Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.

Genome-Wide Characterization of the Cellulose Synthase Gene Superfamily in Tea Plants(Camellia sinensis)

The cellulose synthase gene superfamily,including Cellulose synthase A(CesA)and cellulose synthase-like(Csl)gene families,is responsible for the synthesis of cellulose and hemicellulose,respectively.The CesA/Csl genes are vital for abiotic stress resistance and shoot tenderness regulation of tea plants(Camellia sinensis).However,the CesA/Csl gene family has not been extensively studied in tea plants.Here,we identified 53 CsCesA/Csl genes in tea plants.These genes were grouped into five subfamilies(CsCesA,CsCslB,CsCslD,CsCslE,CsCslG)based on the phylogenetic relationships with Arabidopsis and rice.The analysis of chromosome distribution,gene structure,protein domain and motif revealed that most genes in CsCesA,CsCslD and CsCslE subfamilies were conserved,whereas CsCslB and CsCslG subfamily members are highly diverged.The transcriptome analysis showed that most CsCesA/Csl genes displayed tissue-specific expression pattern.In addition,members of CsCslB4,CsCesA1/3/6,CsCslB3/4,CsCslD3,CsCslE1 and CsCslG2/3 subfamilies were up-regulated under drought and cold stresses,indicating their potential roles in regulating stress tolerance in tea plants.Furthermore,the expression levels of CsCslG2_6 and CsCslD3_5 in different tissues and cultivars,respectively,were positively correlated with the cellulose content that is negatively related with shoot tenderness.Thus,these two genes were speculated to be involved in the regulation of shoot tenderness in tea plants.Our findings may help elucidate the evolutionary relationships and expression patterns of the CsCesA/Csl genes in tea plants,and provide more candidate genes responsible for stress tolerance and tenderness regulation in tea plants for future functional research.

Cloning,Characterization,and Gene Annotation of Cellulose Synthase Genes from Arabidopsis thaliana

The mechanistic basis of cellulose biosynthesis in plants has gained ground during last decade or so.The isolation of plant cDNA clones encoding cotton homologs of the bacterial cellulose

Phylogenetically Distinct Cellulose Synthase Genes Support Secondary Wall Thickening in Arabidopsis Shoot Trichomes and Cotton Fiber

Through exploring potential analogies between cotton seed trichomes （or cotton fiber） and arabidopsis shoot trichomes we discovered that CesAs from either the primary or secondary wall phylogenetic clades can support secondary wall thickening. CesA genes that typically support primary wall synthesis, AtCesA 1,2,3,5, and 6, underpin expansion and secondary wall thickening of arabidopsis shoot trichomes. In contrast, apparent orthologs of CesA genes that support secondary wall synthesis in arabidopsis xylem, AtCesA4,7, and 8, are up-regulated for cotton fiber secondary wall deposition. These conclusions arose from： （a） analyzing the expression of CesA genes in arabidopsis shoot trichomes; （b） observing birefringent secondary walls in arabidopsis shoot trichomes with mutations in AtCesA4, 7, or 8; （c） assaying up-regulated genes during different stages of cotton fiber development; and （d） comparing genes that were co.expressed with primary or secondary wall CesAs in arabidopsis with genes up- regulated in arabidopsis trichomes, arabidopsis secondary xylem, or cotton fiber during primary or secondary wall deposition. Cumulatively, the data show that： （a） the xylem of arabidopsis provides the best model for secondary wall cellulose synthesis in cotton fiber; and （b） CesA genes within a ＂cell wall toolbox＂ are used in diverse ways for the construction of particular specialized cell walls.

Molecular studies of cellulose synthase supercomplex from cotton fiber reveal its unique biochemical properties

Cotton fiber is a highly elongated and thickened single cell that produces large quantities of cellulose,which is synthesized and assembled into cell wall microfibrils by the cellulose synthase complex(CSC).In this study,we report that in cotton(Gossypium hirsutum)fibers harvested during secondary cell wall(SCW)synthesis,GhCesA 4,7,and 8 assembled into heteromers in a previously uncharacterized 36-mer-like cellulose synthase supercomplex(CSS).This super CSC was observed in samples prepared using cotton fiber cells harvested during the SCW synthesis period but not from cotton stem tissue or any samples obtained from Arabidopsis.Knock-out of any of GhCesA 4,7,and 8 resulted in the disappearance of the CSS and the production of fiber cells with no SCW thickening.Cotton fiber CSS showed significantly higher enzyme activity than samples prepared from knock-out cotton lines.We found that the microfibrils from the SCW of wild-type cotton fibers may contain 72 glucan chains in a bundle,unlike other plant materials studied.GhCesA4,7,and 8 restored both the dwarf and reduced vascular bundle phenotypes of their orthologous Arabidopsis mutants,potentially by reforming the CSC hexamers.Genetic complementation was not observed when non-orthologous CesA genes were used,indicating that each of the three subunits is indispensable for CSC formation and for full cellulose synthase function.Characterization of cotton CSS will increase our understanding of the regulation of SCW biosynthesis.

Rab-H_1b is essential for trafficking of cellulose synthase and for hypocotyl growth in Arabidopsis thaliana

Ceil-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells, cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis; however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana, Rab-H1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6 （CESA6） to the plasma membrane. Loss of Rab-Hlb function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls.Exocytosis of CESA6 was impaired in rab-Mb cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-Mb cells, suggesting a defect in cisternal homeostasis caused by Rab-Hlb loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypo- cotyl growth, and suggest Rab-Hlb is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.

The trafficking and behavior of cellulose synthase and a glimpse of potential cellulose synthesis regulators

Cellulose biosynthesis is a topic of intensive research not only due to the significance of cellulose in the integrity of plant cell walls,but also due to the potential of using cellulose,a natural carbon source,in the production of biofuels.Characterization of the composition,regulation,and trafficking of cellulose synthase complexes(CSCs)is critical to an understanding of cellulose biosynthesis as well as the characterization of additional proteins that contribute to the production of cellulose either through direct interactions with CSCs or through indirect mechanisms.In this review,a highlight of a few proteins that appear to affect cellulose biosynthesis,which includes:KORRIGAN(KOR),Cellulose Synthase-Interactive Protein 1(CSI1),and the poplar microtubule-associated protein,PttMAP20,will accompany a description of cellulose synthase(CESA)behavior and a discussion of CESA trafficking compartments that might act in the regulation of cellulose biosynthesis.

SIMYB1 and SIMYB2, two new MYB genes from tomato, transcriptionally regulate cellulose biosynthesis in tobacco

Cellulose, a major constituent of plant biomass, is synthesized by a cellulose synthase complex. It has been demonstrated that MYB genes transcriptionally regulate cellulose synthase in Arabidopsis. However, little is known about this process in tomato. Here, two MYB （SIMYB1/2） and three cellulose synthase （CESA） （SICESA41516） genes were isolated. SIMYB1/2 and SICESA4/5/6 accumulation was found to correspond to cellulose accumulation in different tissues of tomato. Dual luciferase assays indicated that these two MYBs were transcriptional activators that interact with promoters of SICESA4/5/6. Moreover, SIMYB2 could also activate promoters of SIMYB1/2, suggesting the possible underlying auto-activation mech- anisms for MYB transcription factors. Transient over-expression of SlMYB1/2 in Nicotiana tabacum up-regulated tobacco endogenous NtCESA genes and increased cellulose accumulation. The function of SIMYB112 was further investigated using stable transformation and the results indicated that N. tabacum lines heterologous expressing SIMYB1/2 displayed a pleiotropic phenotype, long and narrow leaves, with NtCESA induced and significant increase of cellulose. In conclusion, our data suggest that tomato SIMYB1/2 have transcriptional regulatory roles in cellulose biosynthesis and SIMYB2 was more effective than SIMYB1, which may due to the transcriptional activation by SIMYB2 on SIMYB1 and itself.

Cellulose synthesis in land plants

All plant cells are surrounded by a cell wall that provides cohesion,protection,and a means of directional growth to plants.Cellulose microfibrils contribute the main biomechanical scaffold for most of these walls.The biosynthesis of cellulose,which typically is the most prominent constituent of the cell wall and therefore Earth’s most abundant biopolymer,is finely attuned to developmental and environmental cues.Our understanding of the machinery that catalyzes and regulates cellulose biosynthesis has substantially improved due to recent technological advances in,for example,structural biology and microscopy.Here,we provide a comprehensive overview of the structure,function,and regulation of the cellulose synthesis machinery and its regulatory interactors.We aim to highlight important knowledge gaps in the field,and outline emerging approaches that promise a means to close those gaps.

Cell Wall, Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.

The Cooperative Activities of CSLD2, CSLD3, and CSLD5 Are Required for Normal Arabidopsis Development

Glycosyltransferases of the Cellulose Synthase Like D （CS/D） subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were coexpressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant.

Thirteen Dipterocarpoideae genomes provide insights into their evolution and borneol biosynthesis

Dipterocarpoideae,the largest subfamily of the Dipterocarpaceae,is a dominant component of Southeast Asian rainforests and is widely used as a source of wood,damar resin,medicine,and essential oil.However,many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance.Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes,providing clues to the function and evolution of these species.Here,we report on 13 high-quality Dipterocarpoideae genome assemblies,ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae.Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae.Based on evolutionary analysis,we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes.We discovered an expansion of genes encoding cellulose synthase(CesA),which is essential for cellulose biosynthesis and secondary cell-wall formation.We functionally identified five bornyl diphosphate synthase(BPPS)genes,which specifically catalyze the biosynthesis of borneol,a natural medicinal compound extracted from damar resin and oils,thus providing a basis for large-scale production of natural borneol in vitro.