Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall

It has not yet been reported how the secondary CESA (cellulose synthase) proteins are organized in the rosette structure. A membrane-based yeast two-hybrid (MbYTH) approach was used to analyze the interactions between the CESA proteins involved in secondary cell wall synthesis of Arabidopsis and the findings were confirmed in planta by bimolecular fluorescence complementation (BiFC) assay. Results indicated that although all CESA proteins can interact with each other, only CESA4 is able to form homodimers. A model is proposed for the secondary rosette structure. The RING-motif proved not to be essential for the interaction between the CESA proteins.

Structured summary

MINT-6951243: PIP2-1 (uniprotkb:P43286) physically interacts (MI:0218) with PIP2-1 (uniprotkb:P43286) by bimolecular fluorescence complementation (MI:0809)MINT-6950816: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) withCESA4 (uniprotkb:Q84JA6) by membrane bound complementation assay (MI:0230)MINT-6951056, MINT-6951071, MINT-6951088, MINT-6951103: CESA7 (uniprotkb:Q9SWW6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6950949, MINT-6950990: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by membrane bound complementation assay (MI:0230)MINT-6950909, MINT-6951030: CESA4 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951042: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6951004, MINT-6951016: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951217, MINT-6951230: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)MINT-6951120, MINT-6951140, MINT-6951156, MINT-6951170, MINT-6951185: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA7 (uniprotkb:Q9SWW6) by bimolecular fluorescence complementation (MI:0809)MINT-6951199: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)     

The cellulose/lignin assembly assessed by molecular modeling. Part 2: Seeking for evidence of organization of lignin molecules at the interface with cellulose.

We have extended our previous computational investigation of the cellulose lignin assembly by considering more complex systems. Surface coverage of cellulose, structural parameters such as molecular mass and structural features of the lignin models and the presence of an explicit hydrated environment have been taken into account to examine their influence on the associative interactions between cellulose and lignin. To this end, different lignin molecular models, from beta-O-4 dimers up to a 20-units oligomer, were considered. Independently of the system studied, the key feature of the adsorption is globally preserved: aromatic rings of lignin adopt a preferential parallel orientation relative to the cellulose surface. Such structural order appears to be limited to the first shell of lignin units adsorbed on the cellulose. The pre-organization of the lignin monolayer at the surface of cellulose is not significantly changed at the interface with water. However, adsorption significantly depends on the molecular mass and the structure of lignin. The structural order is significantly hindered by the presence of branching or some particular inter-units linkages in the structure of lignin. Such results rationalize the apparent contradiction between the available experimental results.     

Regulation of secondary cell wall development by cortical microtubules during tracheary element differentiation in Arabidopsis cell suspensions

Cortical microtubules participate in the deposition of patterned secondary walls in tracheary element differentiation. In this study, we established a system to induce the differentiation of tracheary elements using a transgenic Arabidopsis (Arabidopsis thaliana) cell suspension stably expressing a green fluorescent protein-tubulin fusion protein. Approximately 30% of the cells differentiated into tracheary elements 96 h after culture in auxin-free media containing 1 mum brassinolide. With this differentiation system, we have been able to time-sequentially elucidate microtubule arrangement during secondary wall thickening. The development of secondary walls could be followed in living cells by staining with fluorescein-conjugated wheat germ agglutinin, and the three-dimensional structures of the secondary walls could be simultaneously analyzed. A single microtubule bundle first appeared beneath the narrow secondary wall and then developed into two separate bundles locating along both sides of the developing secondary wall. Microtubule inhibitors affected secondary wall thickening, suggesting that the pair of microtubule bundles adjacent to the secondary wall played a crucial role in the regulation of secondary wall development.     

Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall

Several brittle culm mutations of rice (Oryza sativa) causing fragility of plant tissues have been identified genetically but not characterized at a molecular level. We show here that the genes responsible for three distinct brittle mutations of rice, induced by the insertion of the endogenous retrotransposon Tos17, correspond to CesA (cellulose synthase catalytic subunit) genes, OsCesA4, OsCesA7 and OsCesA9. Three CesA genes were expressed in seedlings, culms, premature panicles, and roots but not in mature leaves, and the expression profiles were almost identical among the three genes. Cellulose contents were dramatically decreased (8.9%-25.5% of the wild-type level) in the culms of null mutants of the three genes, indicating that these genes are not functionally redundant. Consistent with these results, cell walls in the cortical fiber cells were shown to be thinner in all the mutants than in wild-type plants. Based on these observations, the structure of a cellulose-synthesizing complex involved in the synthesis of the secondary cell wall is discussed.     

DIMINUTO 1 affects the lignin profile and secondary cell wall formation in Arabidopsis

Brassinosteroids (BRs) play a crucial role in plant growth and development and DIMINUTO 1 (DIM1), a protein involved in BR biosynthesis, was previously identified as a cell elongation factor in Arabidopsis thaliana. Through promoter expression analysis, we showed that DIM1 was expressed in most of the tissue types in seedlings and sectioning of the inflorescence stem revealed that DIM1 predominantly localizes to the xylem vessels and in the interfascicular cambium. To investigate the role of DIM1 in cell wall formation, we generated loss-of-function and gain-of-function mutants. Disruption of the gene function caused a dwarf phenotype with up to 38 and 23% reductions in total lignin and cellulose, respectively. Metabolite analysis revealed a significant reduction in the levels of fructose, glucose and sucrose in the loss-of-function mutant compared to the wild type control. The loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type, but no changes were detected in the gain-of-function mutant. Phloroglucinol and toluidine blue staining showed a size reduction of the vascular apparatus with smaller and disintegrated xylem vessels in the inflorescence stem of the loss-of-function mutant. Taken together, these data indicate a role for DIM1 in secondary cell wall formation. Moreover, this study demonstrated the potential role of BR hormones in modulating cell wall structure and composition.     

Mechanism for formation of the secondary wall thickening in tracheary elements: Microtubules and microfibrils of tracheary elements of Pisum sativum L. and Commelina communis L. and the effects of amiprophosmethyl

Arrangements of microfibrils (MFs) and microtubules (MTs) were examined in tracheary elements (TEs) of Pisum sativum L. and Commelina communis L. by production of replicas of cryo-sections, and by immunofluorescence microscopy, respectively. The secondary wall thickenings of TEs of Pisum and Commelina roots have pitted and latticed patterns, respectively. Most MFs in the pitted thickening of Pisum TEs retain a parallel alignment as they pass around the periphery of pits. However, some groups of MFs grow into the pits but then terminate at the edge of the thickening, indicating that cellulose-synthase complexes are inactivated in the plasma membrane under the pit. Microtubules of TEs of both Pisum and Commelina are localized under the secondary thickening and few MTs are detected in the areas between wall thickenings. In the presence of the MT-disrupting agent, amiprophosmethyl, cellulose and hemicellulose, which is specific to secondary thickening, are deposited in deformed patterns in TEs of Pisum roots, Pisum epicotyls and Commelina roots. This indicates that the localized deposition of hemicellulose as well as cellulose involves MTs. The deformed, but heterogeneous pattern of secondary thickening is still visible, indicating that MTs are involved in determining and maintaining the regular patterns of the secondary thickening but not the spatial heterogeneous pattern of the wall deposition. A working hypothesis for the formation of the secondary thickening is proposed.Abbreviations APM amiprophosmethyl - DMSO dimethyl sulfoxide - F-WGA fluorescein-conjugated wheat-germ agglutinin - M F microfibril - MT microtubule - PEG polyethyleneglycol - TE tracheary element I thank Ms. Aiko Hirata (Institute of Applied Microbiology, University of Tokyo, Japan) for help in taking stereomicrographs. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.     

Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins

Summary Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.Abbreviations BSL-II Bandeiraea simplicifolia lectin II - DSL Datura stramonium lectin - F fluorescein-conjugated - LEL Lycopersicon esculentum lectin - MT microtubule - STL Solanum tuberosum lectin - TE tracheary element - WGA wheat-germ agglutinin     

Whole-mount silver staining of Arabidopsis and maize: a new method for staining secondary wall thickenings in tracheary elements

Secondary wall thickenings in tracheary elements were specifically stained by incubation of Arabidopsis and maize in Silver Stain Plus (Bio-Rad) staining solution, after pretreatment with SDS and ethanol solution. Scanning electron microscopic analysis of sections of celery revealed that silver particles were deposited on the secondary wall thickenings, indicating that the staining was due to the deposition of silver through the interaction of the stain with lignin. This method is more sensitive than the acidified phloroglucinol method.     

Habituation to thaxtomin A in hybrid poplar cell suspensions provides enhanced and durable resistance to inhibitors of cellulose synthesis

Background  

Thaxtomin A (TA), a phytotoxin produced by the phytopathogen Streptomyces scabies, is essential for the development of potato common scab disease. TA inhibits cellulose synthesis but its actual mode of action is unknown. Addition of TA to hybrid poplar (Populus trichocarpa x Populus deltoides) cell suspensions can activate a cellular program leading to cell death. In contrast, it is possible to habituate hybrid poplar cell cultures to grow in the presence of TA levels that would normally induce cell death. The purpose of this study is to characterize TA-habituated cells and the mechanisms that may be involved in enhancing resistance to TA.     

Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by α-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.     

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors.

Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells. A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins [PBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes characterize normal cells. Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymes was biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs.     

The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.

The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.     

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements

Summary. The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.Correspondence and reprints: Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A.Present address: Biology Editors Co., Peacedale, Rhode Island, U.S.A.Present address: Department of Biology and Marine Biology, Roger Williams University, Bristol, Rhode Island, U.S.A.Present address: Department of Crop Science and Department of Botany, North Carolina State University, Raleigh, North Carolina, U.S.A.     

Vitrification of carnation in vitro: Changes in cell wall mechanical properties,cellulose and lignin content

Vitrification of internodes of carnation was brought about by culturing in liquid medium. Cell wall extensibility of these internodes was kinetically followed in comparison to that of normal plants using the constant stress method. Liquid culture induced increased immediate and total deformation capacities of the walls from the second day. Measurements indicated that these deformation capacities involved plastic properties rather than elastic ones. These changes were paralleled by decreased relative levels of cellulose and lignin.     

Zinnia elegans uses the same peroxidase isoenzyme complement for cell wall lignification in both single-cell tracheary elements and xylem vessels

The nature of the peroxidase isoenzyme complement responsible for cell wall lignification in both Zinnia elegans seedlings and Z. elegans tracheary single-cell cultures have been studied. Results showed that both hypocotyls and stems from lignifying Z. elegans seedlings express a cell wall-located basic peroxidase of pI approximately 10.2, which was purified to homogeneity. Molecular mass determination under non-denaturing conditions showed an M(r) of about 43 000, similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496-501 and 632-635 (alpha and beta absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group being ferric protoporphyrin IX. The N-terminal amino acid sequence of this Z. elegans basic peroxidase was KVAVSPLS (peptide motif in bold), which shows strong homologies with the N-amino acid terminus of other strongly basic plant peroxidases. Isoenzyme and western blot analyses showed that this peroxidase isoenzyme is also expressed in trans-differentiating Z. elegans tracheary single-cell cultures. The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. From these results, it may be concluded that Z. elegans uses a single programme, i.e. an identical peroxidase isoenzyme complement, for lignification of the xylem, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels).     

Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.     

A receptor-like kinase controls the amplitude of secondary cell wall synthesis in rice

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Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall.

Recessive mutations at three loci cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis. These irregular xylem (irx) mutations were identified by screening plants from a mutagenized population by microscopic examination of stem sections. The xylem cell defect was associated with an up to eightfold reduction in the total amount of cellulose in mature inflorescence stems. The amounts of cell wall-associated phenolics and polysaccharides were unaffected by the mutations. Examination of the cell walls by using electron microscopy demonstrated that the decreases in cellulose content of irx lines resulted in an alteration of the spatial organization of cell wall material. This suggests that a normal pattern of cellulose deposition may be required for assembly of lignin or polysaccharides. The reduced cellulose content of the stems also resulted in a decrease in stiffness of the stem material. This is consistent with the irregular xylem phenotype and suggests that the walls of irx plants are not resistant to compressive forces. Because lignin was implicated previously as a major factor in resistance to compressive forces, these results suggest either that cellulose has a direct role in providing resistance to compressive forces or that it is required for the development of normal lignin structure. The irx plants had a slight reduction in growth rate and stature but were otherwise normal in appearance. The mutations should be useful in facilitating the identification of factors that control the synthesis and deposition of cellulose and other cell wall components.     

Effect of silicon deficiency on secondary cell wall synthesis in rice leaf

Rice (Oryza sativa L.) is a typical Si-accumulating plant and is able to accumulate Si up to >10?% of shoot dry weight. The cell wall has been reported to become thicker under Si-deficient condition. To clarify the relationship between Si accumulation and cell wall components, the physical properties of, and macromolecular components and Si content in, the pectic, hemicellulosic, and cellulosic fractions prepared from rice seedlings grown in hydroponics with or without 1.5?mM silicic acid were analyzed. In the absence of Si (the ?Si condition), leaf blades drooped, but physical properties were enhanced. Sugar content in the cellulosic fraction and lignin content in the total cell wall increased under ?Si condition. After histochemical staining, there was an increase in cellulose deposition in short cells and the cell layer just beneath the epidermis in the ?Si condition, but no significant change in the pattern of lignin deposition. Expression of the genes involved in secondary cell wall synthesis, OsCesA4, OsCesA7, OsPAL, OsCCR1 and OsCAD6 was up-regulated under ?Si condition, but expression of OsCesA1, involved in primary cell wall synthesis, did not increase. These results suggest that an increase in secondary cell wall components occurs in rice leaves to compensate for Si deficiency.