Studies on Xyloglucanase during the Germination of Seeds of Tamarindus indica

Germinating seeds of Tamarindus indica contain endo-β-1, 4-xyloglucanases which degrade tamarind xyloglucan, but not carboxymethylcellulose (CMC). The xyloglucanases are isolated from the germinating tamarind seeds using 50 mM acetate buffer, pH 5.5 containing 0.5 M NaCl. The Km value is 0.667 g/liter and the enzyme is optimally active at pH 5.5 and stable between pH 4 - 6.5. The optimum temperature is 45?C and is quite stable upto 50?C. The activity declined by 50% at 60?C and is completely inactivated at 70?C. Highest xyloglucanase activity and specific activity are observed on the 23rd day of germination. The polyacrylamide gel electrophoresis (PAGE) indicated the presence of five isozymes of xyloglucanases which are visualized by activity staining separately with congo red and grams iodine. Isozyme 2 is the major xyloglucanase present throughout the germination period.

Identification of EDTA-Soluble Polysaccharides from Pea Epicotyl Cell Walls and Their Interaction with Xyloglucan

Nascent pectin and glucuronoxylan were prepared from membrane-bound enzymes obtained from pea epicotyls. They had previously been shown to exhibit a protein- and pH-dependent pattern of binding to cell wall ghosts and to xy-loglucan extracted from cell walls prepared from pea epicotyls;maximum binding required a pH of 3-4, and the pres-ence of cell wall proteins, namely assemblins. To determine whether wall polysaccharides deposited in cell walls be-have in the same manner as nascent polymers, radioactively labeled EDTA-soluble polymers were prepared from newly-deposited pea epicotyl cell walls. Different enzyme treatments followed by column chromatography, in addition to complete acid hydrolysis followed by paper and thin layer chromatography, indicated the presence of pectin, to-gether with smaller amounts of glucuronoxylan, in this EDTA-soluble extract. These radioactively labeled polysaccha-rides were found to bind to cell wall ghosts and to xyloglucan extracted from the second and third internodes of pea epicotyls cell walls in a pH-dependent manner, similar to the binding pattern obtained with nascent polymers. Maxi-mum binding occurred at pH 3-4, and also required the presence of protein.

Influence of Xyloglucan Molar Mass on Rheological Properties of Cellulose Nanocrystal/Xyloglucan Hydrogels

Plant components are an inexhaustible source for the construction of bio-based materials.Here we report,for the first time,the elaboration of biobased cellulose nanocrystals(CNC)/xyloglucan(XG)hydrogels.XG is a hemicellulose displaying a great affinity for cellulose surface and can be thus irreversibly adsorbed on CNC.Properties of the hydrogels were investigated by varying the molar mass of XG either by enzymatic treatment with Endo-glucanase(EG2)or physical fractionation by ultrasound(US).Fractions were characterised by high-performance size exclusion chromatography(HPSEC)and their monosacchari decompositions were determined.Three fractions with high,average and small molar mass,(800,300 and 100103 g/mol respectively),were selected in order to tune the properties of the hydrogel.Sol-gel transition conditions were determined for each fraction by achieving phase diagram using the inverted tube method.Mechanical properties,assessed by rheology,are improved by increasing XG molar mass since elastic modulus is higher for hydrogels formed with higher molar mass fractions as well as the strain at break.Gel formation is likely due to the adsorption of XG fractions on CNC which increases the effective hydrodynamic volume of CNC leading to steric stabilization and interactions between loops and tails of XG adsorbed.

Functions of Xyloglucan in Plant Cells

While an increase in the number of xyloglucan tethers between the cellulose microfibrils in plant cell walls increases the walls＇ rigidity, the degradation of these tethers causes the walls to loosen. Degradation can occur either through the integration of xyloglucan oligosaccharides due to the action of xyloglucan endotransglucosylase or through direct hydrolysis due to the action of xyloglucanase. This is why the addition of xyloglucan and its fragment oligosac- charides causes plant tissue tension to increase and decrease so dramatically. Experiments involving the overexpression of xyloglucanase and cellulase have revealed the roles of xyloglucans in the walls. The degradation of wall xyloglucan in poplar by the transgenic expression of xyloglucanase, for example, not only accelerated stem elongation in the primary wall, but also blocked upright-stem gravitropism in the secondary wall. Overexpression of cellulase also reduced xylo- glucan content in the walls as cellulose microfibrils were trimmed at their amorphous region, resulting in increased cell volume in Arabidopsis leaves and in sengon with disturbed leaf movements. The hemiceflulose xylogiucan, in its function as a tether, plays a key role in the loosening and tightening of cellulose microfibrils： it enables the cell to change its shape in growth and differentiation zones and to retain its final shape after cell maturation.

Xyloglucans of Monocotyledons Have Diverse Structures

Except in the Poaceae, little is known about the structures of the xyloglucans in the primary walls of monocotyledons. Xyloglucan structures in a range of monocotyledon species were examined. Wall preparations were isolated, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-（1→4）-β-glucanase preparation. The oligosaccharides released were analyzed by high-performance anion-exchange chromatography and by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Oligosaccharide profiles of the non-commelinid monocotyledons were similar to those of most eudicotyledons, indicating the xyloglucans were fucogalactoxyloglucans, with a XXXG a core motif and the fucosylated units XXFG and XLFG. An exception was Lemna minor （Araceae）, which yielded no fucosylated oligosaccharides and had both XXXG and XXGn core motifs. Except for the Arecales （palms） and the Dasypogonaceae, which had fucogalactoxyloglucans, the xyloglucans of the commelinid monocotyledons were structurally different. The Zingiberales and Commelinales had xyloglucans with both XXGn and XXXG core motifs; small proportions of XXFG units, but no XLFG units, were present. In the Poales, the Poaceae had xyloglucans with a XXGn core motif and no fucosylated units. In the other Poales families, some had both XXXG and XXGn core motifs, others had only XXXG; XXFG units were present, but XLFG units were not.

Xyloglucan for Generating Tensile Stress to Bend Tree Stem

In response to environmental variation, angiosperm trees bend their stems by forming tension wood, which consists of a cellulose-rich G （gelatinous）-Iayer in the walls of fiber cells and generates abnormal tensile stress in the secondary xylem. We produced transgenic poplar plants overexpressing several endoglycanases to reduce each specific polysaccharide in the cell wall, as the secondary xylem consists of primary and secondary wall layers. When placed horizontally, the basal regions of stems of transgenic poplars overexpressing xyloglucanase alone could not bend upward due to low strain in the tension side of the xylem. In the wild-type plants, xyloglucan was found in the inner surface of G-layers during multiple layering. In situ xyloglucan endotransglucosylase （XET） activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, began at the inner surface layers S1 and S2 and was retained throughout G-layer development, while the incorporation of xyloglucan heptasaccharide （XXXG） for wall loosening occurred in the primary wall of the expanding zone. We propose that the xyloglucan network is reinforced by XET to form a further connection between wall-bound and secreted xyloglucans in order to withstand the tensile stress created within the cellulose G-layer micro fibrils.

RNA-Seq Analysis of Developing Nasturtium Seeds （Tropaeolum majus）： Identification and Characterization of an Additional Galactosyltransferase Involved in Xyloglucan Biosynthesis

A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum majus） seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was iden- tified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog （AT5G62220） revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 （mur3.1） and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.

A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis

Salinity is an abiotic stress that substantially limits crop production worldwide. To identify salt stress tolerance determinants, we screened for Arabidopsis mutants that are hypersensitive to salt stress and designated these mutants as short root in salt medium （rsa）. One of these mutants, rsa3-1, is hypersensitive to NaCI and LiCI but not to CsCI or to general osmotic stress. Reactive oxygen species （ROS） over-accumulate in rsa3-1 plants under salt stress. Gene expression profiling with Affymetrix microarray analysis revealed that RSA3 controls expression of many genes including genes encoding proteins for ROS detoxification under salt stress. Map-based cloning showed that RSA3 encodes a xyloglucan galactosyltransferase, which is allelic to a gene previously named MUR3/KAM1. The RSA3/ MUR3/KAMl-encoded xylogluscan galactosyltransferase regulates actin microfilament organization （and thereby con- tributes to endomembrane distribution） and is also involved in cell wall biosynthesis. In rsa3-1, actin cannot assemble and form bundles as it does in the wild-type but instead aggregates in the cytoplasm. Furthermore, addition of phal- Ioidin, which prevents actin depolymerization, can rescue salt hypersensitivity of rsa3-1. Together, these results sug- gest that RSA3/MUR3/KAM1 along with other cell wall-associated proteins plays a critical role in salt stress tolerance by maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS.

海巴戟果实软化相关基因McXTH的克隆和表达分析

【目的】了解诺丽果实软化规律,克隆木葡聚糖内转糖苷酶/水解酶(XTH)基因,初步明确其表达模式。【方法】用果实硬度计测量后熟阶段的诺丽果实在25℃放置及乙烯利处理两种状态下的硬度变化。从诺丽果实采后0~48 h转录组数据中筛选差异表达的XTH候选基因。通过生物信息学分析,克隆目的基因全长,并利用荧光定量PCR技术分析基因表达模式,同时用便携式乙烯测量仪测定内源乙烯释放量。【结果】后熟阶段的海巴戟果实采后表皮硬度随贮藏时间增加而下降,且乙烯利处理后的软化趋势较室温自然放置更为显著。从海巴戟果实采后0~48 h转录组数据中筛选出基因XTH(DN16278g1)的表达量高、差异大。该基因具有完整的开放阅读框(ORF),克隆测序后将其命名为McXTH,并将其提交Genbank获得登录号为ON512442。McXTH基因ORF全长1062 bp,共编码353个氨基酸,与其他植物中的XTH基因氨基酸序列的相似性为75%~88%。RT-qPCR结果和内源乙烯释放量表明,在海巴戟果实成熟阶段McXTH的表达量与同时期的内源乙烯释放量在趋势上大体相同。【结论】海巴戟果实的软化受乙烯和关键基因XTH调控,Mc XTH的克隆为进一步进行XTH基因在海巴戟果实成熟软化过程的功能研究夯实基础。

XTH家族基因在植物生长发育及胁迫响应中的研究进展

木葡聚糖内转糖基酶/水解酶(xyloglucan endotransglucosylase/hydrolase, XTH)是参与植物细胞壁的重塑的关键酶之一,可以切割和重新连接木葡聚糖。许多物种中都已经鉴定出了XTH基因家族,发现其广泛参与植物生命周期各种生命活动的调节。因此,研究XTH基因家族对理解植物的生长发育有着重要的意义。在这里,我们综述了XTH家族基因在植物生长发育中的作用,主要包括参与植物根、茎、叶的生长,花器官的开放与脱落,果实的成熟与软化等;还讨论了XTHs在非生物胁迫和生物胁迫响应中的作用,为深入研究XTHs的功能和作用机制提供参考。

Pectin May Hinder the Unfolding of Xyloglucan Chains during Cell Deformation： Implications of the Mechanical Performance of Arabidopsis Hypocotyls with Pectin Alterations

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on tour1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants （Col-0） and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels （-tensile strength） of the hypocotyls of the mutants with pectin alterations （mur1, qua2） were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for tour1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

Hetero-trans-β-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglu-can or mixed-linkageβ-D-glucan(MLG).However,Equisetum hetero-trans-β-glucanase(HTG)grafts cellu-lose onto xyloglucan oligosaccharides(XGOs)-and,we now show,xyloglucan polysaccharide-in vitro,thus exhibiting CXE(cellulose:xyloglucan endotransglucosylase)activity.In addition,HTG also catalyzes MLG-to-XGO bonding(MXE activity).In this study,we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds.To quantify and visu-alize CXE and MXE action,we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics.We demonstrated covalent cellulose--xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15%and 60%of total transglucanase action,respectively,and peaked in aging,strengthening tissues:CXE in xylem and cells bordering intercellular canals and MXE in scleren-chyma.Recombinant bacterial expansin(EXLX1)strongly augmented CXE activity in vitro.CXE and MXE ac-tion in living Equisetum structural tissues potentially strengthens stems,while expansin might augment the HTG-catalyzed CXE reaction,thereby allowing efficient CXE action in muro.Our methods will enable surveys for comparable reactions throughout the plant kingdom.Furthermore,engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.

Plasticity of Xyloglucan Composition in Bean （Phaseolus vulgaris）-Cultured Cells during Habituation and Dehabituation to Lethal Concentrations of Dichlobenil

Bean cells that have been habituated to grow in a lethal concentration （12 μM） of 2,6-dichlorobenzonitrile （dichlobenil or DCB, a cellulose biosynthesis inhibitor） are known to have decreased cellulose content in their cell walls. Xyloglucan, which is bound to cellulose and together with it forms the main loading network of plant cell walls, has also been described to decrease in habituated cells, but whether the change on cellulose affects the xyloglucan structure besides its abundance has not been analyzed. Fragmentation analysis with xyloglucan-specific endoglucanase （XEG） and endocellulase revealed that habituation to DCB caused a change in the fine structure of xyloglucan, namely a decrease in fucosyl residues attached to the galactosyl-xylosyl residues along the glucan backbone. After the removal of herbicide from the medium （dehabituated cells）, xyloglucan recovered its fucosyl residues. In addition, some cello-oligosaccharides could be detected only in habituated cells＇ xyloglucan digested by XEG and endocellulase, corresponding to a glucan co- valently bound or co-precipitated with the hemicelluloses. These results show that structural flexibility of cell walls relies in part on the plasticity of xyloglucan composition and opens up new perspectives to further research in this field.

Characterization of Kiwifruit Xyloglucan

Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit （Actinidia deliciosa） throughout fruit enlargement and maturation. Most of the xyioglucan （XG） persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose Ⅱ （HC-Ⅱ） with 4.28 M KOH containing 0.02% NaBH4, and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-Ⅱ fraction to 62 mol% in the purified XG powder. The molar ratio of glucose： xylose： galactose： fucose in the purified XG was 10： 6.9： 2.1： 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa Mr determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low Mr glucomannan remained associated with this fraction.

Acceleration of Cell Growth by Xyloglucan Oligosaccharides in Suspension-Cultured Tobacco Cells

The incorporation of xyloglucan oligosaccharide （XXXG） into the walls of suspension-cultured tobacco cells accelerated cell expansion followed by cell division, changed cell shape from cylindrical to spherical, decreased cell size, and caused cell aggregation. Fluorescent XXXG added to the culture medium was found to be incorporated into the surface of the entire wall, where strong incorporation occurred not only on the surface, but also in the interface walls between cells during cell division. Cell expansion was always greater in the transverse direction than in the longitudinal direction and then, immediately, expansion led to cell division in the presence of XXXG; this process might result in the high level of cell aggregation seen in cultured tobacco cells. We concluded that the integration of this oligosaccharide into the walls could accelerate not only cell expansion, but also cell division in cultured cells.

Distribution of Fucosylated Xyloglucans among the Walls of Different Cell Types in Monocotyledons Determined by Immunofluorescence Microscopy

Xyloglucans in the non-lignified primary cell walls of different species of monocotyledons have diverse struc- tures, with widely varying proportions of oligosaccharide units that contain fucosylated side chains （F side chains）. To determine whether fucosylated xyloglucans occur in all non-lignified walls in a range of monocotyledon species, we used immunofluorescence microscopy with the monoclonal antibody CCRC-M1. The epitope of this antibody, α-L-FUCp-（1 →2）- β-D-Galp, occurs in F side chains. In most non-commelinid monocotyledons, the epitope was found in all non-lignified walls. A similar distribution was found in the palm Phoenix canariensis, which is a member of the basal commelinid order Arecales. However, in the other commelinid orders Zingiberales, Commelinales, and Poales, the occurrence of the epitope was restricted, sometimes occurring in only the phloem walls, but often also in walls of other cell types including stomatal guard and subsidiary cells and raphide idioblasts. No epitope was found in the walls of the commelinids Tradescantia virginiana （Commelinaceae, Commelinales） and Zea mays （Poaceae, Poales）, but it occurred in the phloem walls of two other Poaceae species, Lolium multiflorum and L. perenne. The distribution of the epitope is discussed in relation to xyloglucan structures in the different taxa. However, the functional significance of the restricted distributions is unknown.

Elucidating the role of SlXTH5 in tomato fruit softening

Fruit softening in tomato(Solanum lycopersicum)is closely associated with cell wall disassembly,which is brought about through the action of a range of cell wall structure-related enzymes and other proteins such as expansins.Xyloglucan endotransglucosylase/hydrolase(XTH)(EC 2.4.1.207 and/or EC 3.2.1.151)has been proposed to be key player involved in xyloglucan metabolism.SlXTH5 showed the highest expression level among all SlXTHs during tomato ripening.In this study,the role of SlXTH5 involved in tomato softening was investigated in CRISPR-based knockout mutants of SlXTH5.Loss-of-function of SlXTH5 in transgenic tomato lines resulted in slightly firmer fruit pericarp,but significantly decreased their color index compared with azygous wild type(WT)control fruits.Increased paste viscosity was detected in CRISPR mutants,indicating that the activity of SlXTH5 is responsible for maintaining cell wall structural integrity.Immunocytochemistry studies were performed using the monoclonal antibody probe LM25 to examine the localization and distribution of xyloglucan in the pericarp cells of the CRISPR mutant fruits.The data indicated more xyloglucan was retained in the pericarp of CRISPR mutant fruit than in WT control fruit.This study revealed the link between SlXTH5 and xyloglucan metabolism and indicated the potential of manipulating SlXTH5 to regulate fruit softening.

超声及酶解处理的罗望子木葡聚糖流变特性研究

研究了超声处理1.5和2.5 h和半乳糖苷酶酶解16、32和48 h对罗望子木葡聚糖(Xyloglucan,XG)流变特性的影响。使用旋转流变仪对处理前后的木葡聚糖在静态剪切和动态剪切下的黏度和模量进行了分析,并利用凝胶色谱-多角度激光光散射和动态激光光散射仪对木葡聚糖分子量分布和平均粒径进行了分析,同时使用扫描电子显微镜对木葡聚糖的微观结构进行了观察。温度扫描结果表明,在5℃时,超声和酶解处理过的木葡聚糖的黏度显著降低(P<0.05),但不同处理时间的样品差异较小。在5~60℃范围内,木葡聚糖的弹性模量与黏性模量均接近于7 Pa。超声处理的样品黏度随温度增加而降低,且总体呈现出以黏性为主的流变特性。48 h酶解处理的木葡聚糖弹性特征高于16及32 h酶解木葡聚糖的弹性特性。分子量分析结果显示,超声处理1.5和2.5 h的木葡聚糖平均分子量从原885 kDa分别降低至780和520 kDa;而酶解的木葡聚糖由低分子量(101~478 kDa)和高分子量(4591~12173 kDa)两部分组成,其中低分子量组分比例占80%以上。动态光散射结果表明,经半乳糖苷酶酶解的木葡聚糖平均粒径增大,而超声对样品的粒径影响较小。本研究为物理及生物法改性木葡聚糖,以获得具有不同流变特性的多糖胶体提供了一定经验,有利于进一步拓展其在食品工业中的应用。

罗望子种子中木葡聚糖的提取工艺优化

以罗望子种子为原料,采用酸、碱处理,结合活性炭吸附、透析的方法去除罗望子种子中木葡聚糖中的蛋白质。基于单因素试验结果,进行三因素三水平正交试验设计,研究料液比、酸处理pH值、碱处理pH值对木葡聚糖纯度的影响。结果表明,提取木葡聚糖的最优条件为料液比1∶3(g∶mL),酸处理pH值3.5,碱处理pH值12,在此条件下,获得的精制木葡聚糖的蛋白质含量为0.2%,细菌内毒素含量为150 EU/g,总糖含量为99.63%。

Analysis on Tissue-specific Expression of Dp XTH1 and Dp XTH2 Genes in Dahlia

[Objective] This study aimed to investigate the functions and related mechanisms of xyloglucan Endotransglycosylase/hydrolases （XTHs） during the growth and development of dahlia. [Method] Using /3-actin as the reference gene, the rela- tive transcription levels of DpXTH1 and DpXTH2 genes in roots, stems, leaves and petals of dahlia were analyzed by real-time RT-PCR. [Result] The DpXTH1 and DpXTH2 were not expressed in the roots, but expressed abundantly in the petals of dahlia. There were little expressions in the stems and leaves of dahlia. [Conclusion] The DpXTH1 and DpXTH2 were petal-specific genes and closely related to the growth and development of petals in dahlia.