棉花漆酶基因在转基因新疆杨中的表达及其对木质素合成的影响

以新疆杨叶柄为外植体,利用农杆菌法将棉花漆酶基因GaLAC1导入新疆杨.PCR,Soutllern杂交证明外源基因已经整合到杨树基因组中.漆酶活性分析表明转基因植株中漆酶活性较非转基因对照显著提高.与对照植株相比,转基因新疆杨茎段中总木质素的含量有不同程度的增加,最高达21.5%.木质素的组织化学染色进一步证实了GaLAC1的过量表达能够导致转基因植株中总木质素含量的增加.实验结果表明GaLAC1参与了植物体内木质素的合成,这是首次成功利用转基因植物证实植物漆酶基因参与木质素合成的报道.     

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.     

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand‐full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.     

PbrmiR397a regulates lignification during stone cell development in pear fruit

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.     

MiR397b regulates both lignin content and seed number in Arabidopsis via modulating a laccase involved in lignin biosynthesis

Plant laccase (LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of microRNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a microRNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b (OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b‐resistant laccase mRNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397‐mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin.     

Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar

Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2- to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants.     

Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis

Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H₂O₂. Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were β-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The effect of lignin model compound structure on the rate of oxidation catalyzed by two different fungal laccases

Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize various substrates, such as phenolic subunits of lignin. The substrate range can be widened to non-phenolic units by the use of mediators. Since discovery of the laccase-mediator system, direct reactions of lignin and laccase without mediated electron-transfer have gained much less attention. The objective of this study was to investigate lignin as a substrate for fungal laccases by using lignin model compounds. These model compounds contained guaiacylic and syringylic moieties and also compounds of guaiacylic origin at a higher oxidation level. Some of these compounds are commercially available, but most of them were synthesized. The oxidation reaction rates of the lignin model compounds were studied by monitoring consumption of the co-substrate oxygen, in reactions catalyzed by laccases from two different fungi; Melanocarpus albomyces and Trametes hirsuta, possessing different molecular and catalytic properties. These reaction rate studies were compared to physicochemical properties of the lignin model compounds: relative redox potentials determined using cyclic voltammetry and pK_a-values. Docking of syringylic and biphenylic compounds to the active sites of both laccases was performed and the resulting model complex structures were used to further interpret the reaction rate results. Reaction rates of laccases are mainly affected by the ability of a lignin model compound to be oxidized and the pK_a-value of the substrate seems to be less important. As a consequence, syringylic compounds are oxidized with the highest rates and compounds at a higher oxidation level and redox-potential, such as vanillin, are oxidized at a much lower rate. Both guaiacylic and syringylic type compounds fit well in the active sites of both laccases. Only for a biphenylic compound, steric clashes were observed, and they are likely to have an effect on the reaction rate. When the oxidation rates on the selected model compounds with the two different laccases were compared, the redox-potential difference between laccases T1 copper and the lignin model compound (ΔE) was not the only property that determined the oxidation rate. In the case of lignin model substrates, also the selectivity of a specific laccase, reflected in the k_cat/K_m value, plays an important role.     

Improving retting of fibre through genetic modification of flax to express pectinases

Flax (Linum usitatissimum L.) is a raw material used for important industrial products. Linen has very high quality textile properties, such as its strength, water absorption, comfort and feel. However, it occupies less than 1% of the total textile market. The major reason for this is the long and difficult retting process by which linen fibres are obtained. In retting, bast fibre bundles are separated from the core, the epidermis and the cuticle. This is accomplished by the cleavage of pectins and hemicellulose in the flax cell wall, a process mainly carried out by plant pathogens like filamentous fungi. The remaining bast fibres are mainly composed of cellulose and lignin. The aim of this study was to generate plants that could be retted more efficiently. To accomplish this, we employed the novel approach of transgenic flax plant generation with increased polygalacturonase (PGI ) and rhamnogalacturonase (RHA) activities. The constitutive expression of Aspergillus aculeatus genes resulted in a significant reduction in the pectin content in tissue-cultured and field-grown plants. This pectin content reduction was accompanied by a significantly higher (more than 2-fold) retting efficiency of the transgenic plant fibres as measured by a modified Fried’s test. No alteration in the lignin or cellulose content was observed in the transgenic plants relative to the control. This indicates that the over-expression of the two enzymes does not affect flax fibre composition. The growth rate and soluble sugar and starch contents were in the range of the control levels. It is interesting to note that the RHA and PGI plants showed higher resistance to Fusarium culmorum and F. oxysporum attack, which correlates with the increased phenolic acid level. In this report, we demonstrate for the first time that over-expression of the A. aculeatus genes results in flax plants more readily usable for fibre production. The biochemical parameters of the cell wall components indicated that the fibre quality remains similar to that of wild-type plants, which is an important pre-requisite for industrial applications. Magdalena Musialak and Magdalena Wróbel-Kwiatkowska participated equally in the preparation of this paper     

小麦盐华南象草PpCAD基因的亚细胞定位及其在转基因烟草中的异源表达迫相关基因的克隆与表达分析

该研究根据已克隆的华南象草(Pennisetum purpureum cv.Huanan)肉桂醇脱氢酶(CAD)基因PpCAD的cDNA序列,构建亚细胞定位载体pAN580-PpCAD,用PEG介导法转化象草原生质体,以探究PpCAD蛋白在细胞内的定位;同时构建植物过表达载体pBA002-PpCAD,通过农杆菌介导法在烟草中异源表达,以研究PpCAD基因与植物木质素合成的关系。结果显示:(1)PpCAD定位在象草原生质体的细胞质内;(2)过表达载体pBA002-PpCAD转化烟草后获得27株转基因烟草,其中25株PCR鉴定为阳性;(3)半定量RT-PCR检测6株转基因烟草后发现,PpCAD基因在不同植株的表达量存在差异,通过Southern杂交检测后发现该差异与目的基因插入的拷贝数有关;(4)6株转基因烟草和野生型烟草表型上没有明显差异,除目的基因多拷贝插入的植株OEC6外,木质素含量有不同程度的提高,最高比野生型提高了56.50%。研究表明,PpCAD是一个细胞质蛋白,在烟草中过表达PpCAD能够提高植株木质素含量,表明PpCAD基因参与了植物的木质素合成,可用于象草的木质素调控研究。     

Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for β-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen (Populus sieboldii ×P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.     

Degradation of aromatic compounds through the β‐ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β‐ketoadipate pathway. In this study, we investigated the role of 3‐carboxy‐cis,cis‐muconate lactonizing enzyme (CMLE), an enzyme of the β‐ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β‐ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β‐ketoadipate pathway in plant‐pathogenic, soil‐borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

A new role for veratryl alcohol: regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.     

Decreased peroxidase activity in transgenic tobacco and its effect on lignification

An antisense gene construct of a peroxidase gene (Shpx6a) from a tropical pasture legume Stylosanthes humilis was transferred into tobacco cells via Agrobacterium tumefaciens to test whether peroxidase activity could be decreased and what effect this would have on lignification. A large number of tobacco cell lines were regenerated on selective media and stable integration of the transgene was confirmed in randomly selected putative transformants. Analyses of the primary transgenic plants and their progeny (T ₁) demonstrated that the total peroxidase activity was significantly decreased (up to 36%) as compared to that measured in untransformed control plants. Importantly, reduction in peroxidase activity is accompanied by decreases (up to 23%) in lignin content in several transgenic lines.     

Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I.