参与植物细胞壁半纤维素木聚糖合成的糖基转移酶

木聚糖是双子叶植物次生细胞壁中最主要的半纤维素,含有木聚糖的次生壁是最丰富的植物生物质,广泛应用于能源、制浆、造纸和纺织业中,但其主要组分戊糖对细胞壁生物质利用具有较大影响。揭示木聚糖合成的分子机制,为遗传修饰细胞壁组成,更好地利用细胞壁生物质提供新的策略。近年来对模式植物拟南芥中多个木聚糖合成有缺陷的突变体的分析表明：GT43家族的IRX9、IRX9-L、IRX14、IRX14-L,GT47家族的FRA8、F8H、IRX10、IRX10-L,GT8家族的IRX8、PARVUS、QUA1、GUX1、GUX2等参与了木聚糖主链、还原末端序列和侧链的合成。本文主要对这些研究进展做一综述,并讨论了木聚糖合成的机制及亟待解决的问题,展望了其发展趋势。     

高等植物细胞壁中纤维素的合成

植物细胞壁主要由纤维素、半纤维素、木质素和果胶质等构成.近年来,在细胞壁形成,如纤维素合成方面的研究取得了一系列非常令人鼓舞的进展.本文就高等植物细胞壁中纤维素合成机制的研究进展作一介绍.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl Esterases Involved in its Release and Their Applications

ABSTRACT

Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.     

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

DHA合成途径中关键酶基因在毕赤酵母中的表达研究

DHA（二十二碳六烯酸）是人体所需的一种非常重要的多不饱和脂肪酸,主要存在于深海鱼类和海洋微藻类生物中。由于高等植物自身不能合成DHA,因此通过基因工程方法在作物(如玉米)中表达DHA,将会成为最健康的DHA来源,同时也能够减轻人类对海洋资源的破坏。从深海藻类中挑选了5个DHA合成途径中的关键酶基因,分别是Δ4脱饱和酶基因（D4）、Δ5脱饱和酶基因（D5）、Δ6脱饱和酶基因（D6）、C18延长酶基因（E18）和C20延长酶基因（E20）。密码子优化并合成这5个基因,为确定优化后的核苷酸序列是否能在真核生物中正确表达,以pPIC9K为基础载体连入目的基因转化到毕赤酵母GS115 (His4+MUts)中进行表达分析。结果表明,这些基因在甲醇诱导96 h后蛋白表达量最大,Western Blot结果表明表达产物为目的蛋白。该结果为进一步培育能够自身合成DHA的作物奠定了基础。     

Functional Analysis of Sinorhizobium meliloti Genes Involved in Biotin Synthesis and Transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

Identification of Candida glabrata Genes Involved in pH Modulation and Modification of the Phagosomal Environment in Macrophages

Candida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D₃ stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.     

Oxidoreductases Involved in Cell Carbon Synthesis of Methanobacterium thermoautotrophicum

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60°C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; α-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V_max and K_M values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective α-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the α-ketoacid and CO₂. The data indicate that the two enzymes are similar to pyruvate synthase and α-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.     

Reversal of the Vancomycin Inhibition of Peptidoglycan Synthesis by Cell Walls

Addition of cell walls to the peptidoglycan synthetase-acceptor system containing vancomycin (50 μg/ml) prevented the inhibition by the antibiotic. In addition, the inhibition of incorporation of [¹⁴C]muramyl-pentapeptide into peptidoglycan in the presence of vancomycin was reversed by the addition of cell walls to the assay mixture at 60 min. Cell walls previously saturated with vancomycin lost their ability to reverse the inhibition by the antibiotic. The inhibition of peptidoglycan synthesis by ristocetin was partially reversed by the addition of cell walls. The initial stage in peptidoglycan synthesis is catalyzed by phospho-N-acetyl(NAc)muramyl-pentapeptide translocase (uridine 5′-phosphate) according to the reaction: UDP-NAc-muramyl-pentapeptide + acceptor acceptor-phospho-NAc-muramyl-pentapeptide + UMP where acceptor is C₅₅-isoprenoid alcohol phosphate. Vancomycin stimulates the transfer of phospho-NAc-muramyl-pentapeptide to the acceptor, and the addition of cell walls to this assay mixture prevented the stimulation of transfer. In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate (UMP) with UDP-NAc-muramyl-pentapeptide. The exchange reaction is effectively inhibited by vancomycin. For example, 60 μg of vancomycin per ml inhibited the rate of exchange by 50%. Addition of cell walls restored the exchange of UMP with the UMP moiety of UDP-NAc-muramyl-pentapeptide. Thus, cell walls appeared to have a higher affinity for vancomycin than did either the peptidoglycan synthetase-acceptor system or phospho-NAc-muramyl-pentapeptide translocase. These results provide support for the proposal made by Best and Durham that the effective binding of vancomycin to the cell wall could result in the inhibition of transfer of membrane-associated peptidoglycan chains to the growing wall.     

Large Scale Identification of Genes Involved in Cell Surface Biosynthesis and Architecture in Saccharomyces Cerevisiae

The sequenced yeast genome offers a unique resource for the analysis of eukaryotic cell function and enables genome-wide screens for genes involved in cellular processes. We have identified genes involved in cell surface assembly by screening transposon-mutagenized cells for altered sensitivity to calcofluor white, followed by supplementary screens to further characterize mutant phenotypes. The mutated genes were directly retrieved from genomic DNA and then matched uniquely to a gene in the yeast genome database. Eighty-two genes with apparent perturbation of the cell surface were identified, with mutations in 65 of them displaying at least one further cell surface phenotype in addition to their modified sensitivity to calcofluor. Fifty of these genes were previously known, 17 encoded proteins whose function could be anticipated through sequence homology or previously recognized phenotypes and 15 genes had no previously known phenotype.     

Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide in Streptococcus mutans

We identified in Streptococcus mutans six new genes (rgpA through rgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in the rgpE mutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.     

与木葡聚糖合成的糖基转移酶基因研究进展

半纤维素多糖木葡聚糖(XyG)存在于大多数植物的初生细胞壁中, 对细胞壁的结构组织和生长发育具有重要的调控作用。XyG在植物进化中存在结构的多样性。该文概述了参与XyG合成的糖基转移酶的最新研究进展, XyG合成需要多种糖基转移酶参与, 这些酶类很可能以蛋白酶复合体的形式存在并发挥作用, XyG的结构和组成的改变对植物的生长发育也产生影响。     

参与木葡聚糖合成的糖基转移酶基因研究进展

半纤维素多糖木葡聚糖(XyG)存在于大多数植物的初生细胞壁中, 对细胞壁的结构组织和生长发育具有重要的调控作用。XyG在植物进化中存在结构的多样性。该文概述了参与XyG合成的糖基转移酶的最新研究进展, XyG合成需要多种糖基转移酶参与, 这些酶类很可能以蛋白酶复合体的形式存在并发挥作用, XyG的结构和组成的改变对植物的生长发育也产生影响。