Regulation Of Cellulose Biosynthesis Research Articles

Genome-wide identification of the COBRA-Like gene family in Pinus tabuliformis and the role of PtCOBL12 in the regulation of cellulose biosynthesis

COBRA-Like (COBL) genes encoding a plant-specific glycosylphosphatidylinositol (GPI) anchored protein, which have been proven to be key regulators in the cell expansion process and cellulose crystallinity. However, the function of COBL genes remains to be further elucidated in Pinus tabuliformis. We analyzed the P. tabuliformis genome and identified 15 COBL genes, which were anchored on 12 chromosomes and divided into three subgroups. Furthermore, we selected four COBL genes to explore their expression patterns in different seasons. The quantitative PCR results showed that COBL genes were mainly highly expressed in the active stage. Meanwhile, we analyzed the P. tabuliformis at different ages, and the expression level of the COBL genes in adult P. tabuliformis was much higher than that in juvenile. Most importantly, we found that PtCOBL12 overexpression in Arabidopsis thaliana improved plant growth and increased the cellulose content and relative crystallinity. Under drought stress, the germination rate and root length of transgenic plants were significantly higher than those of wild-type. Single-particle tracking analysis results showed that the particle velocity, dwell time, and motion range of PtCOBL12 on the plasma membrane increased significantly under drought conditions. Taken together, these results further clarify the molecular mechanism of COBL genes in the growth and development of conifers and provide a new view of the molecular dynamic model of COBL genes in response to drought stress.

Disruption of Very-Long-Chain-Fatty Acid Synthesis Has an Impact on the Dynamics of Cellulose Synthase in Arabidopsis thaliana.

In higher plants, cellulose is synthesized by membrane-spanning large protein complexes named cellulose synthase complexes (CSCs). In this study, the Arabidopsis PASTICCINO2 (PAS2) was identified as an interacting partner of cellulose synthases. PAS2 was previously characterized as the plant 3-hydroxy-acyl-CoA dehydratase, an ER membrane-localized dehydratase that is essential for very-long-chain-fatty acid (VLCFA) elongation. The pas2-1 mutants show defective cell elongation and reduction in cellulose content in both etiolated hypocotyls and light-grown roots. Although disruption of VLCFA synthesis by a genetic alteration had a reduction in VLCFA in both etiolated hypocotyls and light-grown roots, it had a differential effect on cellulose content in the two systems, suggesting the threshold level of VLCFA for efficient cellulose synthesis may be different in the two biological systems. pas2-1 had a reduction in both CSC delivery rate and CSC velocity at the PM in etiolated hypocotyls. Interestingly, Golgi but not post-Golgi endomembrane structures exhibited a severe defect in motility. Experiments using pharmacological perturbation of VLCFA content in etiolated hypocotyls strongly indicate a novel function of PAS2 in the regulation of CSC and Golgi motility. Through a combination of genetic, biochemical and cell biology studies, our study demonstrated that PAS2 as a multifunction protein has an important role in the regulation of cellulose biosynthesis in Arabidopsis hypocotyl.

The Photoperiodic Flowering Time Regulator FKF1 Negatively Regulates Cellulose Biosynthesis.

Cellulose synthesis is precisely regulated by internal and external cues, and emerging evidence suggests that light regulates cellulose biosynthesis through specific light receptors. Recently, the blue light receptor CRYPTOCHROME 1 (CRY1) was shown to positively regulate secondary cell wall biosynthesis in Arabidopsis (Arabidopsis thaliana). Here, we characterize the role of FLAVIN-BINDING KELCH REPEAT, F-BOX 1 (FKF1), another blue light receptor and well-known photoperiodic flowering time regulator, in cellulose biosynthesis. A phenotype suppression screen using a cellulose deficient mutant cesa1aegeus,cesa3ixr1-2 (c1,c3), which carries nonlethal point mutations in CELLULOSE SYNTHASE A 1 (CESA1) and CESA3, resulted in identification of the phenotype-restoring large leaf (llf) mutant. Next-generation mapping using the whole genome resequencing method identified the llf locus as FKF1 FKF1 was confirmed as the causal gene through observation of the llf phenotype in an independent triple mutant c1,c3,fkf1-t carrying a FKF1 T-DNA insertion mutant. Moreover, overexpression of FKF1 in llf plants restored the c1,c3 phenotype. The fkf1 mutants showed significant increases in cellulose content and CESA gene expression compared with that in wild-type Columbia-0 plants, suggesting a negative role of FKF1 in cellulose biosynthesis. Using genetic, molecular, and phenocopy and biochemical evidence, we have firmly established the role of FKF1 in regulation of cellulose biosynthesis. In addition, CESA expression analysis showed that diurnal expression patterns of CESAs are FKF1 independent, whereas their circadian expression patterns are FKF1 dependent. Overall, our work establishes a role of FKF1 in the regulation of cell wall biosynthesis in Arabidopsis.

The Regulation of Cellulose Biosynthesis in Plants.

Cell walls define the shape of plant cells, controlling the extent and orientation of cell elongation, and hence organ growth. The main load-bearing component of plant cell walls is cellulose, and how plants regulate its biosynthesis during development and in response to various environmental perturbations is a central question in plant biology. Cellulose is synthesized by cellulose synthase (CESA) complexes (CSCs) that are assembled in the Golgi apparatus and then delivered to the plasma membrane (PM), where they actively synthesize cellulose. CSCs travel along cortical microtubule paths that define the orientation of synthesis of the cellulose microfibrils. CSCs recycle between the PM and various intracellular compartments, and this trafficking plays an important role in determining the level of cellulose synthesized. In this review, we summarize recent findings in CESA complex organization, CESA posttranslational modifications and trafficking, and other components that interact with CESAs. We also discuss cell wall integrity maintenance, with a focus on how this impacts cellulose biosynthesis.

Molecular cloning and expression analysis of seven sucrose synthase genes in bamboo (Bambusa emeiensis): investigation of possible roles in the regulation of cellulose biosynthesis and response to hormones

ABSTRACTSucrose synthase (SUS, EC 2.4.1.13) is a key enzyme involved in sucrose metabolism. In this study, amplification by polymerase chain reaction confirmed that there are at least seven SUS genes (BeSUS1-7) in the Bambusa emeiensis genome. The expression patterns of the BeSUSs genes differed from each other in various bamboo tissues. Among these BeSUSs, BeSUS2 was expressed predominately in the root and BeSUS5 was most abundant in developing shoots, indicating that these two isozymes might play more important physiological roles in root and shoot than the other BeSUSs genes, respectively. In addition, BeSUSs response to abscisic acid (ABA), methyl jasmonate (MeJA) and 2,6-dichlorobenzonitrile treatment (2,6-DCB, an inhibitor of cellulose synthesis) was also investigated in leaves. The results showed that the expression levels of the BeSUS1, 5 and 7 genes were not only conspicuously induced by ABA treatment but also up-regulated by exogenous application of MeJA, indicating that these three isozymes are most likely involved in ABA and MeJA stress responses and are important in meeting the increased glycolytic demand that occurs during these stresses. Moreover, after 2,6-DCB treatment, the cellulose content of leaves was decreased and the transcripts of BeSUS2, 3 and 7 were markedly decreased, while those of BeSUS 5 and 6 were conspicuously induced. The results suggest that the possible roles of BeSUSs genes in the pathway of sucrose metabolism, bamboo resistance to abiotic stresses and the regulation of cellulose biosynthesis may be divergent.

The Emerging Role of Protein Phosphorylation as a Critical Regulatory Mechanism Controlling Cellulose Biosynthesis.

Plant cell walls are extracellular matrices that surround plant cells and critically influence basic cellular processes, such as cell division and expansion. Cellulose is a major constituent of plant cell walls, and this paracrystalline polysaccharide is synthesized at the plasma membrane by a large protein complex known as the cellulose synthase complex (CSC). Recent efforts have identified numerous protein components of the CSC, but relatively little is known about regulation of cellulose biosynthesis. Numerous phosphoproteomic surveys have identified phosphorylation events in CSC associated proteins, suggesting that protein phosphorylation may represent an important regulatory control of CSC activity. In this review, we discuss the composition and dynamics of the CSC in vivo, the catalog of CSC phosphorylation sites that have been identified, the function of experimentally examined phosphorylation events, and potential kinases responsible for these phosphorylation events. Additionally, we discuss future directions in cellulose synthase kinase identification and functional analyses of CSC phosphorylation sites.

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.

In silico and functional characterization of the promoter of a Eucalyptussecondary cell wall associated cellulose synthase gene (EgCesA1)

Background Cellulose is an important biopolymer produced by all plants and is used in a number of different industries, including for pulp and paper production. Cellulose is deposited into the plant cell wall by a large membranebound protein complex, which is composed of different cellulose synthase (CESA) proteins. The cellulose content and pattern of deposition in plant cell walls is highly variable depending on the function of the cell. All plant cells have a thin primary cell wall, but a number of plant cell types, including xylem cells, also deposit a secondary cell wall to give these tissues mechanical strength required to perform their function. Different cellulose synthase (CesA) genes have been shown to be involved in the deposition of primary and secondary walls. In Arabidopsis, three CesA (AtCesA4, 7 and 8) genes have consistently been associated with cells depositing secondary cell walls, while a different set of CesA genes have been shown to function during primary cell wall formation [Reviewed in 1]. These findings have been mirrored by studies of CesA gene orthologs in Populus and Eucalyptus[2-4]. While there have been a number of studies on CesA genes and their functions, much less is known about the regulation of these genes. In a previous study, we investigated the promoters of CesA genes involved in primary and secondary cell wall formation by performing a phylogenetic footprinting analysis to identify cis-elements conserved in the promoters from orthologous Arabidopsis, Populus and Eucalyptus cellulose synthase genes [5]. We identified a number of putative cis-regulatory elements that may play a role in the regulation of cellulose biosynthesis during primary and secondary cell wall formation. In the current study our aim is to further validate the ciselements identified in the CesA gene promoters by investigating their conservation across different Eucalyptus species and to determine the regulatory function of these promoter regions and the proteins which bind to them.

Characterising the role of the Eucalyptus grandis SND2promoter in secondary cell wall biosynthesis

Background NAC and MYB transcription factors (TFs) have been shown to play prominent roles in the regulation of plant developmental processes. Two Arabidopsis thaliana NAC domain TFs (AtSND2, AtSND3) and one MYB domain TF (AtMYB103) were shown to be downstream targets of two master regulators of xylem fibre cell development, NST1 and SND1 [1,2]. These TFs were able to induce the expression of a GUS reporter gene under the control of the AtCesA8 promoter [3], indicating that they may be involved in the regulation of cellulose biosynthesis in the secondary cell walls of A. thaliana xylem fibres. It is hypothesized that putative orthologs of these TFs will also play important roles in regulating fibre secondary cell wall biosynthesis in woody plants such as Eucalyptus grandis. The transcriptional network regulating wood fibre development is uncharacterized in E. grandis, therefore it would be beneficial to identify upstream components of this transcriptional network in E. grandis. In this ongoing study we aim to identify TFs which bind to the promoter of the putative ortholog of AtSND2 in E. grandis (EgSND2) and possibly also to the promoters of the orthologs of AtSND3 and AtMYB103. In parallel, we are characterizing the detailed heterologous expression patterns of the EgSND2, EgSND3 and MYB103 promoter regions in A. thaliana plants. This work forms part of an effort to elucidate the transcriptional network regulating wood fibre development in E. grandis. Methods A reverse BLAST approach was used to identify candidate orthologs of AtSND2, AtSND3 and AtMYB103 in Eucalyptus, Populus and Vitis. In silico cis-element analysis was performed on the promoter regions of the putative orthologs to identify previously characterised and novel cis-elements. The 1.5 kb regions upstream of the translational start site (TSS) of EgSND2, EgSND3 and EgMYB103 were isolated from E. grandis genomic DNA. The amplified fragments were cloned into pMDC162, a GUS reporter vector, and introduced into A. thaliana Col-O plants for heterologous GUS expression analysis. The same reporter constructs were also transformed directly into the vascular cambium of potted E. grandis plants to determine endogenous promoter activity by Induced Somatic Sector Analysis (ISSA, [4]). Qualitative GUS reporter analyses were performed on the EgSND2promoter::GUS, EgSND3promoter::GUS and EgMYB103promoter::GUS constructs in Arabidopsis plants at 1, 3 and 6 weeks. A 500 bp truncation upstream of the TSS of EgSND2 was generated and cloned into the pHIS2.1 vector along with the 1.5 kb EgSND2 promoter sequence for use in yeast onehybrid (Y1-H) screening. Candidate proteins which bind to the cloned promoter sequences will be identified using Y1-H analysis and further characterised.

Forest and fibre genomics: biotechnology tools for applied tree improvement

Eucalyptus tree breeders and geneticists stand to benefit tremendously from a recently announced effort to produce the first complete genome sequence for a eucalypt tree by 2010. A milestone for eucalypt research, the project will facilitate the development of new biotechnology tools that will accelerate the domestication, improvement and conservation of eucalypt tree species. We have embarked on a research venture in South Africa to study the molecular genetics of wood formation in fast-growing Eucalyptus trees and to develop biotechnology tools that can be integrated into applied tree-breeding programmes. The Wood and Fibre Molecular Genetics (WFMG) Programme, initiated in 2003, is a joint research programme of the University of Pretoria and the two largest pulp and paper companies in South Africa, Sappi and Mondi South Africa. Early objectives of the programme included the identification and isolation of candidate genes for cellulose production in Eucalyptus trees and the assessment of allelic diversity in candidate wood-formation genes. The application of DNA fingerprinting in eucalypt breeding programmes represented an early technology delivery to industry with practical, short-term benefits, which have served to offset the long-term nature of the research programme. Current research projects in the WFMG programme focus on the genetic control of carbon allocation, the transcriptional regulation of cellulose biosynthesis and the role of miRNAs and other small RNAs in wood formation in Eucalyptus. This paper provides a brief overview of research progress in the WFMG programme and discusses realistic time frames for the delivery of genome-based tree improvement tools.

New approaches to the study of cellulose biosynthesis

Examples of a variety of approaches for studying the mechanism and regulation of cellulose biosynthesis are presented. Attempts to demonstrate conclusively a cellulose synthase activity using membrane preparations derived from higher plants have not been successful; the predominant UDP-glucose: beta-glucan-beta-glucosyltransferase detected in these preparations is a beta-(1----3)-glucan synthase that is dependent upon Ca2+ and a beta-glucobiose, such as laminaribiose or cellobiose, for activity. Xyloglucan glucosyltransferase activity is detected in all plant preparations examined and, in cotton fibres, the activity of this enzyme correlates well with the level of xyloglucan found in the fibre. Low activity for a Mg2+-dependent beta-(1----4)-glucan synthase is found in extracts from soybeans and mung beans, but not cotton fibres; this enzyme could represent either a dissociated form of xyloglucan glucosyltransferase or a partially latent form of cellulose synthase. In vitro translation of RNA from developing cotton fibres shows notable increases in the level of several relatively abundant translatable mRNAs associated with the time of onset of secondary-wall cellulose synthesis. In order to determine whether these apparent changes in gene expression represent enhanced expression of specific genes required for cellulose synthesis, several strategies are being developed for identification of specific polypeptides required for this process. One strategy involves the successful development of a technique for detection of glucan synthase activity in acrylamide gels, a technique that should prove useful for characterization of the polypeptide composition of such enzymes. We have also synthesized a photoaffinity analogue of 2,6-dichlorobenzonitrile (DCB), a potent and specific inhibitor of cellulose synthesis. The analogue is also an effective inhibitor in vivo, and upon ultraviolet irradiation of extracts in the presence of the radioactive analogue, we observe a relatively specific labelling of a polypeptide of 18 X 10(3)Mr. Finally, we have studied the spatial regulation and structural requirements for cellulose synthesis in internode cells of the alga Chara corallina. Cellulose deposition in vivo shows spatial localization, which correlates with acid and base bands along the cell. Using internodes perfused with solutions containing UDP-[14C]glucose and subsequently ligated, we were able to demonstrate synthesis of a highly insoluble cell-wall-localized glucan, thus offering hope that Chara can be developed as another useful system for studying the mechanism and regulation of cellulose synthesis.