Differing structures of galactoglucomannan in eudicots and non-eudicot angiosperms.

The structures of cell wall mannan hemicelluloses have changed during plant evolution. Recently, a new structure called ß-galactoglucomannan (ß-GGM) was discovered in eudicot plants. This galactoglucomannan has ß-(1,2)-Gal-α-(1,6)-Gal disaccharide branches on some mannosyl residues of the strictly alternating Glc-Man backbone. Studies in Arabidopsis revealed ß-GGM is related in structure, biosynthesis and function to xyloglucan. However, when and how plants acquired ß-GGM remains elusive. Here, we studied mannan structures in many sister groups of eudicots. All glucomannan structures were distinct from ß-GGM. In addition, we searched for candidate mannan ß-galactosyltransferases (MBGT) in non-eudicot angiosperms. Candidate AtMBGT1 orthologues from rice (OsGT47A-VII) and Amborella (AtrGT47A-VII) did not show MBGT activity in vivo. However, the AtMBGT1 orthologue from rice showed MUR3-like xyloglucan galactosyltransferase activity in complementation analysis using Arabidopsis. Further, reverse genetic analysis revealed that the enzyme (OsGT47A-VII) contributes to proper root growth in rice. Together, gene duplication and diversification of GT47A-VII in eudicot evolution may have been involved in the acquisition of mannan ß-galactosyltransferase activity. Our results indicate that ß-GGM is likely to be a eudicot-specific mannan.

Plasmodesmata callose binding protein 2 contributes to the regulation of cambium/phloem formation and auxin response during the tissue reunion process in incised Arabidopsis stem.

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1-3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response.

Maintenance of Methyl-Esterified Pectin Level in Pollen Mother-Cell Stages Is Required for Microspore Development.

Pectin modification and degradation are vital for plant development, although the underlying mechanisms are still not well understood. Furthermore, reports on the function of pectin in early pollen development are limited. We generated OsPME-FOX rice lines with little methyl-esterified pectin even in the early-pollen mother-cell stage due to overexpression of the gene encoding pectin-methylesterase. Overexpression of OsPME1 in rice increased the activity of PME, which decreased the degree of pectin methyl esterification in the cell wall. OsPME1-FOX grew normally and showed abnormal phenotypes in anther and pollen development, especially in terms of the pollen mother-cell stage. In addition, we examined modifications of cell-wall polysaccharides at the cellular level using antibodies against polysaccharides. Immunohistochemical staining using LM19 and LM20 showed that methyl-esterified pectin distribution and the pectin contents in pollen mother-cell wall decreased in OsPME1-FOX compared with the wild type. Thus, the maintenance of methyl-esterified pectin plays a role in degrading and maintaining the pollen mother-cell wall during microspore development.

Effects of polygalacturonase overexpression on pectin distribution in the elongation zones of roots under aluminium stress.

The roots of many plant species contain large amounts of pectin and it contributes to the formation of the rhizosphere. In the present study, the relationship between the root-tip pectin content and aluminium (Al) tolerance in wild-type (WT) and demethylesterified pectin degradation enzyme gene overexpressor (OsPG2-FOX) rice lines was compared. OsPG2-FOX rice showed reduced pectin content in roots, even under control conditions; Al treatment reduced root elongation and the pectin content in the root elongation zone. Wild-type rice showed more pectin accumulation in the root elongation zone after Al treatment. Relative to WT rice, OsPG2-FOX rice showed more Al accumulation in the root elongation zone. These results indicate that the amount of pectin influences Al tolerance and that the distribution of pectin in the root elongation zone inhibits Al accumulation in rice roots. Pectin accumulation in cell walls in the root elongation zone may play a role in protecting rice plants from the Al-induced inhibition of root elongation by regulating pectin distribution.

Virtual issue: cell wall functions in plant growth and environmental responses.

Plant cell walls have multiple functions, including determining cell shape and size, cell-cell adhesion, controlling cell differentiation and growth, and promoting abiotic and biotic stress tolerance. This virtual issue introduces the physiological functions of cell walls in growth and environmental responses. The articles detail research on (1) embryogenesis and seed development, (2) vegetative growth, (3) reproductive growth, and (4) environmental responses. These articles, published in the Journal of Plant Research, will provide valuable information for future research on the function and dynamics of cell walls at various growth stages, and in response to environmental factors.

Rice Putative Pectin Methyltransferase Gene OsPMT10 Is Required for Maintaining the Cell Wall Properties of Pistil Transmitting Tissues via Pectin Modification.

Precise directional control of pollen tube growth via mechanical guidance by pistil tissue is critical for the successful fertilization of flowering plants and requires active cell-to-cell communication and maintenance of softness in the transmitting tissue. However, the regulation of transmitting tissue softness as controlled by cell wall properties, especially pectin, has not been reported. Here we report that regulation of pectin methylesterification supports pollen elongation through pistil transmitting tissues in Oryza sativa. The rice pectin methylesterase gene OsPMT10 was strongly expressed in reproductive tissues, especially the pistil. The ospmt10 mutant did not have a significant effect on vegetative growth, but the fertility rate was reduced by approximately half. In the ospmt10 mutant, pollen tube elongation was observed in the transmitting tissue of the style, but approximately half of the pollen tubes did not extend all the way to the ovule. Tissue cross-sections of the upper ovary were prepared, and immunohistochemical staining using LM19 and LM20 showed that methylesterified pectin distribution was decreased in ospmt10 compared with the wild type. The decreased expression of methylesterified pectins in ospmt10 may have resulted in loss of fluidity in the apoplast space of the transmitting tissue, rendering it difficult for the pollen tube to elongate in the transmitting tissue and thereby preventing it from reaching the ovule.

Structural Alteration of Rice Pectin Affects Cell Wall Mechanical Strength and Pathogenicity of the Rice Blast Fungus Under Weak Light Conditions.

Pectin, a component of the plant cell wall, is involved in cell adhesion and environmental adaptations. We generated OsPG-FOX rice lines with little pectin due to overexpression of the gene encoding a pectin-degrading enzyme [polygalacturonase (PG)]. Overexpression of OsPG2 in rice under weak light conditions increased the activity of PG, which increased the degradation of pectin in the cell wall, thereby reducing adhesion. Under weak light conditions, the overexpression of OsPG decreased the pectin content and cell adhesion, resulting in abnormally large intercellular gaps and facilitating invasion by the rice blast fungus. OsPG2-FOX plants had weaker mechanical properties and greater sensitivity to biotic stresses than wild-type (WT) plants. However, the expression levels of disease resistance genes in non-infected leaves of OsPG2-FOX were more than twice as high as those of the WT and the intensity of disease symptoms was reduced, compared with the WT. Under normal light conditions, overexpression of OsPG2 decreased the pectin content, but did not affect cell adhesion and sensitivity to biotic stresses. Therefore, PG plays a role in regulating intercellular adhesion and the response to biotic stresses in rice.

Cell wall Glycine-rich Protein2 is involved in tapetal differentiation and pollen maturation.

The tapetum plays important roles in anther development by providing materials for pollen-wall formation and nutrients for pollen development. Here, we report the characterization of a male-sterile mutant of glycine-rich protein 2 (OsGRP2), which exhibits irregular cell division and dysfunction of the tapetum. GRP is a cellwall structural protein present in the cell walls of diverse plant species, but its function is unclear in pollen development. We found that few GRP genes are expressed in rice and thus focused on one highly expressed gene, OsGRP2. The tapetal cell walls of an OsGRP2 mutant did not thicken at the pollen mothercell stage, as a result, pollen maturation and fertility rate decreased. High OsGRP2 expression was detected in male-floral organs, and OsGRP2 was distributed in the tapetum. OsGRP2 participated in establishment of the cellwall network during early tapetum development. In conclusion, our results indicate that OsGRP2 plays important roles in the differentiation and function of the tapetum.

Rice Putative Methyltransferase Gene OsPMT16 Is Required for Pistil Development Involving Pectin Modification.

Pectin synthesis and modification are vital for plant development, although the underlying mechanisms are still not well understood. Furthermore, reports on the function of pectin in the pistil are limited. Herein, we report the functional characterization of the OsPMT16 gene, which encodes a putative pectin methyltransferase (PMT) in rice. The cell walls of rice leaves contain less pectin, and chemical analysis of pectin in the flower organ had not been previously performed. Therefore, in the present study, the amount of pectin in the reproductive tissues of rice was investigated. Of the reproductive tissues, the pistil was especially rich in pectin; thus, we focused on the pistil. OsPMT16 expression was confirmed in the pistil, and effects of pectin methylesterification regulation on the reproductive stage were investigated by studying the phenotype of the T-DNA insertion mutant. The ospmt16 mutant showed significantly reduced fertility. When the flowers were observed, tissue morphogenesis was abnormal in the pistil. Immunofluorescence staining by pectin-specific monoclonal antibodies of the pistil revealed that total pectin and esterified pectin were decreased among ospmt16 mutants. These results indicate that OsPMT16 contributes significantly to pistil development during reproductive growth.

Changes in the Distribution of Pectin in Root Border Cells Under Aluminum Stress.

Root border cells (RBCs) surround the root apices in most plant species and are involved in the production of root exudates. We tested the relationship between pectin content in root tips and aluminum (Al) tolerance by comparing these parameters in wild-type (WT) and sensitive-to-Al-rhizotoxicity (star1) mutant rice plants. Staining for demethylesterified pectin decreased after Al treatment in the WT. A high level of pectin was observed in RBCs of the root tips. The level of total pectin was increased by about 50% compared with the control. In the Al-sensitive star1 mutant, Al treatment decreased root elongation and pectin content, especially in RBCs. In addition, almost no Al accumulation was observed in the control, whereas more Al was accumulated in the RBCs of STAR1 roots. These results show that the amount of pectin influences Al tolerance; that Al accumulation in rice roots is reduced by the distribution of pectin in root-tip RBCs; and that these reactions occur in the field around the RBCs, including the surrounding mucilage. Al accumulation in rice roots is reduced by the distribution of pectin in root tips, and pectin in the root cell walls contributes to the acquisition of Al tolerance in rice by regulating its distribution. The release of Al-binding mucilage by RBCs could play a role in protecting root tips from Al-induced cellular damage.

UDP-arabinopyranose mutase gene expressions are required for the biosynthesis of the arabinose side chain of both pectin and arabinoxyloglucan, and normal leaf expansion in Nicotiana tabacum.

Plant cell walls are composed of polysaccharides such as cellulose, hemicelluloses, and pectins, whose location and function differ depending on plant type. Arabinose is a constituent of many different cell wall components, including pectic rhamnogalacturonan I (RG-I) and II (RG-II), glucuronoarabinoxylans (GAX), and arabinoxyloglucan (AXG). Arabinose is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. The UDP-arabinopyranose mutases (UAMs) have been demonstrated to convert UDP-arabinopyranose (UDP-Arap) to UDP-arabinofuranose (UDP-Araf) in rice (Oryza sativa L.). The UAMs have been implicated in polysaccharide biosynthesis and developmental processes. Arabinose residues could be a component of many polysaccharides, including branched (1→5)-α-arabinans, arabinogalactans in pectic polysaccharides, and arabinoxyloglucans, which are abundant in the cell walls of solanaceous plants. Therefore, to elucidate the role of UAMs and arabinan side chains, we analyzed the UAM RNA interference transformants in tobacco (Nicotiana tabacum L.). The tobacco UAM gene family consists of four members. We generated RNAi transformants (NtUAM-KD) to down-regulate all four of the UAM members. The NtUAM-KD showed abnormal leaf development in the form of a callus-like structure and many holes in the leaf epidermis. A clear reduction in the pectic arabinan content was observed in the tissue of the NtUAM-KD leaf. The arabinose/xylose ratio in the xyloglucan-rich cell wall fraction was drastically reduced in NtUAM-KD. These results suggest that UAMs are required for Ara side chain biosynthesis in both RG-I and AXG in Solanaceae plants, and that arabinan-mediated cell wall networks might be important for normal leaf expansion.

Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

The AMOR Arabinogalactan Sugar Chain Induces Pollen-Tube Competency to Respond to Ovular Guidance.

Precise directional control of pollen-tube growth by pistil tissue is critical for successful fertilization of flowering plants [1-3]. Ovular attractant peptides, which are secreted from two synergid cells on the side of the egg cell, have been identified [4-6]. Emerging evidence suggests that the ovular directional cue is not sufficient for successful guidance but that competency control by the pistil is critical for the response of pollen tubes to the attraction signal [1, 3, 7]. However, the female molecule for this competency induction has not been reported. Here we report that ovular methyl-glucuronosyl arabinogalactan (AMOR) induces competency of the pollen tube to respond to ovular attractant LURE peptides in Torenia fournieri. We developed a method for assaying the response capability of a pollen tube by micromanipulating an ovule. Using this method, we showed that pollen tubes growing through a cut style acquired a response capability in the medium by receiving a sufficient amount of a factor derived from mature ovules of Torenia. This factor, named AMOR, was identified as an arabinogalactan polysaccharide, the terminal 4-O-methyl-glucuronosyl residue of which was necessary for its activity. Moreover, a chemically synthesized disaccharide, the ß isomer of methyl-glucuronosyl galactose (4-Me-GlcA-ß-(1→6)-Gal), showed AMOR activity. No specific sugar-chain structure of plant extracellular matrix has been identified as a bioactive molecule involved in intercellular communication. We suggest that the AMOR sugar chain in the ovary renders the pollen tube competent to the chemotropic response prior to final guidance by LURE peptides.

Distribution of XTH, expansin, and secondary-wall-related CesA in floral and fruit abscission zones during fruit development in tomato (Solanum lycopersicum).

After fruit development is triggered by pollination, the abscission zone (AZ) in the fruit pedicel strengthens its adhesion to keep the fruit attached. We previously reported that xyloglucan and arabinan accumulation in the AZ accompanies the shedding of unpollinated flowers. After the fruit has developed and is fully ripened, shedding occurs easily in the AZ due to lignin accumulation. Regulation of cell wall metabolism may play an important role in these processes, but it is not well understood. In the present report, we used immunohistochemistry to visualize changes in the distributions of xyloglucan and arabinan metabolism-related enzymes in the AZs of pollinated and unpollinated flowers, and in ripened fruits. During floral abscission, we observed a gradual increase in polyclonal antibody labeling of expansin in the AZ. The intensities of LM6 and LM15 labeling of arabinan and xyloglucan, respectively, also increased. However, during floral abscission, we observed a large 1 day post anthesis (DPA) peak in the polyclonal antibody labeling of XTH in the AZ, which then decreased. These results suggest that expansin and XTH play important, but different roles in the floral abscission process. During fruit abscission, unlike during floral abscission, no AZ-specific expansin and XTH were observed. Although lignification was seen in the AZ of over-ripe fruit pedicels, secondary cell wall-specific cellulose synthase signals were not observed. This suggests that cellulose metabolism-related enzymes do not play important roles in the AZ prior to fruit abscission.

Effects of tooth root contact on the stability of orthodontic anchor screws in the maxilla: Comparison between self-drilling and self-tapping methods.

INTRODUCTION: We evaluated the effects of screw placement angle on the frequency of root contact and the effects of root contact on screw stability, comparing self-drilling and self-tapping methods. METHODS: In total, 80 patients with 142 screws (diameter, 1.6 mm; length, 8.0 mm) were included. Cone-beam computed tomography images were taken. Cortical bone thickness, interroot distance, shortest distance between the screw and adjacent tooth root, and screw placement angle were measured. RESULTS: The success rates of the screws were 91.5% for the self-drilling method and 94.4% for the self-tapping method (P >0.05). The self-drilling screws tended to contact the distal tooth roots in the right maxilla. In the self-drilling method, the failure rate was significantly higher in the root contact group than in the no-contact group (P <0.05). CONCLUSIONS: The success rate was not significantly different between the self-drilling and the self-tapping methods in the maxilla. Avoidance of tooth root contact may improve the success rate more in the self-drilling method than in the self-tapping method.

The matrix polysaccharide (1;3,1;4)-ß-D-glucan is involved in silicon-dependent strengthening of rice cell wall.

Poales [represented by rice (Oryza sativa L.)] in angiosperms and Equisetum (horsetails) in Pteridophytes are two major groups of heavy silicon (Si) accumulators. In rice, Si is polymerized preferentially in the epidermal cell wall, forming Si-cuticle double layers and Si-cellulose double layers beneath the cuticle. This Si layer is thought to exert various beneficial effects on the growth and development of land plants. Although the recent discovery of the influx and efflux transporters of silicic acid has shed some light on the molecular mechanisms of Si uptake and transport in rice, the mechanism underlying the final incorporation of polymerized Si into the cell wall remains elusive. Despite their phylogenetic distance, the cell walls of the two Si accumulators, Poales and Equisetum, share another common component, i.e. (1;3,1;4)-ß-D-glucan, also known as mixed-linkage glucan (MLG), a matrix polysaccharide not found in other plants. Based on this coincidence, a possible correlation between the functions of Si and MLG in the cell wall has been suggested, but no experimental evidence has been obtained in support of this functional correlation. Here, we present an analysis of the correlative action of Si and MLG on the mechanical properties of leaf blades using a transgenic rice line in which the MLG level was reduced by overexpressing EGL1, which encodes (1;3,1;4)-ß-D-glucanase. The reduction in MLG did not affect total Si accumulation, but it significantly altered the Si distribution profile and reduced the Si-dependent mechanical properties of the leaf blades, strongly suggesting a functional correlation between Si and MLG.

UDP-arabinopyranose mutase 3 is required for pollen wall morphogenesis in rice (Oryza sativa).

l-Arabinose is one of the main constituents of cell wall polysaccharides such as pectic rhamnogalacturonan I (RG-I), glucuronoarabinoxylans and other glycoproteins. It is found predominantly in the furanose form rather than in the thermodynamically more stable pyranose form. UDP-L-arabinofuranose (UDP-Araf), rather than UDP-L-arabinopyranose (UDP-Arap), is a sugar donor for the biosynthesis of arabinofuranosyl (Araf) residues. UDP-arabinopyranose mutases (UAMs) have been shown to interconvert UDP-Araf and UDP-Arap and are involved in the biosynthesis of polysaccharides including Araf. The UAM gene family has three members in Oryza sativa. Co-expression network in silico analysis showed that OsUAM3 expression was independent from OsUAM1 and OsUAM2 co-expression networks. OsUAM1 and OsUAM2 were expressed ubiquitously throughout plant development, but OsUAM3 was expressed primarily in reproductive tissue, particularly at the pollen cell wall formation developmental stage. OsUAM3 co-expression networks include pectin catabolic enzymes. To determine the function of OsUAMs in reproductive tissues, we analyzed RNA interference (RNAi)-knockdown transformants (OsUAM3-KD) specific for OsUAM3. OsUAM3-KD plants grew normally and showed abnormal phenotypes in reproductive tissues, especially in terms of the pollen cell wall and exine. In addition, we examined modifications of cell wall polysaccharides at the cellular level using antibodies against polysaccharides including Araf. Immunolocalization of arabinan using the LM6 antibody showed low levels of arabinan in OsUAM3-KD pollen grains. Our results suggest that the function of OsUAM3 is important for synthesis of arabinan side chains of RG-I and is required for reproductive developmental processes, especially the formation of the cell wall in pollen.

XTH20 and XTH19 regulated by ANAC071 under auxin flow are involved in cell proliferation in incised Arabidopsis inflorescence stems.

One week after partial incision of Arabidopsis inflorescence stems, the repair process in damaged tissue includes pith cell proliferation. Auxin is a key factor driving this process, and ANAC071, a transcription factor gene, is upregulated in the distal region of the incised stem. Here we show that XTH20 and the closely related XTH19, members of xyloglucan endotransglucosylase/hydrolases family catalyzing molecular grafting and/or hydrolysis of cell wall xyloglucans, were also upregulated in the distal part of the incised stem, similar to ANAC071. XTH19 was expressed in the proximal incision region after 3 days or after auxin application to the decapitated stem. Horizontal positioning of the plant with the incised side up resulted in decreased ProDR 5 :GUS, ANAC071, XTH20, and XTH19 expression and reduced pith cell proliferation. In incised stems of Pro35S :ANAC071-SRDX plants, expression of XTH20 and XTH19 was substantially and moderately decreased, respectively. XTH20 and XTH19 expression and pith cell proliferation were suppressed in anac071 plants and were increased in Pro35S :ANAC071 plants. Pith cell proliferation was also inhibited in the xth20xth19 double mutant. Furthermore, ANAC071 bound to the XTH20 and XTH19 promoters to induce their expression. This study revealed XTH20 and XTH19 induction by auxin via ANAC071 in the distal part of an incised stem and their involvement in cell proliferation in the tissue reunion process.

Regulatory specialization of xyloglucan (XG) and glucuronoarabinoxylan (GAX) in pericarp cell walls during fruit ripening in tomato (Solanum lycopersicum).

Disassembly of cell wall polysaccharides by various cell wall hydrolases during fruit softening causes structural changes in hemicellulose and pectin that affect the physical properties and softening of tomato fruit. In a previous study, we showed that the changes in pectin during tomato fruit ripening were unique in each fruit tissue. In this study, to clarify the changes in hemicellulose in tissues during tomato fruit ripening, we focused on glucuronoarabinoxylan (GAX) and xyloglucan (XG). GAX was detected only in the skin and inner epidermis of the pericarp using LM11 antibodies, whereas a large increase in XG was detected in all fruit tissues using LM15 antibodies. The activity of hemicellulose degradation enzymes, such as ß-xylosidase and α-arabinofuranosidase, decreased gradually during fruit ripening, although the tomato fruits continued to soften. In contrast, GAX and XG biosynthesis-related genes were expressed in all tomato fruit tissues even during ripening, indicating that XG was synthesized throughout the fruit and that GAX may be synthesized only in the vascular bundles and the inner epidermis. Our results suggest that changes in the cell wall architecture and tissue-specific distribution of XG and GAX might be required for the regulation of fruit softening and the maintenance of fruit shape.