Plant callose synthase complexes

Synthesis of callose (-1,3-glucan) in plants has been a topic of much debate over the past several decades. Callose synthase could not be purified to homogeneity and most partially purified cellulose synthase preparations yielded -1,3-glucan in vitro, leading to the interpretation that cellulose synthase might be able to synthesize callose. While a rapid progress has been made on the genes involved in cellulose synthesis in the past five years, identification of genes for callose synthases has proven difficult because cognate genes had not been identified in other organisms. An Arabidopsis gene encoding a putative cell plate-specific callose synthase catalytic subunit (CalS1) was recently cloned. CalS1 shares high sequence homology with the well-characterized yeast -1,3-glucan synthase and transgenic plant cells over-expressing CalS1 display higher callose synthase activity and accumulate more callose. The callose synthase complex exists in at least two distinct forms in different tissues and interacts with phragmoplastin, UDP-glucose transferase, Rop1 and, possibly, annexin. There are 12 CalS isozymes in Arabidopsis, and each may be tissue-specific and/or regulated under different physiological conditions responding to biotic and abiotic stresses.     

温度对棉纤维糖代谢相关酶活性的影响

以棉纤维比强度高的科棉1号和中等强度的美棉33B 2个基因型棉花品种为材料,于2005年在江苏南京(长江流域下游棉区)和徐州(黄河流域黄淮棉区)设置不同播期(4月25日和5月25日)试验,研究了不同温度下棉纤维发育过程中蔗糖酶、蔗糖合成酶、磷酸蔗糖合成酶和β-1,3-葡聚糖酶等糖代谢相关酶活性的动态变化特征及其与纤维长度和比强度形成的关系.结果表明：棉纤维伸长发育期,蔗糖酶、β-1,3-葡聚糖酶活性较高;纤维加厚发育期,蔗糖合成酶和磷酸蔗糖合成酶活性上升速度快、活性高,蔗糖酶和β-1,3-葡聚糖酶活性下降速度快.纤维伸长期,蔗糖酶活性升高对纤维的伸长具有明显促进作用;纤维加厚发育期,提高蔗糖合成酶、磷酸蔗糖合成酶活性及加快蔗糖酶和β-1,3-葡聚糖酶活性下降速度有利于提高纤维比强度.科棉1号前期蔗糖酶、β-1,3-葡聚糖酶活性及中后期蔗糖合成酶、磷酸蔗糖合成酶活性均较美棉33B高.在本试验条件下,23.3 ℃是高强纤维形成的适宜温度,23.3 ℃～25.5 ℃是纤维长度形成的适宜温度.     

An Arabidopsis callose synthase

-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic -1,4-glucan is the predominant polysaccharide in plant cell walls. Plant -1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce -1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast -1,3-glucan synthase whose expression partially complements a yeast -1,3-glucan synthase mutant. AtGsl5 is developmentally expressed at highest levels in flowers, consistent with flowers having high -1,3-glucan synthase activities for deposition of callose in pollen. A role for AtGsl5 in callose synthesis is also indicated by AtGsl5expression in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated -1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5mRNA accumulation is induced by SA in wild-type plants, while expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant.     

低温对棉纤维比强度形成的生理机制影响

通过设置播期试验使棉纤维加厚发育过程(铃龄25~50 d)处于不同的温度条件下,研究低温对棉花纤维比强度形成的内在生理机制影响,为采取调控措施解决目前棉花(Gossypium)生产中存在的晚熟劣质问题提供理论依据。两年试验结果表明:棉纤维加厚发育期24.0 ℃左右的日均温是高强纤维形成的最佳温度,其内在生理机制表现为棉纤维蔗糖合成酶活性最高,β_1,3_葡聚糖酶活性最低,纤维素的累积量和累积速率均明显高于其它低温条件,纤维超分子结构取向参数角较小,处于优化状态,最终表现为纤维比强度亦最大;低于21.0 ℃时即对棉纤维加厚发育相关酶活性产生明显影响,纤维比强度降低。当温度降到15.0 ℃左右时,棉纤维蔗糖合成酶活性显著降低,而β-1,3_葡聚糖酶活性显著升高,同时纤维素累积量和累积速率均显著降低,纤维超分子结构取向参数角明显宽化,棉纤维不能正常发育,不利于高强纤维的形成(铃重仅为3.22 g,纤维比强度仅为15.73 cN·tex^-1)。     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers

Summary.  Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick. Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers. Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A. E-mail: candace.haigler@ttu.edu     

Response of the enzymes to nitrogen applications in cotton fiber (Gossypium hirsutum L.) and their relationships with fiber strength

To investigate the response of key enzymes to nitrogen (N) rates in cotton fiber and its relationship with fiber strength, experiments were conducted in 2005 and 2006 with cotton cultivars in Nanjing. Three N rates 0, 240 and 480 kgN/hm2, signifying optimum and excessive nitrogen application levels were applied.The activities and the gene expressions of the key enzymes were affected by N, and the characteristics of cellulose accumulation and fiber strength changed as the N rate varied. Beta-1,3-glucanase activity in cotton fiber declined from 9 DPA till boll opening, and the beta-1, 3-glucanase coding gene expression also followed a unimodal curve in 12—24 DPA. In 240 kgN/hm² condition, the characteristics of enzyme activity and gene expression manner for sucrose synthase and beta-1,3-glucanase in developing cotton fiber were more favorable for forming a longer and more steady cellulose accumulation process, and for high strength fiber development.     

Sucrose Utilization via Invertase and Sucrose Synthase with Respect to Accumulation of Cellulose and Callose Synthesis in Wheat Roots under Oxygen Deficiency

The sucrose cleavage by sucrose synthase (SuSy) and neutral invertase was studied in wheat roots (Triticum aestivum L.) subjected to hypoxia or anoxia for 4 days. By in situ activity staining, increased SuSy activity was observed in the tip region and stele of root axes while the activity of invertase decreased. Cellulose content significantly increased in hypoxically treated roots. The cellulose deposition was correlated with regions of high SuSy activity, being mainly located in the pericycle and endodermis. Invertase activity was distributed along the root without clear difference between cortex and stele. Under root hypoxia, a significant increase in the structural carbohydrates, callose and especially cellulose, was shown. Increasing levels of soluble carbohydrates were partially used to synthesize cellulose for secondary wall thickening and callose to counteract the tissue injury following low-oxygen stress. Under strict anoxia, the roots were much more injured but sustained a high level of cellulose and callose while the soluble carbohydrates almost disappeared.     

南荻纤维素合成的相关生理特性

为探明南获纤维素合成相关的生理特性,研究了盆栽条件下南荻生长过程中IAA、ABA含量和纤维素合成关键酶（蔗糖合成酶、β-1,3-葡聚糖酶）活性的变化及其与纤维素含量间的关系。结果显示,在整个生育期,叶片中IAA含量、蔗糖合成酶活性和β-1,3-葡聚糖酶活性变化趋势一致,均呈先升高后降低的单峰曲线变化;叶片中ABA含量与茎秆蔗糖含量均先降低后增加再降低;茎秆纤维素、半纤维素和木质素含量均随生育进程呈现升高趋势。生育前期为调控纤维素合成的关键时期,此时ABA／IAA比值下降,蔗糖合成酶和β-1,3-葡聚糖酶活性快速上升,有利于纤维素的快速积累。     

Interaction between Cytokinesis-Related Callose and Cortical Microtubules in Dividing Cells of the Liverwort Riella helicophylla

Abstract: In juvenile walls of dividing cells of the liverwort Riella helicophylla the nitroso-derivative of photolysed Nifedipine (a calcium antagonist) stimulates the deposition of callose. This enhanced biosynthesis of β-1,3-glucan can only be observed in the cell plate, the juvenile cell walls and the walls of adjacent cells. An immunocytological analysis of this effect revealed that no cortical microtubules occurred at the sites of callose deposition. The cells of the control displayed a normal distribution of cortical microtubules at the plasma membrane as long as no callose was deposited along the corresponding walls. In a second set of experiments, inhibitors of microtubule polymerization and depolymerization (amiprophosmethyl and taxol, respectively) were used. At low concentrations, these substances also caused a significant stimulation of callose deposition in the plane of cell division. Based on these findings, we propose a regulatory model of callose and cellulose biosynthesis that depends on the binding of the cellulose/callose synthase complex to cortical microtubules that may be mediated by unknown binding protein(s).     

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (βglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml⁻¹ inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of β-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.     

<Emphasis Type="Italic">Neurospora crassa</Emphasis> FKS Protein Binds to the (1,3)β-Glucan Synthase Substrate,UDP-Glucose

The essential fungal cell-wall polymer (1,3)β-glucan is synthesized by the enzyme (1,3)β-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)β-glucan synthase. We have purified (1,3)β-glucan synthase from Neurospora crassa ∼400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[β-³²P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H⁺-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)β-glucan synthase. Received: 31 May 2002 / Accepted: 27 July 2002     

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5–GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.     

Changes in the sugar composition and molecular mass distribution of matrix polysaccharides during cotton fiber development

Cotton (Gossypium herbaceum L.) fiber development consists of a fiber elongation stage (up to 20 d post-anthesis) and a subsequent cell wall thickening stage. Cell wall analysis revealed that the extractable matrix (pectic and hemicellulosic) polysaccharides accounted for 30-50% of total sugar content in the fiber elongation stage but less than 3% in the cell wall thickening stage. By contrast, cellulose increased dramatically after the fiber elongation ceased. The amounts of extractable xyloglucans and arabinose- and galactose-containing polymers per seed increased in the early fiber elongation stage and decreased thereafter. The amounts of extractable acidic polymers and non-cellulosic beta-glucans (mainly composed of beta-1,3-glucans) increased in parallel with fiber elongation and then decreased. The molecular masses of extractable non-cellulosic beta-glucans, and arabinose- and galactose-containing polymers decreased during both fiber elongation and cell wall thickening stages. The molecular mass of extractable xyloglucans also decreased during the fiber elongation stage, but this decrease ceased during the cell wall thickening stage. Conversely, the molecular size of acidic polymers in the extractable pectic fraction increased during both stages. Thus, not only the amounts but also the molecular size of the extractable matrix polysaccharides showed substantial changes during cotton fiber development.     

Callose in Frankia-Infected Tissue of Datisca glomerata is an Artifact of Specimen Preparation

Abstract: Callose, or β-1,3-glucan, is a plant cell wall polysaccharide that occurs endogenously at distinct sites in a variety of tissues. Callose is also formed in response to stress involving cell membrane perturbation. In sections of chemically-fixed nodule tissue of the actinorhizal host, Datisca glomerata, callose was cytochemically detected within the Frankia -infected cortical cells, as an extensive network of wall material surrounding the microsymbiont, but not in uninfected cortical cells. Callose formation was completely inhibited within the infected cells when 2-deoxy-D-glucose, an inhibitor of callose formation, was included in the tissue fixative. The study concludes that callose deposition in the Datisca nodule infected zone is apparently a stress response to tissue preparation and fixation. However, the rapidity and extent of callose deposition primarily at the symbiotic interface in Frankia -infected cells suggests an unusual predisposition to biosynthesis of β-1,3-glucan in the nodule cortical cells that is related to their interaction with the microsymbiont.     

Distribution of callose synthase, cellulose synthase, and sucrose synthase in tobacco pollen tube is controlled in dissimilar ways by actin filaments and microtubules

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules.     

Cotton fiber annexins: a potential role in the regulation of callose synthase

Cotton fibers contain a characteristic set of proteins which interact with plasma membranes in a Ca²⁺-dependent manner. The association of these proteins with the membrane is correlated with a reduced level of UDP-glucose: (1→3)-β-glucan (callose) synthase activity. Analysis of the proteins released from membranes by EDTA treatment shows that the most abundant proteins comprise a family of at least three polypeptides (p34) which resemble annexins. This resemblance includes similarity in size (about 34 kDa), sequence homology, Ca²⁺-dependent precipitation or interaction with the plasma membrane, and ability to serve as a substrate for phosphorylation by endogenous protein kinase(s) which also bind to the membranes in a Ca²⁺-dependent manner. A purified fraction of these annexins binds to, and inhibits, the activity of a partially purified cotton fiber callose synthase. These findings suggest that one possible function of annexin(s) in plants is to modulate the activity and/or localization of callose synthase.     

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG uridinediphosphoglucose - GDPG guanosinediphosphoglucose - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - DMSO dimethyl-sulfoxide - DNP 2,4-dinitrophenol - DPA days post anthesis     

Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 μm, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for β-1,4-glucan and β-1,3-glucan.