibited chlorosis and stunted growth, even death. There were marked changes in malondialdehyde content and relative membrane permeability caused by alkaline stress in non-transgenic lines compared to transgenic lines. The results indicate that the gene GsGST14 could enhance alkaline resistance in transgenic alfalfa.     

Integrative analysis of transgenic alfalfa (Medicago sativa L.) suggests new metabolic control mechanisms for monolignol biosynthesis

The entanglement of lignin polymers with cellulose and hemicellulose in plant cell walls is a major biological barrier to the economically viable production of biofuels from woody biomass. Recent efforts of reducing this recalcitrance with transgenic techniques have been showing promise for ameliorating or even obviating the need for costly pretreatments that are otherwise required to remove lignin from cellulose and hemicelluloses. At the same time, genetic manipulations of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on lignin composition, thus raising the question of whether the current understanding of the pathway is indeed correct. To address this question systemically, we developed and applied a novel modeling approach that, instead of analyzing the pathway within a single target context, permits a comprehensive, simultaneous investigation of different datasets in wild type and transgenic plants. Specifically, the proposed approach combines static flux-based analysis with a Monte Carlo simulation in which very many randomly chosen sets of parameter values are evaluated against kinetic models of lignin biosynthesis in different stem internodes of wild type and lignin-modified alfalfa plants. In addition to four new postulates that address the reversibility of some key reactions, the modeling effort led to two novel postulates regarding the control of the lignin biosynthetic pathway. The first posits functionally independent pathways toward the synthesis of different lignin monomers, while the second postulate proposes a novel feedforward regulatory mechanism. Subsequent laboratory experiments have identified the signaling molecule salicylic acid as a potential mediator of the postulated control mechanism. Overall, the results demonstrate that mathematical modeling can be a valuable complement to conventional transgenic approaches and that it can provide biological insights that are otherwise difficult to obtain.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell types

* Independent antisense down-regulation of 10 individual enzymes in the monolignol pathway has generated a series of otherwise isogenic alfalfa (Medicago sativa) lines with varying lignin content and composition. These plants show various visible growth phenotypes, and possess significant differences in vascular cell size and number. * To better understand the phenotypic consequences of lignin modification, the distributions of lignin content and composition in stems of the various alfalfa lines at the cellular level were studied by confocal microscopy after staining for specific lignin components, and by chemical analysis of laser capture dissected tissue types. * Although all antisense transgenes were driven by the same promoter with specificity for vascular, fiber and parenchyma tissues, the impact of down-regulating a specific transgene varied in the different tissue types. For example, reducing expression of ferulate 5-hydroxylase reduced accumulation of syringyl lignin in fiber and parenchyma cells, but not in vascular elements. * The results support a model for cell type-specific regulation of lignin content and composition at the level of the monolignol pathway, and illustrate the use of laser capture microdissection as a new approach to spatially resolved lignin compositional analysis.     

MicroRNA156 as a promising tool for alfalfa improvement

A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa (Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13) contain miR156 cleavage sites and their expression was down‐regulated in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, transgenic alfalfa plants had increased root length, while nodulation was maintained. The multitude of traits affected by miR156 may be due to the network of genes regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.     

Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene

Laccases are copper-containing glycoproteins, which are widespread in higher plants as multigene families. To gain more insight in the function of laccases in plants, especially potential role in lignification, we produced transgenic poplar plants overexpressing a cotton laccase cDNA (GaLAC1) under the control of the cauliflower mosaic virus 35S promoter. As compared with untransformed control plants, transgenic plants exhibited a 2.1- to 13.2-fold increased laccase activity, whereas plant growth rate and morphological characters remained similar to control plants. A 2.1–19.6% increase in total lignin content of the stem was found in transgenic plants. Moreover, transgenic plants showed a dramatically accelerated oxidation rate of phenolics, without obvious change in total phenolic content. Our data suggested that GaLAC1 may participate in lignin synthesis and phenolic metabolism in plants. The present work provided a new genetic evidence for the involvement of plant laccases in lignification.