A new cellulose synthase gene (PtrCesA2) from aspen xylem is orthologous to Arabidopsis AtCesA7 (irx3) gene associated with secondary cell wall synthesis

We report here the molecular cloning and characterization of a new full-length cellulose synthase (CesA) cDNA, PtrCesA2 from aspen (Populus tremuloides) trees. The predicted PtrCesA2 protein shows a high degree of identity/similarity (87%/91%) to the predicted gene product of Arabidopsis AtCesA7 gene that has been associated with secondary cell wall development. Previously, a mutation in AtCesA7 gene (irx3) was correlated with a significant decrease in the amount of cellulose synthesized (about 70%) and genetic complementation of irx3 mutant with a wild-type AtCesA7 gene restored the normal phenotype. This is the first report of a full-length AtCesA7 ortholog from any non-Arabidopsis species. Interestingly, PtrCesA2 shares only 64% identity with our earlier reported PtrCesA1 from aspen suggesting its structural distinctness from the only other known CesA member from the aspen genome. PtrCesA1 is a xylem-specific and tension stress responsive gene that is highly similar to another Arabidopsis gene, AtCesA8 which also has been associated with secondary wall development. Moreover, AtCesA7 and AtCesA8 are suggested to be part of the same cellulose synthase complex. Isolation of PtrCesA2 from a xylem library enriched in cells with active secondary wall synthesis, PtrCesA2 expression levels similar to PtrCesA1 and high similarity of PtrCesA1 and PtrCesA2 to AtCesA8 and AtCesA7, respectively, suggest that both these aspen genes might be involved in the secondary wall development in aspen woody tissues. Availability of two aspen CesA orthologs will now enable us to examine if PtrCesA1 and PtrCesA2 functionally interact during aspen wood development that has long-term implications on genetic improvement of forest trees.     

AtCSLD3, a cellulose synthase-like gene important for root hair growth in arabidopsis

A member of the cellulose synthase-like (subfamily D) gene family of Arabidopsis, AtCSLD3, has been identified by T-DNA tagging. The analysis of the corresponding mutant, csld3-1, showed that the AtCSLD3 gene plays a role in root hair growth in plants. Root hairs grow in phases: First a bulge is formed and then the root hair elongates by polarized growth, the so-called "tip growth." In the mutant, root hairs were initiated at the correct position and grew into a bulge, but their elongation was severely reduced. The tips of the csld3-1 root hairs easily leaked cytoplasm, indicating that the tensile strength of the cell wall had changed at the site of the tip. Based on the mutant phenotype and the functional conservation between CSLD3 and the genuine cellulose synthase proteins, we hypothesized that the CSLD3 protein is essential for the synthesis of polymers for the fast-growing primary cell wall at the root hair tip. The distinct mutant phenotype and the ubiquitous expression pattern indicate that the CSLD3 gene product is only limiting at the zone of the root hair tip, suggesting particular physical properties of the cell wall at this specific site of the root hair cell.     

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.     

Gene expression profiles in different stem internodes reveal the genetic regulation of primary and secondary stem development in <Emphasis Type="Italic">Betula platyphylla</Emphasis>

Secondary growth of stems is an important process for the radial increase of trees. To gain an insight into the molecular mechanisms underlying stem development from primary to secondary growth and to provide information for molecular research and breeding in Betula platyphylla (birch), the gene expression profiles of material from the first, third, and fifth internodes (IN) of 3-month-old seedlings were analyzed. Compared with the first IN, 177 genes were up-regulated and 157 genes down-regulated in the third IN; in the fifth IN, 180 genes were up-regulated and 275 genes were down-regulated. The expressions of 24 genes were up-regulated and 6 genes were down-regulated in the fifth IN relative to the third IN. The differentially expressed genes were annotated as having roles in cambium, xylem, and phloem development and formation; including cell wall expansion, cellulose biosynthesis, lignin biosynthesis and deposition, xylem extension, cell wall modification, and growth hormone responses. The expressions of genes related to cell wall expansion and cellulose biosynthesis in the primary cell wall were down-regulated in the third and fifth IN relative to the first IN. Genes involved in lignin biosynthesis, xylem extension, and cellulose synthesis in the secondary cell wall were up-regulated in the third and fifth IN relative to the first IN. These results described the patterns of gene expression during stem development in birch and provided candidate genes for further functional characterization.     

Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant

Key message

Overexpression of cotton cellulose synthase like D3 (GhCSLD3) gene partially rescued growth defect of atcesa6 mutant with restored cell elongation and cell wall integrity mainly by enhancing primary cellulose production.

Abstract

Among cellulose synthase like (CSL) family proteins, CSLDs share the highest sequence similarity to cellulose synthase (CESA) proteins. Although CSLD proteins have been implicated to participate in the synthesis of carbohydrate-based polymers (cellulose, pectins and hemicelluloses), and therefore plant cell wall formation, the exact biochemical function of CSLD proteins remains controversial and the function of the remaining CSLD genes in other species have not been determined. In this study, we attempted to illustrate the function of CSLD proteins by overexpressing Arabidopsis AtCSLD2, -3, -5 and cotton GhCSLD3 genes in the atcesa6 mutant, which has a background that is defective for primary cell wall cellulose synthesis in Arabidopsis. We found that GhCSLD3 overexpression partially rescued the growth defect of the atcesa6 mutant during early vegetative growth. Despite the atceas6 mutant having significantly reduced cellulose contents, the defected cell walls and lower dry mass, GhCSLD3 overexpression largely restored cell wall integrity (CWI) and improved the biomass yield. Our result suggests that overexpression of the GhCSLD protein enhances primary cell wall synthesis and compensates for the loss of CESAs, which is required for cellulose production, therefore rescuing defects in cell elongation and CWI.

     

Root hair-specific disruption of cellulose and xyloglucan in <Emphasis Type="Italic">AtCSLD3</Emphasis> mutants,and factors affecting the post-rupture resumption of mutant root hair growth

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5oC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.     

OsCD1 encodes a putative member of the cellulose synthase-like D sub-family and is essential for rice plant architecture and growth

The cell wall plays important roles in plant architecture and morphogenesis. The cellulose synthase-like super-families were reported to contain glycosyltransferases motif and are required for the biosynthesis of cell wall polysaccharides. Here, we describe a curled leaf and dwarf mutant, cd1, in rice, which exhibits multiple phenotypic traits such as the reduction of plant height and leaf width, curled leaf morphology and a decrease in the number of grains and in the panicle length. Map-based cloning indicates that a member of the cellulose synthase-like D (CSLD) group is a candidate for OsCD1. RNAi transgenic plants with the candidate CSLD gene display a similar phenotype to the cd1 mutant, suggesting that OsCD1 is a member of the CSLD sub-family. Furthermore, sequence analysis indicates that OsCD1 contains the common D,D,D,QXXRW motif, which is a feature of the cellulose synthase-like super-family. Analysis of OsCD1 promoter with GUS fusion expression shows that OsCD1 exhibits higher expression in young meristem tissues such as fresh roots, young panicle and stem apical meristem. Cell wall composition analysis reveals that cellulose content and the level of xylose are significantly reduced in mature culm owing to loss of OsCD1 function. Take together, the work presented here is useful for expanding the understanding of cell wall biosynthesis.     

Rice plants response to the disruption of OsCSLD4 gene

The plant cell wall is a complex polysaccharide network and performs important developmental and physiological functions far beyond supplying the physical constrains. Plant cells have the ability to react to cell wall defects as exhibited by changes in gene expression, accumulation of ectopic lignin, stress responses and growth arrest. It is a major challenge to understand how plants sense and respond to wall integrity since very little is known about the signaling involved in the responses. Cellulose synthase-like D (CSLD) proteins mediating the biosynthesis of a wall polysaccharide polymer make up a common subfamily to all plants. Recently, we have reported the functional characterization of CSLD4 in rice. Mutations in OsCSLD4 show morphological alterations and pleiotropic effects on wall compositions and structure. Our study demonstrates that OsCSLD4 play a critical role in cell wall formation and plant growth. Here we show the subtle wall alterations by separating the culm residues into five fractions. Quantitative RT-PCR analysis further revealed that the expression of various genes involved in xylan synthesis and cell cycle regulation was altered in mutant plants, as the responses to OsCSLD4 disruption. Therefore, plants may have fine sensory machinery to react to wall defects and modulate growth for adapting to the changes.Key words: OsCSLD4, cell wall biosynthesis, plant development, wall integrity, rice     

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion‐exchange chromatography, could be determined accurately since, xylem‐specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.     

Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls

The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-of-function mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall.