拟南芥RUS4基因沉默对花药药室内壁次生加厚的影响

RUS4是拟南芥DUF647蛋白家族的一个功能未知的成员。沉默RUS4基因引起植株育性严重下降,但育性下降的具体机制尚不清楚。该研究通过观察RUS4基因沉默突变体(称为RUSamiRNA)不同时期花的发育情况,发现其雌蕊发育正常,雄蕊花丝伸长正常,主要缺陷是花药不能正常开裂;通过对RUS4-amiRNA植株花药发育的细胞形态学观察,发现其药室内壁缺乏次生加厚;qRT-PCR分析表明, RUS4-amiRNA花蕾中与植物次生壁加厚相关的转录因子基因NST1、NST2、MYB103和MYB85以及纤维素合成基因IRX1、IRX3、IRX5和IRX8的表达均大幅降低。该研究表明,RUS4可能通过影响次生壁形成相关基因的表达参与花药药室内壁次生壁的形成。     

高效木聚糖降解酶系定制的新进展与挑战

木聚糖是植物细胞壁中含量最丰富的非纤维素多糖,大约占陆地生物质资源的20%-35%。不同物种来源的木聚糖结构因取代方式不同而具有广泛的异质性,这对生物质资源向生物燃料和其他高值产品高效转化提出了重大挑战。因此,需要开发由不同类型酶组成的最佳混合物以有效糖化木聚糖类底物。但是针对特定类型的底物设计高效降解酶系十分困难,应考虑底物的类型、底物的组成和物理性质、多糖的聚合度以及不同降解酶组分的生化性质等。本文从不同植物木聚糖的结构异质性与合成复杂性方面展示了其抗降解屏障,同时介绍了木聚糖主链降解酶系及侧链降解酶系的多样性以及协同降解作用,综述了复杂生境中微生物种群产生的混合酶系、降解菌株产生的高效酶系,以及基于特定木聚糖底物改造并定制简化高效的酶系统。随着不同种类木聚糖精细结构和木聚糖降解酶底物特异性的深入研究,针对特定底物类型进行绿色高效木聚糖酶系定制,加速木聚糖类底物的降解,从而实现木质纤维素资源的绿色高值化利用。     

利用真菌纤维素结合域(CBD)保守性序列进行草菇木聚糖酶cDNA的克隆

木聚糖是植物细胞壁的主要组分,它是木糖以β1 ,4 木糖苷键形成主链,乙酰基,阿拉伯糖基等为附链组成的复合多聚糖.木聚糖酶可以降解木聚糖主链,在木聚糖的生物降解中起着非常重要的作用[1 ] .根据木聚糖酶催化域(catalyticdomain ,CD)氨基酸序列的相似性,木聚糖酶可分为两个家     

杜仲次生木质部分化过程中木质素与半纤维素组分在细胞壁中分布的动态变化

利用紫外光显微镜、透射电子显微镜结合免疫胶体金标记,研究了杜仲（Eucommia ulmoides Oliv.)次生木质部分化过程中木质素与半纤维素组分（木葡聚糖和木聚糖）在细胞壁分布的动态变化。在形成层及细胞伸展区域,细胞壁具有木葡聚糖的分布,而没有木聚糖和木质素沉积,随着次生壁S1层的形成,木质素出现在细胞角隅和胞间层,木聚糖开始出现在S1层中,此时木葡聚糖则分布在初生壁和胞间层;随着次生,壁S2层及S3层的形成和加厚,木质逐逐步由细胞角隅和胞间层扩展到S1、S2和S3层,其沉积呈现出不均匀的块状或片状沉积模式,在次生壁各层形成与其木质化的同时,木聚糖逐渐分布于整个次生壁中,而木糖聚糖仍局限分布于初生壁和胞间层。结果表明,随着细胞次生壁的形成与木质化,细胞壁结构发生较大变化。细胞壁的不同区域,如细胞角隅、胞间层、初生壁和次生壁各层,具有不同的半纤维素组成,其与木质等细胞壁组分结构构成不同的细胞壁分子结构。     

信息库

1　川地曲霉和泡盛曲霉中的两种α L 阿拉伯呋喃糖苷酶　　L 阿拉伯糖是植物细胞壁多糖 ,如阿拉伯聚糖 ,阿拉伯木聚糖和阿拉伯半乳聚糖的共同成分。降解阿拉伯聚糖的两种主要酶活性是 ,内 1,5 α 阿拉伯聚糖酶 [EC3.2 .1.99]和α L 阿拉伯呋喃糖苷酶 [EC3.2 .1.5 5 ]。阿拉伯木聚糖是植物半纤维素的主要成分 ,含有一个 1,4 β连接的木糖主链和不同的取代基。L 阿拉伯呋喃糖是 1,2 α或 1,3 α连接的阿拉伯木聚糖中的一种侧链取代基。酚类化合物 ,如阿魏酰和香豆酰取代基 ,和阿拉伯木聚糖中的部分L 阿拉伯呋喃糖酯化。大麦烧酒中含有…     

木聚糖的酶解和利用

<正> 木聚糖广泛存在于植物细胞壁中,是构成植物半纤维素组分的主要成分。在高等植物和农业废料中约占干重的20％。美每年有5亿吨植物纤维被废弃,其中含10～25％木聚糖形式的木糖,按最低估计也有5000万吨,若将这些木糖发酵,可产生1500万吨酒精。近年来,对于采取酶和微生物步序有效地利用木聚糖已受到了重视。很多微生物能够水解木聚糖,业已分离出真菌、细菌以及放线菌的木聚糖酶。很多微生     

嗜极性木聚糖酶

木聚糖酶内切水解木聚糖主链的1,4-β-D-糖苷键,木聚糖是植物细胞壁中一种主要的多糖。自然界中木聚糖是多种糖类的复合体,这就使得木聚糖酶呈现多态性和多域性,由此需将繁多的木聚糖酶进行归类。木聚糖酶的催化反应属于双置换机制。在已研究的真菌或细菌性木聚糖酶中,大多数在温和的条件下表现出最佳活性,但有很多在极端环境下生长的生物体,为了适应极端环境而产生嗜极性的酶,其中嗜酸的、嗜碱的、嗜热的木聚糖酶,现在已有广泛的研究。对嗜极性木聚糖酶的研究进展作了论述。     

白桦MYB家族基因序列及表达分析

MYB转录因子家族是植物重要的转录因子家族之一,其成员在植物的生长、发育、细胞壁形成及胁迫反应等多方面发挥重要作用。从白桦(Betula platyphylla Suk.)中鉴定了17条MYB家族基因,与拟南芥家族基因进行系统进化分析结果表明,17个白桦MYB基因分属不同亚家族的不同亚类,其中10条属于1R/4R型亚家族,其余7条属于2R型亚家族。BplMYB13与已知的次生细胞壁合成相关基因At MYB46聚为一组,BplMYB15和BplMYB26分别与胁迫响应和非生物胁迫应答相关MYB聚为一组,BplMYB23与硫代葡萄糖苷合成相关MYB聚为一组,BplMYB9、21和22与苯丙烷生物合成相关MYB聚为一组。利用Real time RT-PCR分析白桦MYB家族基因在白桦形成层和木质部组织一个生长季不同发育时期及人工弯曲处理6 h的表达模式。结果显示17个MYB基因在5月中旬至7月中旬白桦形成层活动旺盛,木质部迅速形成期表达量较高,其中Bp MYB13在全生长季形成层和新生木质部中都有较高的表达水平,推测该基因与次生细胞壁的形成相关;在白桦茎干人工弯曲处理6 h时,与直立木和对应木相比,14条基因在应拉木中上调表达;与直立木相比,7条基因在对应木中上调表达。说明这些基因对人工弯曲处理和重力刺激具有应答反应,可能在白桦木质部发育和响应外力刺激等过程的生理变化中起重要作用。     

拟南芥纤维素合酶基因时空表达模式与功能预测

纤维素是细胞壁的主要组成成分, 研究纤维素合成可以从源头上解决关于高效降解纤维素的问题。该研究通过综合拟南芥(Arabidopsis thaliana)纤维素合酶基因(AtCESA)家族的进化和芯片表达分析及根据拟南芥全生育期GUS染色结果分析纤维素合酶基因的时空表达模式, 发现拟南芥纤维素合酶基因AtCESA1, 3, 6以及AtCESA4, 7, 8分别参与细胞壁初生壁和次生壁的合成并存在明显的共表达现象。其中, AtCESA1, 3, 6在全生育期表达, AtCESA4, 7, 8主要在根、茎和叶脉等次生壁细胞中表达。AtCESA5和AtCESA6、AtCESA2和AtCESA9以及AtCESA1和AtCESA10等基因对均有基因重复作用。根据AtCESA家族基因表达模式和分子演化关系可以推测, AtCESA5对AtCESA6以及AtCESA9对AtCESA2可能分别存在功能冗余。此外, AtCESA9的表达具明显的组织特异性。上述研究结果为深入认识拟南芥纤维素合酶基因的功能奠定了基础。     

拟南芥种皮粘液质形成及调控机制研究进展

植物细胞壁是地球上储量最丰富的可再生资源,是人类生产和生活中能源、纤维、建筑材料和造纸等原料的主要来源。植物细胞壁的形成机制一直是近年来的研究热点,研究植物细胞壁的形成机制不仅有助于更高效地将细胞壁转化为生物乙醇等可再生能源,也将促进纤维生物质在食品、药品和纺织等领域的更高效利用,对于新能源开发和人类生产生活均具有十分重要的意义。一些十字花科(如拟南芥,Arabidopsis thaliana)和车前科植物的种皮外层细胞在发育过程中会合成和分泌大量的粘液质多糖,其在种子遇水后膨胀并释放,形成透明胶状物质包裹种子周围。拟南芥种皮粘液质的主要成分为果胶质(主要为鼠李半乳糖醛酸聚糖I),同时还含有少量的纤维素和半纤维素成分。种皮粘液质作为一种特化的细胞壁,具有表型容易观察、分离提取简便、组成相对单一、缺失不影响植株生长发育等优点,已成为研究植物细胞壁(果胶)多糖合成、调控及细胞壁组分间互作的理想模式体系,近年来取得了较大的研究进展,本文主要介绍拟南芥种皮粘液质的形成、组成及其调控机制方面的研究进展。     

Enzymatic treatments reveal differential capacities for xylan recognition and degradation in primary and secondary plant cell walls

The capacity of four xylan-directed probes (carbohydrate-binding modules Cf CBM2b-1-2 and Cj CBM15; monoclonal antibodies LM10 and LM11) to recognize xylan polysaccharides in primary and secondary cell walls of tobacco stem sections has been determined. Enzymatic removal of pectic homogalacturonan revealed differential recognition of xylans in restricted regions of cortical primary cell walls. Monoclonal antibody binding to these exposed xylans was more sensitive to xylanase action than carbohydrate-binding module (CBM) binding. In contrast, the recognition of xylans by CBMs in secondary cell walls of the same organ was more sensitive to xylanase action than the recognition of xylans by the monoclonal antibodies. A methodology was developed to quantify indirect immunofluorescence intensities, and to evaluate xylanase impacts. The four xylan probes were also used to detect xylan populations in chromatographic separations of solubilized cell wall materials from tobacco stems. Altogether, these observations reveal the heterogeneity of the xylans in plant cell walls. They indicate that although CBM and antibody probes can exhibit similar specificities against solubilized polymers, they can have differential capacities for xylan recognition in muro , and that the access of molecular probes and enzymes to xylan epitopes/ligands also varies between primary and secondary cell walls that are present in the same organ.     

Gibberellin signaling

A study of stem anatomy and the sclerenchyma fibre cells associated with the phloem tissues of hemp (Cannabis sativa L.) plants is of interest for both understanding the formation of secondary cell walls and for the enhancement of fibre utility as industrial fibres and textiles. Using a range of molecular probes for cell wall polysaccharides we have surveyed the presence of cell wall components in stems of hemp in conjunction with an anatomical survey of stem and phloem fibre development. The only polysaccharide detected to occur abundantly throughout the secondary cell walls of phloem fibres was cellulose. Pectic homogalacturonan epitopes were detected in the primary cell walls/intercellular matrices between the phloem fibres although these epitopes were present at a lower level than in the surrounding parenchyma cell walls. Arabinogalactan-protein glycan epitopes displayed a diversity of occurrence in relation to fibre development and the JIM14 epitope was specific to fibre cells, binding to the inner surface of secondary cell walls, throughout development. Xylan epitopes were found to be present in the fibre cells (and xylem secondary cell walls) and absent from adjacent parenchyma cell walls. Analysis of xylan occurrence in the phloem fibre cells of hemp and flax indicated that xylan epitopes were restricted to the primary cell walls of fibre cells and were not present in the secondary cell walls of these cells.     

Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis

Xylan, the major hemicellulosic polysaccharide in Arabidopsis secondary cell walls, requires a number of glycosyltransferases (GT) to catalyse formation of the various glycosidic linkages found in the polymer. In this study, we characterized IRX10 and IRX10-like ( IRX10-L ), two highly homologous genes encoding members of the glycosyltransferase family 47 (GT47). T-DNA insertions in IRX10 gave a mild irregular xylem (irx) phenotype consistent with a minor defect in secondary cell-wall synthesis, whereas plants containing mutations in IRX10-L showed no change. However, irx10 irx10-L double mutant plants showed a much more severe irx and whole-plant phenotype, suggesting considerable functional redundancy between these two genes. Detailed biochemical analysis of the irx10 irx10-L double mutant showed a large reduction of xylan in the secondary cell walls, consistent with a specific defect in xylan biosynthesis. Furthermore, the irx10 irx10-L mutant retains the unique oligosaccharide found at the reducing end of Arabidopsis xylan, but shows a severe reduction in β(1,4) xylosyltransferase activity. These characteristics are similar to those of irx9 and irx14 , mutants that are believed to be defective in xylan chain elongation, and suggests that IRX10 and IRX10-L also play a role in elongation of the xylan backbone.     

Xylan deposition on secondary wall of Fagus crenata fiber

Summary. Delignified and/or xylanase-treated secondary walls of Fagus crenata fibers were examined by field emission scanning electron microscopy. Microfibrils with a smooth surface were visible in the innermost surface of the differentiating fiber secondary wall. There was no ultrastructural difference between control and delignified sections, indicating that lignin deposition had not started in the innermost surface of the cell wall. There was no ultrastructural difference between control and xylanase-treated sections. Microfibrils on the outer part of the differentiating secondary wall surface had globular substances in delignified sections. These globular substances disappeared following xylanase treatment, indicating that these globules are xylan. The globular substances were not visible near the inner part of the differentiating secondary wall but gradually increased toward the outer part of the secondary wall, indicating that xylan penetrated into the cell wall and continuously accumulated on the microfibrils. Mature-fiber secondary walls were also examined by field emission scanning electron microscopy. Microfibrils were not apparent in the secondary wall in control specimens. Microfibrils with many globular substances were observed in the delignified specimens. Following xylanase treatment, the microfibrils had a smooth surface without any globules, indicating that the globular substance is xylan. These results suggest that cellulose microfibrils synthesized on the plasma membrane are released into the innermost surface of the secondary wall and coated with a thin layer of xylan. Successive deposition of xylan onto the cell wall increases the microfibril diameter. The large amounts of xylan that accumulated on microfibrils appear globular but are covered with lignin after they are deposited. Received February 20, 2001/Accepted September 1, 2001     

Immunolocalization of hemicelluloses in Arabidopsis thaliana stem. Part I: temporal and spatial distribution of xylans

We investigated the microdistribution of xylans in different cell types of Arabidopsis stem using immunolocalization methods with LM10 and LM11 antibodies. Xylan labeling in xylary fibers (fibers) was initially detected at the cell corner of the S(1) layer and increased gradually during fiber maturation, showing correlation between xylan labeling and general secondary cell wall formation processes in fibers. Metaxylem vessels (vessels) showed earlier development of secondary cell walls than fibers, but revealed almost identical labeling patterns to fibers during maturation. No difference in labeling patterns and intensity was detected in the cell wall of fibers, vessels and protoxylem vessels (proto-vessels) between LM10 and LM11, indicating that vascular bundle cells may be chemically composed of a highly homogeneous xylan type. Interestingly, interfascicular fibers (If-fibers) showed different labeling patterns between the two antibodies and also between different developmental stages. LM10 showed no labeling in primary cell walls and intercellular layers of If-fibers at the S(1) formation stage, but some labeling was detected in middle lamella cell corner regions at the S(2) formation stage. In contrast, LM11 revealed uniform labeling across the If-fiber cell wall during all developmental stages. These results suggest that If-fibers have different xylan deposition processes and patterns from vascular bundle cells. The presence of xylan was also confirmed in parenchyma cells following pectinase treatment. Together our results indicate that there are temporal and spatial differences in xylan labeling between cell types in Arabidopsis stem. Differences in xylan labeling between Arabidopsis stem and poplar are also discussed.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

Impact of fine litter chemistry on lignocellulolytic enzyme efficiency during decomposition of maize leaf and root in soil

Residue recalcitrance controls decomposition and soil organic matter turnover. We hypothesized that the complexity of the cell wall network regulates enzyme production, activity and access to polysaccharides. Enzyme efficiency, defined as the relationship between cumulative litter decomposition and enzyme activities over time, was used to relate these concepts. The impact of two contrasting types of cell walls on xylanase, cellulase and laccase efficiencies was assessed in relation to the corresponding changes in residue chemical composition (xylan, glucan, lignin) during a 43-day incubation period. The selected residues were maize roots, which are rich in secondary cell walls that contain lignin and covalent bridges between heteroxylans and lignin, and maize leaves having mostly non-lignified primary cell walls thus making the cellulose and hemicelluloses less resistant to enzymes. Relationships between C mineralization and change in residue quality through decomposition indicated that the level of substitution of arabinoxylans (arabinan to xylan ratio) provides a good explanation of the decomposition process. In leaves enriched in primary cell walls, arabinose substitution of xylan controlled C mineralization rate but hampered polysaccharide decomposition, but to a lesser extent than in roots in which arabinoxylans were mostly cross-linked with lignin. Enzyme activity was higher in leaf than root amended soils while enzyme efficiency was systematically higher in the presence of roots. This apparent paradox suggests that residue quality could preselect the microbial community. Indeed, we found that microorganisms exhibited an initial rapid growth in the presence of a high quality litter and produced enzymes that are not efficient in degrading recalcitrant cell walls while, in the presence of the more recalcitrant maize roots, microbial biomass grew more slowly but produced enzymes of higher efficiency. This high enzyme efficiency could be explained by the synergistic action of hydrolytic and oxidative enzymes even in the early stage of decomposition.     

In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

Background

Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.

Results

Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.

Conclusion

We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.