Cuscuta reflexa invasion induces Ca2+ release in its host

Cuscuta reflexa induces a variety of reaction in its hosts. Some of these are visual reactions, and it is clear that these morphological changes are preceded by events at the molecular level, where signal transduction is one of the early processes. Calcium (Ca²⁺) release is the major second messenger during signal transduction, and we therefore studied Ca²⁺ spiking in tomato during infection with C. reflexa. Bioluminescence in aequorin‐expressing tomato was monitored for 48 h after the onset of Cuscuta infestation. Signals at the attachment sites were observed from 30 to 48 h. Treatment of aequorin‐expressing tomato leaf disks with Cuscuta plant extracts suggested that the substance that induced Ca²⁺ release from the host was closely linked to parasite haustoria.     

Studies on Components of Mammalian Urine

Hexahydrohippuric acid was detected from the urine of cattles together with hippuric and phenaceturic acids as one of the conjugated compounds with glycine. Furthermore, cyclohexanecarboxylic acid was also detected. These experimental results suggest the interesting synthetic processes in vivo or in rumen of the cattle, though both acids have not determined whether they are metabolites of cattle or synthesized by miccroorganisms in rumen.     

Expression of xyloglucan endotransglucosylase/hydrolase (XTH) genes and XET activity in ethylene treated apple and tomato fruits

Xyloglucan endotransglucosylase/hydrolase (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151), a xyloglucan modifying enzyme, has been proposed to have a role during tomato and apple fruit ripening by loosening the cell wall. Since the ripening of climacteric fruits is controlled by endogenous ethylene biosynthesis, we wanted to study whether XET activity was ethylene-regulated, and if so, which specific genes encoding ripening-regulated XTH genes were indeed ethylene-regulated. XET specific activity in tomato and apple fruits was significantly increased by the ethylene treatment, as compared with the control fruits, suggesting an increase in the XTH gene expression induced by ethylene. The 25 SlXTH protein sequences of tomato and the 11 sequences MdXTH of apple were phylogenetically analyzed and grouped into three major clades. The SlXTHs genes with highest expression during ripening were SlXTH5 and SlXTH8 from Group III-B, and in apple MdXTH2, from Group II, and MdXTH10, and MdXTH11 from Group III-B. Ethylene was involved in the regulation of the expression of different SlXTH and MdXTH genes during ripening. In tomato fruit fifteen different SlXTH genes showed an increase in expression after ethylene treatment, and the SlXTHs that were ripening associated were also ethylene dependent, and belong to Group III-B (SlXTH5 and SlXTH8). In apple fruit, three MdXTH showed an increase in expression after the ethylene treatment and the only MdXTH that was ripening associated and ethylene dependent was MdXTH10 from Group III-B. The results indicate that XTH may play an important role in fruit ripening and a possible relationship between XTHs from Group III-B and fruit ripening, and ethylene regulation is suggested.     

Molecular dynamics simulations of a branched tetradecasaccharide substrate in the active site of a xyloglucan endo-transglycosylase

Molecular dynamics simulations of the tetradecasaccharide XXXGXXXG in complex with the hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 have been performed and analysed with respect to structure, dynamics, flexibility and ligand interactions. Notably, the charge state of the so-called ‘helper residue’ aspartate 87 (Asp87), which lies between the catalytic nucleophile [glutamate 85 (Glu85)] and general acid/base (Glu89) residues on the same beta strand, had a significant effect on PttXET16-34 active site structure. When Asp87 was deprotonated, electrostatic repulsion forced the nucleophile away from C1 of the sugar ring in subsite ? 1 and the proton–donating ability of Glu89 was also weakened due to the formation of a hydrogen bond with Asp87, whereas the protonation of Asp87 resulted in the formation of a hydrogen bond with the catalytic nucleophile and correct positioning of the catalytic machinery. The results suggest that catalysis in glycoside hydrolase family 16, and by extension clan GH-B enzymes, is optimal when the catalytic nucleophile is deprotonated for nucleophilic attack on the substrate, whereas the ‘helper residue’ and general acid/base residue are both in their conjugate acid forms to align the nucleophile and deliver a proton to the departing sugar, respectively.     

香蕉果实XET cDNA的克隆及其在成熟软化的果实果皮和果肉中的表达分析

木葡聚糖内糖基转移酶(Xyloglucan endotransglycosylase,XET)通过分解细胞壁半纤维素多糖的主要成分--木葡聚糖而参与果实软化.为了阐明香蕉(Musa acuminata.Colla cv.GrandNain)果实成熟过程中的软化与细胞壁代谢酶XET基因表达模式的关系,采用RT-PCR和RACE-PCR方法,首次从成熟香蕉果实果肉中分离了编码XT基因的全长cDNA(MA-XET1,全长1 095 bp).序列分析表明,MA-XET1的5'端和3'端的非翻译区分别为66 bp和1 89bp,该片段含有一个完整的开放读码框,编码280个氨基酸,推导的MA-XET1蛋白质中存在XET蛋白的催化活性部位DEIDFEFL.Southern杂交表明,MA-XET1在香蕉基因组中由多拷贝基因编码.Northern分析显示,跃变前期的果肉中,不能检测MA-XET1基因的表达,跃变期的果实果肉中MA-XET1表达增加,跃变后期该基因表达略有减弱;在跃变前期的果实果皮中,MA-XET1的积累较低,跃变期的果实果皮中积累大幅增加,而后迅速下降.Propylene(丙烯,乙烯的类似物)处理降低香蕉果实果皮和果肉的硬度,而且propylene促进MA-XET1在果皮和果肉中的积累.这些结果表明,MA-XET1参与香蕉果实成熟过程中的果皮和果肉软化,并且,MA-XET1的表达在转录水平上受乙烯调控.     

欧亚大陆分布的大花菟丝子叶绿体基因组插入缺失分析

运用鸟枪法在Illumina测序仪上对亚洲分布的大花菟丝子(Cuscuta reflexa叶绿体基因组核苷酸全序列进行测定,并与已经发表的分布于欧洲的大花菟丝子进行了比较分析.研究结果表明亚洲分布的大花菟丝子叶绿体基因组总长度为120 972 bp,由79 499 bp的长单拷贝区,8 369 bp的短单拷贝区,以及两个16 552 bp的反向重复区组成,其长度比欧洲分布的大花菟丝子小了549 bp;基因组总GC含量为38.3％,稍高于欧洲分布的大花菟丝子.两地区的大花菟丝子叶绿体全基因组编码的功能基因完全相同,且基因排列顺序也完全一致.另外,经过进一步序列比对后发现亚洲分布的大花菟丝子与欧洲分布的存在251个插入和210个缺失现象,总插入缺失及碱基替换长度分别为7 649 bp和3 720 bp,最大的插入和缺失长度分别为426 bp和435 bp.很多插入缺失都是单碱基,但仍然存在四个长度超过200 bp的大突变,两个大的缺失发生在ycf2基因中,两个大的插入分别发生在trn F-psbE和matK-trnQ间隔区,详细的对比后发现大量的插入缺失都发生在大单拷贝区的基因间隔区,且插入缺失在反向重复区的发生频率较低.本研究首次报道不同大洲分布的同种异养植物的叶绿体全基因组比较分析,为研究这两个区域的居群多样性提供了基础资料.     

Implication of persimmon fruit hemicellulose metabolism in the softening process. Importance of xyloglucan endotransglycosylase

Hemicellulosic polysaccharides from persimmon fruit ( Diospyros kaki L.) pericarp were extracted from depectinated cell walls with 0.5, 1 and 4 M KOH at different stages of development: (I) maximal growth corresponding to the first sigmoidal growth phase; (II) cessation of growth corresponding to the lag between the first and the second sigmoidal phases; (III) maximal growth corresponding to the second sigmoidal phase; and (IV) cessation of growth when the fruit had reached its maximum size and the change in colour (green to red) had taken place. During fruit development the amount of total hemicelluloses per unit dry mass cell wall decreased twofold. Xyloglucan was present in the three hemicellulosic fractions, and also decreased with fruit age, although its amount relative to other hemicelluloses increased. The amount of xyloglucan was especially high in the hemicelluloses extracted with 4 M KOH, representing more than 50% at stages III and IV. The average molecular mass of xyloglucan increased from stage I through stage II (0.5 M hemicellulosic fraction) or through stage III (I and 4 M hemicellulosic fractions) and decreased after that. The xyloglucan endotransglycosylase (XET: EC 2.4.1.-) activity was measured as the incorporation of [³H]XXXGol (reduced xyloglucan heptasaccharide labelled at position 1 of the glucitol moiety) into partially purified persimmon fruit xyloglucan. XET specific activity increased greatly between stages I and II. The importance of this enzyme during fruit ripening is discussed.     

Developmental Expression Patterns of Arabidopsis XTH Genes Reported by Transgenes and Genevestigator

The plant cell wall is the structural basis of cellular form and thus forms a foundation on which morphogenesis builds organs and tissues. Enzymes capable of modifying major wall components are prominent candidates for regulating wall form and function. Xyloglucan endotransglucosylases/hydrolases (XTHs) are predicted to participate in xyloglucan integration and/or restructuring. XTHs are encoded by large gene families in plants; the Arabidopsis genome encodes 33 XTHs. To gain insight into the potential physiological relevance of the distinct members of this family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data reveal that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs show XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes. These data suggest that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

过表达胡杨XTH基因能够提高烟草抗旱性

胡杨是典型的抗旱树种。挖掘和鉴定胡杨的耐旱基因对于提高植物抗旱性具有重要意义。木葡聚糖内转糖苷酶/水解酶（XTH）是植物细胞壁重构过程中的关键酶,在植物逆境胁迫响应中发挥重要作用。我们前期已从胡杨叶片中克隆了PeXTH基因。本文利用Real-time PCR检测PeXTH基因在干旱胁迫下的表达水平。在此基础上,构建植物表达载体pMDC85-PeXTH,通过农杆菌介导法将PeXTH基因转入烟草,分析过表达PeXTH基因烟草的抗旱性。研究发现,胡杨叶片中PeXTH基因的表达受干旱胁迫诱导。干旱处理后,转PeXTH基因烟草的萌发率明显高于野生型烟草;与野生型植株相比,转基因植株的叶片失水速率明显降低。干旱胁迫下,转基因烟草的气孔开度仅为野生型烟草的51.2%～53.6%。结果表明,过表达PeXTH基因能够提高烟草的抗旱性。本研究丰富了对胡杨PeXTH基因功能的认识,为植物抗旱分子育种提供了重要的基因资源。     

滇产菟丝子种子形态的观察

应用解剖镜和扫描电镜观察了产于云南的 3种 12份菟丝子的种子形态 ,结果如下 :( 1)大花菟丝子种脐线形或弧形凹陷 ,晕轮椭圆形具网状纹饰 ,种皮具条状纹饰 ,条纹宽大呈块状 ,绞链状连接 ,排列致密 ;( 2 )中国菟丝子的种脐呈衣领状突起 ,晕轮圆形具不规则网状纹饰 ,种皮具复网状纹饰 ;( 3)日本菟丝子的种脐线形或弧形凹陷 ,晕轮圆形至椭圆形 ,具规则的网状纹饰 ,种皮具规则条状纹饰或网状纹饰 ,条纹狭长 ,排列稀疏 ;( 4 )种子形态不仅可用于菟丝子种及变种的检疫和鉴定 ,而且支持形态聚类分析所得结论。     

Characterization of two partially purified xyloglucan endotransglycosylases from parsley (<Emphasis Type="Italic">Petroselinum crispum</Emphasis>) roots

Two forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from the protein mixture obtained from parsley roots and partially characterized. Both forms were glycoproteins differing in their specific activities but other features were almost the same. Activity and stability of both enzymes in broad pH region were observed with two pH optima, one at acidic pH (5.8) and the second one at basic pH (8.8). The enzymes behaved as typical transglycosylases since no activity was observed in the absence of xyloglucan oligosaccharides in the viscometric assay. Small hetero-transglycosylating activities were observed when hydroxyethyl-or carboxymethyl-celluloses instead of xyloglucan as donor substrate were used as well as when cello-oligosaccharides instead of xyloglucan oligosaccharides were used as the acceptor substrate.     

植物细胞壁松弛因子

植物细胞壁的松弛是细胞伸长必需的一个生理过程,发生于植物生长发育的各个阶段。研究发现参与细胞壁松弛的因子有多种,主要包括膨胀素（expansin）、木葡聚糖内转糖苷酶/水解酶（XTH）、糖基水解酶和羟基自由基（·OH）四大类。本文主要对这些细胞壁松弛因子的结构特征、作用机制及其在植物生理过程中的作用等方面的研究进展进行综述。     

Xyloglucan oligosaccharides with at least two α-d-xylose residues act as acceptor substrates for xyloglucan endotransglycosylase and promote the depolymerisation of xyloglucan

A xyloglucan-derived pentasaccharide. Xyl₂-Glc₃, was shown by viscometry to promote the depolymerisation of xyloglucan by enzyme extracts from bean ( Phaseolus vulgaris L. cv. Canadian Wonder) leaves and pea ( Pisum sativum L. cv. Alaska) stems. Xyl₂-Glc₃ was also shown by a radiochemical assay to act as an acceptor substrate for xyloglucan endotransglycosylase activity (XET: EC 2.4.1.—) present in the same extracts. In both these assays, a heptasaccharide (Xyl₃-Glc₄) was more effective than Xyl₂-Glc₃ whereas two isomeric tetrasaccharides (Xyl₁-Glc₃) were essentially ineffective. The agreement in the structural requirements of the two assays suggests that they share a common basis; we therefore propose that the oligosaccharide-sensitive enzyme that depolymerises xyloglucan is XET rather than cellulase (EC 3.2.1.4). In the viscometric assay, the penta- and heptasaccharides would, according to our interpretation, compete with high molecular weight xyloglucan molecules as acceptor substrates for XET, leading to a decrease in the weight-average molecular weight of the xyloglucan and, therefore, to a decrease in viscosity.
Our results indicate that oligosaccharides have to possess two α- d -xylose residues in order to act as acceptor substrates for XET. The non-reducing end of a high-molecular weight xyloglucan can also act as an acceptor substrate. Therefore, it is likely that exo-hydrolysis by α- d -xylosidase would destroy the ability of a poly saccharide to act as an acceptor, even though α- d -xylosidase may remove only a single xylose residue from each polysaccharide molecule.     

Role of transglycosylation in the integration of xyloglucan into the growing plant cell wall in vivo

Abstract

Xyloglucan endotransglycosylase (XET) activity is widespread in plant cell walls, but its action on xyloglucan in vivo has been difficult to prove because the reaction products are not expected to differ chemically from the reactants. By feeding of cultured Rosa cells with [¹³C]glucose and [³H]arabinose followed by [¹²-C]glucose, and isopyenic centrifugation of the extracted xyloglucan in caesium trifluoroacetate, we have obtained evidence for the annealing of segments of newly-secreted xyloglucan to xyloglucan chains that were already present in the cell wall. This is the first evidence for interpolymeric transglycosylation of xyloglucan in vivo.