Characterization of 25 full-length <Emphasis Type="Italic">S-RNase</Emphasis> alleles,including flanking regions,from a pool of resequenced apple cultivars

Key message

Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell.

Abstract

Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

     

KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery

Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane–cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

The Arabidopsis Cellulose Synthase Complex: A Proposed Hexamer of CESA Trimers in an Equimolar Stoichiometry

Cellulose is the most abundant renewable polymer on Earth and a major component of the plant cell wall. In vascular plants, cellulose synthesis is catalyzed by a large, plasma membrane-localized cellulose synthase complex (CSC), visualized as a hexameric rosette structure. Three unique cellulose synthase (CESA) isoforms are required for CSC assembly and function. However, elucidation of either the number or stoichiometry of CESAs within the CSC has remained elusive. In this study, we show a 1:1:1 stoichiometry between the three Arabidopsis thaliana secondary cell wall isozymes: CESA4, CESA7, and CESA8. This ratio was determined utilizing a simple but elegant method of quantitative immunoblotting using isoform-specific antibodies and ³⁵S-labeled protein standards for each CESA. Additionally, the observed equimolar stoichiometry was found to be fixed along the axis of the stem, which represents a developmental gradient. Our results complement recent spectroscopic analyses pointing toward an 18-chain cellulose microfibril. Taken together, we propose that the CSC is composed of a hexamer of catalytically active CESA trimers, with each CESA in equimolar amounts. This finding is a crucial advance in understanding how CESAs integrate to form higher order complexes, which is a key determinate of cellulose microfibril and cell wall properties.     

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.     

The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.     

Manipulating cellulose biosynthesis by expression of mutant Arabidopsis proM24::CESA3ixr1‐2 gene in transgenic tobacco

Manipulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create modified cellulose for anthropogenic use. Evidence exists that cellulose microfibril structure and its recalcitrance to enzymatic digestion can ameliorated via mis‐sense mutation in the primary cell wall–specific gene AtCELLULOSE SYNTHASE (CESA)3. This mis‐sense mutation has been identified based on conferring drug resistance to the cellulose inhibitory herbicide isoxaben. To examine whether it would be possible to introduce mutant CESA alleles via a transgenic approach, we overexpressed a modified version of CESA3, AtCESA3^ixr1‐2 derived from Arabidopsis thaliana L. Heynh into a different plant family, the Solanceae dicotyledon tobacco (Nicotiana tabacum L. variety Samsun NN). Specifically, a chimeric gene construct of CESA3^ixr1‐2, codon optimized for tobacco, was placed between the heterologous M24 promoter and the rbcSE9 gene terminator. The results demonstrated that the tobacco plants expressing M24‐CESA3^ixr1‐2 displayed isoxaben resistance, consistent with functionality of the mutated AtCESA3^ixr1‐2 in tobacco. Secondly, during enzymatic saccharification, transgenic leaf‐ and stem‐derived cellulose is 54%–66% and 40%–51% more efficient, respectively, compared to the wild type, illustrating translational potential of modified CESA loci. Moreover, the introduction of M24‐AtCESA3^ixr1‐2 caused aberrant spatial distribution of lignified secondary cell wall tissue and a reduction in the zone occupied by parenchyma cells.     

A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1

The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes (“terminal complexes”), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU‐1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High‐efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full‐length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N‐ and C‐terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.     

Cellulose synthesis in two secondary cell wall processes in a single cell type

Plant cells have a rigid cell wall that constrains internal turgor pressure yet extends in a regulated and organized manner to allow the cell to acquire shape. The primary load-bearing macromolecule of a plant cell wall is cellulose, which forms crystalline microfibrils that are organized with respect to a cell''s function and shape requirements. A primary cell wall is deposited during expansion whereas secondary cell wall is synthesized post expansion during differentiation. A complex form of asymmetrical cellular differentiation occurs in Arabidopsis seed coat epidermal cells, where we have recently shown that two secondary cell wall processes occur that utilize different cellulose synthase (CESA) proteins. One process is to produce pectinaceous mucilage that expands upon hydration and the other is a radial wall thickening that reinforced the epidermal cell structure. Our data illustrate polarized specialization of CESA5 in facilitating mucilage attachment to the parent seed and CESA2, CESA5 and CESA9 in radial cell wall thickening and formation of the columella. Herein, we present a model for the complexity of cellulose biosynthesis in this highly differentiated cell type with further evidence supporting each cellulosic secondary cell wall process.     

Spatio‐temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo‐vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP‐labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co‐localisation studies using GFP–CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast‐associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP–CESA from doughnut‐shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP–CESA density diminished, whereas GFP–CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP–CESA in clathrin‐containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose‐deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.     

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.     

The N‐terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6‐like proteins in Arabidopsis. It has been proposed that the CESA6‐like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N‐terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher‐order structures during assembly of the CSC. We found that the mutant phenotypes of prc1‐1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1‐1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N‐terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Our findings suggest that the homodimerization of CESA6 through its N‐terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher‐order structures in CSCs.     

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co‐expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T‐DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.     

Features of the primary wall CESA complex in wild type and cellulose-deficient mutants of Arabidopsis thaliana

Evidence from genetics, co-precipitation and bimolecular fluorescence complementation suggest that three CESAs implicated in making primary wall cellulose in Arabidopsis thaliana form a complex. This study shows the complex has a M(r) of approximately 840 kDa in detergent extracts and that it has undergone distinctive changes when extracts are prepared from some cellulose-deficient mutants. The mobility of CESAs 1, 3, and 6 in a Triton-soluble microsomal fraction subject to blue native polyacrylamide gel electrophoresis was consistent with a M(r) of about 840 kDa. An antibody specific to any one CESA pulled down all three CESAs consistent with their occupying the same 840 kDa complex. In rsw1, a CESA1 missense mutant, extracts of seedlings grown at the permissive temperature have an apparently normal CESA complex that was missing from extracts of seedlings grown at the restrictive temperature where CESAs precipitated independently. In prc1-19, with no CESA6, CESAs 1 and 3 were part of a 420 kDa complex in extracts of light-grown seedlings that was absent from extracts of dark-grown seedlings where the CESAs precipitated independently. Two CESA3 missense mutants retained apparently normal CESA complexes as did four cellulose-deficient mutants defective in proteins other than CESAs. The 840 kDa complex could contain six CESA subunits and, since loss of plasma membrane rosettes accompanies its loss in rsw1, the complex could form one of the six particles which electron microscopy reveals in rosettes.     

Molecular characterization of PoGT8D and PoGT43B, two secondary wall-associated glycosyltransferases in poplar

Dicot wood is mainly composed of cellulose, lignin and glucuronoxylan (GX). Although the biosynthetic genes for cellulose and lignin have been studied intensively, little is known about the genes involved in the biosynthesis of GX during wood formation. Here, we report the molecular characterization of two genes, PoGT8D and PoGT43B, which encode putative glycosyltransferases, in the hybrid poplar Populus alba x tremula. The predicted amino acid sequences of PoGT8D and PoGT43B exhibit 89 and 75% similarity to the Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9, respectively, both of which have been shown to be required for GX biosynthesis. The PoGT8D and PoGT43B genes were found to be expressed in cells undergoing secondary wall thickening, including the primary xylem, secondary xylem and phloem fibers in stems, and the secondary xylem in roots. Both PoGT8D and PoGT43B are predicted to be type II membrane proteins and shown to be targeted to Golgi. Overexpression of PoGT43B in the irx9 mutant was able to rescue the defects in plant size and secondary wall thickness and partially restore the xylose content. Taken together, our results demonstrate that PoGT8D and PoGT43B are Golgi-localized, secondary wall-associated proteins, and PoGT43B is a functional ortholog of IRX9 involved in GX biosynthesis during wood formation.