Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras

The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol . 43 , 1465–1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 ( At4CL1 ) gene in A. thaliana . At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.     

Silencing of 4-coumarate:coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production

? The lignin content of feedstock has been proposed as one key agronomic trait impacting biofuel production from lignocellulosic biomass. 4-Coumarate:coenzyme A ligase (4CL) is one of the key enzymes involved in the monolignol biosynthethic pathway. ? Two homologous 4CL genes, Pv4CL1 and Pv4CL2, were identified in switchgrass (Panicum virgatum) through phylogenetic analysis. Gene expression patterns and enzymatic activity assays suggested that Pv4CL1 is involved in monolignol biosynthesis. Stable transgenic plants were obtained with Pv4CL1 down-regulated. ? RNA interference of Pv4CL1 reduced extractable 4CL activity by 80%, leading to a reduction in lignin content with decreased guaiacyl unit composition. Altered lignification patterns in the stems of RNAi transgenic plants were observed with phloroglucinol-HCl staining. The transgenic plants also had uncompromised biomass yields. After dilute acid pretreatment, the low lignin transgenic biomass had significantly increased cellulose hydrolysis (saccharification) efficiency. ? The results demonstrate that Pv4CL1, but not Pv4CL2, is the key 4CL isozyme involved in lignin biosynthesis, and reducing lignin content in switchgrass biomass by silencing Pv4CL1 can remarkably increase the efficiency of fermentable sugar release for biofuel production.     

Identification and functional analysis of the Pm4CL1 gene in transgenic tobacco plant as the basis for regulating lignin biosynthesis in forest trees

Reducing the lignin content of trees could provide both economic and environmental benefits. To this end, the coumarate:coenzyme A ligase 1 gene (4CL1) was isolated from Pinus massoniana Lamb (Pm4CL1). The sequence of the full-length Pm4CL1 cDNA (accession no. FJ810495) contained an entire open reading frame (ORF) of 1,614 bp, which encoded a polypeptide of 537 amino acid residues. Tobacco (Nicotiana tabacum L.) as a model plant was used for functional characterization of the Pm4CL1 gene in transgenic plants. Results revealed that 4CL1 enzyme activity and lignin content in most antisense Pm4CL1 transgenic tobacco lines were decreased as compared to wild-type; the average 4CL1 enzyme activity was decreased by 48.75% and lignin content was decreased by 24.5%. In contrast, in the sense Pm4CL1 transgenic tobacco lines, average 4CL1 enzyme activity was increased by 72.3% and lignin content was increased by 27.6%. These results suggest that the Pm4CL1 gene from P. massoniana could be applied to regulate lignin biosynthesis in transgenic trees.     

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.     

Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco

The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter. To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco. The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves. The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控

用根癌农杆菌介导法将源于紫穗槐的尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）基因、反义4-香豆酸辅酶A连接酶（4CL）基因以及两者的双价基因分别转移至烟草中。PCR和Southern杂交检测证实外源基因已整合到转基因烟草基因组中。测定全纤维素和Klason木质素含量的结果显示,增强UGPase基因的表达可提高转基因植株的纤维素含量,但对木质素含量没有影响;抑制4CL基因的表达可显著降低转基因植株的木质素含量,但对纤维素含量没有影响;转移双价基因的转基因植株中纤维素含量增加而木质素含量降低。     

华南象草Pp4CL基因的克隆及其转基因烟草木质素含量分析

为探究华南象草(Pennisetum purpureumcv.Huanan)木质素合成关键酶基因的调控机制,通过同源克隆得到华南象草4-香豆酸:CoA连接酶基因(Pp4CL)的cDNA序列,长度为1 943bp,其中编码区序列1 662bp。Pp4CL蛋白由553个氨基酸组成,分子量为59.57kD,等电点为5.2,属于疏水性蛋白。该蛋白含有AMP结合结构域,属于AFD ClassⅠ超家族。在系统进化分析中,Pp4CL与At4CL1、Os4CL1遗传距离最近,聚为一支。Pp4CL氨基酸序列具有SSGTGLPKGV和GEICIRG等2个保守基序,是典型的植物4CL。构建原核表达载体pGEX-4CL,得到约88kD的Pp4CL-GST融合蛋白,为Pp4CL酶活性测定及Western免疫印迹分析奠定了基础。同时构建植物表达载体pBA-4CL,并通过叶盘法对烟草进行了遗传转化,得到3个转基因阳性株系(OX-9、OX-7、OX-4),它们中叶柄木质素总含量分别比非转基因植株(对照)提高了10.0%、16.2%和94.6%,茎秆基部节木质素总含量分别比对照提高了0.9%、4.0%和13.5%。研究结果表明,Pp4CL蛋白与木质素合成有关,过表达Pp4CL基因能够显著提高植株木质素含量。该研究结果为华南象草木质素改良工作打下了基础,同时也为深入开展牧草分子育种提供了依据。     

Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen

Lignins, along with condensed tannins (CTs) and salicylate-derived phenolic glycosides, constitute potentially large phenylpropanoid carbon sinks in tissues of quaking aspen (Populus tremuloides Michx.). Metabolic commitment to each of these sinks varies during development and adaptation, and depends on L-phenylalanine ammonia-lyase (PAL), an enzyme catalyzing the deamination of L-phenylalanine to initiate phenylpropanoid metabolism. In Populus spp., PAL is encoded by multiple genes whose expression has been associated with lignification in primary and secondary tissues. We now report cloning two differentially expressed PAL cDNAs that exhibit distinct spatial associations with CT and lignin biosynthesis in developing shoot and root tissues of aspen. PtPAL1 was expressed in certain CT-accumulating, non-lignifying cells of stems, leaves, and roots, and the pattern of PtPAL1 expression varied coordinately with that of CT accumulation along the primary to secondary growth transition in stems. PtPAL2 was expressed in heavily lignified structural cells of shoots, but was also expressed in non-lignifying cells of root tips. Evidence of a role for Pt4CL2, encoding 4-coumarate:coenzyme A ligase, in determining CT sink strength was gained from cellular co-expression analysis with PAL1 and CTs, and from experiments in which leaf wounding increased PAL1 and 4CL2 expression as well as the relative allocation of carbon to CT with respect to phenolic glycoside, the dominant phenolic sink in aspen leaves. Leaf wounding also increased PAL2 and lignin pathway gene expression, but to a smaller extent. The absence of PAL2 in most CT-accumulating cells provides in situ support for the idea that PAL isoforms function in specific metabolic milieus.     

Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a "bonsai tree-like" appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated beta-aryl ether (beta-O-4 unit) and spirodienone (beta-1) levels, accompanied by lower phenylcoumaran (beta-5), resinol (beta-beta), and dibenzodioxocin (5-5/beta-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.     

Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-μm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm⁻¹, differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Differential substrate inhibition couples kinetically distinct 4-coumarate:coenzyme a ligases with spatially distinct metabolic roles in quaking aspen

4-Coumarate:coenzyme A ligase (4CL) activates hydroxycinnamates for entry into phenylpropanoid branchways that support various metabolic activities, including lignification and flavonoid biosynthesis. However, it is not clear whether and how 4CL proteins with their broad substrate specificities fulfill the specific hydroxycinnamate requirements of the branchways they supply. Two tissue-specific 4CLs, Pt4CL1 and Pt4CL2, have previously been cloned from quaking aspen (Populus tremuloides Michx.), but whether they are catalytically adapted for the distinctive metabolic roles they are thought to support is not apparent from published biochemical data. Therefore, single- and mixed-substrate assays were conducted to determine whether the 4CLs from aspen exhibit clear catalytic identities under certain metabolic circumstances. Recombinant Pt4CL1 and Pt4CL2 exhibited the expected preference for p-coumarate in single-substrate assays, but strong competitive inhibition favored utilization of caffeate and p-coumarate, respectively, in mixed-substrate assays. The Pt4CL1 product, caffeoyl-CoA, predominated in mixed-substrate assays with xylem extract, and this was consistent with the near absence of Pt4CL2 expression in xylem tissue as determined by in situ hybridization. It is interesting that the Pt4CL2 product p-coumaroyl-CoA predominated in assays with developing leaf extract, although in situ hybridization revealed that both genes were coexpressed. The xylem extract and recombinant 4CL1 data allow us to advance a mechanism by which 4CL1 can selectively utilize caffeate for the support of monolignol biosynthesis in maturing xylem and phloem fibers. Loblolly pine (Pinus taeda), in contrast, possesses a single 4CL protein exhibiting broad substrate specificity in mixed-substrate assays. We discuss these 4CL differences in terms of the contrasts in lignification between angiosperm trees and their gymnosperm progenitors.     

Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane

Sugarcane (Saccharum spp. hybrids) is a major feedstock for commercial bioethanol production. The recent integration of conversion technologies that utilize lignocellulosic sugarcane residues as well as sucrose from stem internodes has elevated bioethanol yields. RNAi suppression of lignin biosynthetic enzymes is a successful strategy to improve the saccharification of lignocellulosic biomass. 4-coumarate:coenzyme A ligase (4CL) is a key enzyme in the biosynthesis of phenylpropanoid metabolites, such as lignin and flavonoids. Identifying a major 4CL involved in lignin biosynthesis among multiple isoforms with functional divergence is key to manipulate lignin biosynthesis. In this study, two full length 4CL genes (Sh4CL1 and Sh4CL2) were isolated and characterized in sugarcane. Phylogenetic, expression and RNA interference (RNAi) analysis confirmed that Sh4CL1 is a major lignin biosynthetic gene. An intragenic precision breeding strategy may facilitate the regulatory approval of the genetically improved events and was used for RNAi suppression of Sh4CL1. Both, the RNAi inducing cassette and the expression cassette for the mutated ALS selection marker consisted entirely of DNA sequences from sugarcane or the sexually compatible species Sorghum bicolor. Field grown sugarcane with intragenic RNAi suppression of Sh4CL1 resulted in reduction of the total lignin content by up to 16.5?% along with altered monolignol ratios without reduction in biomass yield. Mature, field grown, intragenic sugarcane events displayed 52–76?% improved saccharification efficiency of lignocellulosic biomass compared to wild type (WT) controls. This demonstrates for the first time that an intragenic approach can add significant value to lignocellulosic feedstocks for biofuel and biochemical production.     

Expression of a bacterial, phenylpropanoid-metabolizing enzyme in tobacco reveals essential roles of phenolic precursors in normal leaf development and growth

Tobacco plants (Nicotiana tabacum cv XHFD 8) were genetically modified to express a bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) enzyme which is active with intermediates of the phenylpropanoid pathway. We have previously shown that HCHL expression in tobacco stem resulted in various pleiotropic effects, indicative of a reduction in the carbon flux through the phenylpropanoid pathway, accompanied by an abnormal phenotype. Here, we report that in addition to the reduction in lignin and phenolic biosynthesis, HCHL expression also resulted in several gross morphological changes in poorly lignified tissue, such as abnormal mesophyll and palisade. The effect of HCHL expression was also noted in lignin-free single cells, with suspension cultures displaying an altered shape and different growth patterns. Poorly/non-lignified cell walls also exhibited a greater ease of alkaline extractability of simple phenolics and increased levels of incorporation of vanillin and vanillic acid. However, HCHL expression had no significant effect on the cell wall carbohydrate chemistry of these tissues. Evidence from this study suggests that changes in the transgenic lines result from a reduction in phenolic intermediates which have an essential role in maintaining structural integrity of low-lignin or lignin-deprived cell walls. These results emphasize the importance of the intermediates and products of phenylpropanoid pathway in modulating aspects of normal growth and development of tobacco. Analysis of these transgenic plants also shows the plasticity of the lignification process and reveals the potential to bioengineer plants with reduced phenolics (without deleterious effects) which could enhance the bioconversion of lignocellulose for industrial applications.     

Transgenic poplars with reduced lignin show impaired xylem conductivity, growth efficiency and survival

We studied xylem anatomy and hydraulic architecture in 14 transgenic insertion events and a control line of hybrid poplar (Populus spp.) that varied in lignin content. Transgenic events had different levels of down-regulation of two genes encoding 4-coumarate:coenzyme A ligase (4CL). Two-year-old trees were characterized after growing either as free-standing trees in the field or as supported by stakes in a greenhouse. In free-standing trees, a 20 to 40% reduction in lignin content was associated with increased xylem vulnerability to embolism, shoot dieback and mortality. In staked trees, the decreased biomechanical demands on the xylem was associated with increases in the leaf area to sapwood area ratio and wood specific conductivity (k(s)), and with decreased leaf-specific conductivity (k(l)). These shifts in hydraulic architecture suggest that the bending stresses perceived during growth can affect traits important for xylem water transport. Severe 4CL-downregulation resulted in the patchy formation of discoloured, brown wood with irregular vessels in which water transport was strongly impeded. These severely 4CL-downregulated trees had significantly lower growth efficiency (biomass/leaf area). These results underscore the necessity of adequate lignification for mechanical support of the stem, water transport, tree growth and survival.     

Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents

? Reduced lignin content in perennial crops has been sought as a means to improve biomass processability for paper and biofuels production, but it is unclear how this could affect wood properties and tree form. ? Here, we studied a nontransgenic control and 14 transgenic events containing an antisense 4-coumarate:coenzyme A ligase (4CL) to discern the consequences of lignin reduction in poplar (Populus sp.). During the second year of growth, trees were grown either free-standing in a field trial or affixed to stakes in a glasshouse. ? Reductions in lignin of up to 40% gave comparable losses in wood strength and stiffness. This occurred despite the fact that low-lignin trees had a similar wood density and up to three-fold more tension wood. In free-standing and staked trees, the control line had twice the height for a given diameter as did low-lignin trees. Staked trees had twice the height for a given diameter as free-standing trees in the field, but did not differ in wood stiffness. ? Variation in tree morphogenesis appears to be governed by lignin x environment interactions mediated by stresses exerted on developing cells. Therefore our results underline the importance of field studies for assessing the performance of transgenic trees with modified wood properties.     

Alterations in the Biosynthesis of Lignin in Transgenic Plants with Chimeric Genes for 4-Coumarate: Coenzyme A Ligase

The introduction of chimeric sense and antisense gene constructsfor 4-coumarate:coenzyme A ligase into tobacco plants causedthe reduction of the 4CL activity in the transgenic plants.In the transgenic plants, the cell walls of the xylem tissuein stems were brown and the molecular structure of lignin inthe colored cell walls was dramatically different from thatin the control plants. Analysis with different types of stainrevealed that levels of cinnamyl aldehyde residues and syringylunits in lignin were depressed in the brownish cell walls. Furthermore,the lignin content in colored tissue was lower than that inthe normal tissue. Our results indicate that 4CL has importantroles in the determination of the composition and the amountof lignin in tobacco plants. (Received December 27, 1995; Accepted July 23, 1996)     

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.