Cellulose Synthesis In Plants Research Articles

Genome-wide characterization of the cellulose synthase gene family in Ziziphus jujuba reveals its function in cellulose biosynthesis during fruit development

The cellulose synthase (Ces/Csl) is a key enzyme in plant cellulose synthesis. Jujube fruits are rich in cellulose. 29 ZjCesA/Csl genes were identified in jujube genome and showed tissue-specific expression. 13 genes highly expressed in jujube fruit exhibited obviously sequential expressions during the fruit development, indicating that they might play distinct roles during the process. Meanwhile, the correlation analysis showed the expressions of ZjCesA1 and ZjCslA1 were significant positive related to the cellulose synthase activities. Furthermore, transient overexpressions of ZjCesA1 or ZjCslA1 in jujube fruits significantly increased cellulose synthase activities and contents, whereas silencing of ZjCesA1 or ZjCslA1 in jujube seedlings obviously reduced cellulose levels. Moreover, the Y2H assays verified that ZjCesA1 and ZjCslA1 may participate in cellulose synthesis by forming protein complexes. The study not only reveals the bioinformatics characteristics and functions of cellulose synthase genes in jujube, but also provides clues for studying cellulose synthesis in other fruits.

Screening cellulose synthesis related genes of EgrEXP and EgrHEX in Eucalyptus grandis

Eucalyptus (including Eucalyptus grandis) is an excellent wood forest tree species that provides a large number of plant fiber raw materials for the paper and timber industries. Cellulose, an essential structural component in plant cell walls, is a renewable biomass resource that plays a very important role in nature. There is still a lack of research on the role of gene regulation in cellulose synthesis. To study the genes of cellulose synthesis, the wood chemical indexes of Eucalyptus grandis were analyzed by taking three different parts from the main stem of Eucalyptus grandis as raw materials. The results showed that the cellulose content in the middle of the trunk was significantly higher than that at the chest diameter and at the upper part of the trunk. A total of 296 differentially expressed genes (DEGs) were obtained from the three site by transcriptome, and 19 key candidate genes were related to the synthesis of cellulose in Eucalyptus grandis. EgrEXP1 and EgrHEX4 were overexpressed in 84 K poplar, the content of cellulose and lignin in genetically modified plants was significantly higher than that of wild type 84 K poplar. Also, the average plant height and average root count were significantly higher than those of control plants, and the average diameter of the middle and stem bases were significantly larger than those of control plants. In this study, the genes related to cellulose synthesis in Eucalyptus grandis are studied, which serve as a strong foundation for understanding the molecular regulation of cellulose synthesis in plants.

Stepwise increase of thaxtomins production in Streptomyces albidoflavus J1074 through combinatorial metabolic engineering

Herbicide-resistance in weeds has become a serious threat to agriculture across the world. Thus, there is an urgent need for the discovery and development of herbicides with new modes of action. Thaxtomin phytotoxins are a group of nitrated diketopiperazines produced by potato common scab-causing phytopathogen Streptomyces scabies and other actinobacterial pathogens. They are generally considered to function as inhibitors of cellulose synthesis in plants, and thus have great potential to be used as natural herbicides. Generation of an overproducing strain is crucial for the scale-up production of thaxtomins and their wide use in agriculture. In the present study, we employed a stepwise strategy by combining heterologous expression, repressor deletion, activator overexpression, and optimization of fermentation media for high-level production of thaxtomins. The maximum yield of 728 mg/L thaxtomins was achieved with engineered Streptomyces albidoflavus J1074 strains in shake-flask cultures, and it was approximately 36-fold higher than S. albidoflavus J1074 carrying the unmodified cluster. Moreover, the yield of thaxtomins could reach 1973 mg/L when the engineered strain was cultivated in a small-scale stirred-tank bioreactor. This is the highest titer reported to date, representing a significant leap forward for the scale-up production of thaxtomins. Our study presents a robust, easy-to-use system that will be broadly useful for improving titers of bioactive compounds in many Streptomyces species.

TRANVIA (TVA) facilitates cellulose synthase trafficking and delivery to the plasma membrane

Cellulose is synthesized at the plasma membrane by cellulose synthase (CESA) complexes (CSCs), which are assembled in the Golgi and secreted to the plasma membrane through the trans-Golgi network (TGN) compartment. However, the molecular mechanisms that guide CSCs through the secretory system and deliver them to the plasma membrane are poorly understood. Here, we identified an uncharacterized gene, TRANVIA (TVA), that is transcriptionally coregulated with the CESA genes required for primary cell wall synthesis. The tva mutant exhibits enhanced sensitivity to cellulose synthesis inhibitors; reduced cellulose content; and defective dynamics, density, and secretion of CSCs to the plasma membrane as compared to wild type. TVA is a plant-specific protein of unknown function that is detected in at least two different intracellular compartments: organelles labeled by markers for the TGN and smaller compartments that deliver CSCs to the plasma membrane. Together, our data suggest that TVA promotes trafficking of CSCs to the plasma membrane by facilitating exit from the TGN and/or interaction of CSC secretory vesicles with the plasma membrane.

Structure of Arabidopsis CESA3 catalytic domain with its substrate UDP-glucose provides insight into the mechanism of cellulose synthesis

Cellulose is synthesized by cellulose synthases (CESAs) from the glycosyltransferase GT-2 family. In plants, the CESAs form a six-lobed rosette-shaped CESA complex (CSC). Here we report crystal structures of the catalytic domain of Arabidopsis thaliana CESA3 (AtCESA3CatD) in both apo and uridine diphosphate (UDP)-glucose (UDP-Glc)-bound forms. AtCESA3CatD has an overall GT-A fold core domain sandwiched between a plant-conserved region (P-CR) and a class-specific region (C-SR). By superimposing the structure of AtCESA3CatD onto the bacterial cellulose synthase BcsA, we found that the coordination of the UDP-Glc differs, indicating different substrate coordination during cellulose synthesis in plants and bacteria. Moreover, structural analyses revealed that AtCESA3CatD can form a homodimer mainly via interactions between specific beta strands. We confirmed the importance of specific amino acids on these strands for homodimerization through yeast and in planta assays using point-mutated full-length AtCESA3. Our work provides molecular insights into how the substrate UDP-Glc is coordinated in the CESAs and how the CESAs might dimerize to eventually assemble into CSCs in plants.

Temperature-Dependent Oxygen Isotope Fractionation in Plant Cellulose Biosynthesis Revealed by a Global Dataset of Peat Mosses

The oxygen isotope composition (δ18O) of plant cellulose has been widely used to study ecohydrological processes of ecosystems as well as to reconstruct past climate conditions in terrestrial climate archives. These applications are grounded on a key assumption that the biochemical fractionation during cellulose synthesis is a constant around +27‰ and is not affected by environmental factors. Here we revisit the influence of temperature on biochemical fractionation factor during cellulose synthesis using a global compilation of Sphagnum cellulose δ18O data. Sphagnum (peat mosses) are known for inhabiting waterlogged peatlands and possessing unique physiological strategy in that their cellulose δ18O could closely reflect growing-season precipitation δ18O. Although within-site cellulose δ18O variability shows a median standard deviation of 0.7–0.8‰ resulting from different degree of evaporative enrichment of 18O in metabolic leaf water, this evaporative enrichment is a small quantity due to the external capillary “water buffer” in Sphagnum mosses. Using site-specific minimum cellulose δ18O data that most likely reflect the signal of unevaporated source water, we show that the apparent enrichment factor between cellulose and precipitation δ18O increases with decreasing air temperature. In particular, the apparent enrichment factor could reach as high as 32‰ when growth temperature is below 5 °C. This observational dataset extends the support for the temperature-dependent oxygen isotope fractionation in plant cellulose synthesis previously demonstrated in laboratory experiment, with implications for paleoclimate and plant physiology studies that employ cellulose δ18O measurements particularly in alpine regions.

In silico structure prediction of full-length cotton cellulose synthase protein (GhCESA1) and its hierarchical complexes

Cellulose synthase (CESA) polymerizes glucose into β-1,4-glucan chains that assemble to form cellulose microfibrils. Cellulose is the most abundant natural polymer in the world and a major structural component of the plant cell wall. An understanding of cellulose synthesis in plants is crucial for advancement in biofuels research and requires high-resolution 3D CESA structures. However, the determination of 3D structures of plant CESAs and their specific hierarchical arrangement into cellulose synthesis complexes (CSCs) has been a challenge. The prediction of CESA structures using computational methods presents a challenge due to poor sequence homology with resolved structures, long sequence, and structural complexity due to a mixture of globular, transmembrane, and intrinsically disordered regions. Herein, we present a 3D atomic-resolution model of a full-length (974-aa) cotton CESA (GhCESA1) structure using a variety of computational techniques with a reasonable ProSA-web z-score of − 8.32 (PDB available in SI). The overall fold of the CESA model indicates that there are similarities to BcsA bacterial cellulose synthase, such as the transmembrane topology and the internalization of conserved catalytic motifs. The plant-specific regions (CSR, P-CR, and N-term) fold into distinct subdomains, indicating the importance of these regions in CESA assembly into plant CSCs. We further examined possible assemblies of CESA monomers forming trimers and 18-mer CSCs, and we compared our results to those obtained by freeze fracture transmission electron microscopy. We observed that there are numerous competing ways in which CESAs may be arranged into homotrimers and CSCs. Our predicted structure can be used to probe CESA structure–activity relationships, select and subsequently test possible mutants, and investigate CESA aggregation into CSCs and microfibril formation to optimize biomass properties.

Endosidin20 Targets the Cellulose Synthase Catalytic Domain to Inhibit Cellulose Biosynthesis

Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.

Structure/function relationships in the rosette cellulose synthesis complex illuminated by an evolutionary perspective

Cellulose microfibrils are a key component of plant cell walls, which in turn compose most of our renewable biomaterials. Consequently, there is considerable interest in understanding how cellulose microfibrils are made in living cells by the plant cellulose synthesis complex (CSC). This remarkable multi-subunit complex contains cellulose synthase (CESA) proteins, and it is often called a rosette due to its six-lobed shape. Each CSC moves within the plasma membrane as it spins a strong cellulose microfibril in its wake. To accomplish this biological manufacturing process, the CESAs harvest an activated sugar substrate from the cytoplasm for use in the polymerization of glucan chains. An elongating glucan is simultaneously translocated across the plasma membrane by each CESA, where the group of chains emanating from one CSC co-crystallizes into a cellulose microfibril that becomes part of the assembling cell wall. Here we review major advances in understanding CESA and CSC structure/function relationships since 2013, when ground-breaking insights about the structure of cellulose synthases in bacteria and plants were published. We additionally discuss: (a) the relationship of CSC substructure to the size of the fundamental cellulose fibril; (b) an evolutionary perspective on the driving force behind the existence of hetero-oligomeric CSCs that currently appear to dominate in land plants; and (c) how cellulose properties may be regulated by CESA and CSC activity. We also pose major questions that still remain in this rapidly changing and exciting research field.

SHOU4 Proteins Regulate Trafficking of Cellulose Synthase Complexes to the Plasma Membrane

Cell walls play critical roles in plants, regulating tissue mechanics, defining the extent and orientation of cell expansion, and providing a physical barrier against pathogen attack [1]. Cellulose microfibrils, which are synthesized by plasma membrane-localized cellulose synthase (CESA) complexes, are the primary load-bearing elements of plant cell walls [2]. Cell walls are dynamic structures that are regulated in part by cell wall integrity (CWI)-monitoring systems that feed back to modulate wall properties and the synthesis of new wall components [3]. Several receptor-like kinases have been implicated as sensors of CWI [3-5], including the FEI1/FEI2 receptor-like kinases [4]. Here, we characterize two genes encoding novel plant-specific plasma membrane proteins (SHOU4 and SHOU4L) that were identified in a suppressor screen of the cellulose-deficient fei1 fei2 mutant. shou4 shou4l double mutants display phenotypes consistent with elevated levels of cellulose, and elevated levels of non-crystalline cellulose are present in this mutant. Disruption of SHOU4 and SHOU4L increases the abundance of CESA proteins at the plasma membrane as a result of enhanced exocytosis. The SHOU4/4L N-terminal cytosolic domains directly interact with CESAs. Our results suggest that the SHOU4 proteins regulate cellulose synthesis in plants by influencing the trafficking of CESA complexes to the cell surface.

Hydrogen isotope ratios of moss cellulose and source water in wetlands of Lake Superior, United States reveal their potential for quantitative paleoclimatic reconstructions

The hydrogen isotopic composition of organic substrates provides valuable information on past hydrological changes. To explore whether quantitative information can be obtained from this proxy, we measured D/H ratios of cellulose extracted from mosses inhabiting both hollow and hummock sites in three swales located near Lake Superior, United States. These isotope ratios were compared to those of potential water sources, namely swale, moss, and ground water, to evaluate whether cellulose D/H ratios reflect those of its water sources. For that evaluation, we relied on a better mechanistic understanding of the isotopic fractionation occurring during cellulose synthesis in plants as well as the relative simplicity of moss physiology in regard to water use strategies and a dataset that combines published D/H ratios for modern moss cellulose. We found that moss cellulose δD values for hollow species (−118.1±8.5‰) were significantly depleted (p=0.016) in D by 8‰ in relation to those for hummock species (−110.1±8.1‰), possibly due to an averaging effect taking place in hollow species. In addition, we found that 40% of the hydrogen in cellulose from hollow species probably experienced isotopic exchange with the surrounding cell water, and this percentage is not statistically different from that (42%) obtained from an analysis of published data on modern moss cellulose. This analysis also reveals that the fraction of hydrogen in modern moss cellulose that is subjected to isotope exchange falls within a relatively narrow range (±3%). Using this range and a mechanistic model, we were able to estimate source water δD values using those measured for moss cellulose, and these estimates were statistically indistinguishable from the measured δD values of moss and swale water. Further, the ratios of δD/δ18O of moss cellulose of hollow species yielded values that are similar to those reported for the local meteoric water line, thus adding a potential source of paleo-hydrological reconstructions from moss cellulose. Consequently, our combined results suggest not only that moss cellulose D/H ratios reflect those of source water, but that the latter values can be estimated fairly precisely from cellulose D/H ratios using a relatively simple mechanistic model. However, the accuracy of this model hinges on assumptions that need to be tested further to corroborate its widespread applicability.

T-DNA alleles of the receptor kinase THESEUS1 with opposing effects on cell wall integrity signaling.

Perturbation of cellulose synthesis in plants triggers stress responses, including growth retardation, mediated by the cell wall integrity-sensing receptor-like kinase (RLK) THESEUS1 (THE1). The analysis of two alleles carrying T-DNA insertions at comparable positions has led to conflicting conclusions concerning the impact of THE1 signaling on growth. Here we confirm that, unlike the1-3 and other the1 alleles in which cellular responses to genetic or pharmacological inhibition of cellulose synthesis are attenuated, the1-4 showed enhanced responses, including growth inhibition, ectopic lignification, and stress gene expression. Both the1-3 and the1-4 express a transcript encoding a predicted membrane-associated truncated protein lacking the kinase domain. However, the1-3, in contrast to the1-4, strongly expresses antisense transcripts, which are expected to prevent the expression of the truncated protein as suggested by the genetic interactions between the two alleles. Seedlings overexpressing such a truncated protein react to isoxaben treatment similarly to the1-4 and the full-length THE overexpressor. We conclude that the1-4 is a hypermorphic allele; that THE1 signaling upon cell wall damage has a negative impact on cell expansion; and that caution is required when interpreting the phenotypic effects of T-DNA insertions in RLK genes.

BRASSINOSTEROID INSENSITIVE2 negatively regulates cellulose synthesis in Arabidopsis by phosphorylating cellulose synthase 1

The deposition of cellulose is a defining aspect of plant growth and development, but regulation of this process is poorly understood. Here, we demonstrate that the protein kinase BRASSINOSTEROID INSENSITIVE2 (BIN2), a key negative regulator of brassinosteroid (BR) signaling, can phosphorylate Arabidopsis cellulose synthase A1 (CESA1), a subunit of the primary cell wall cellulose synthase complex, and thereby negatively regulate cellulose biosynthesis. Accordingly, point mutations of the BIN2-mediated CESA1 phosphorylation site abolished BIN2-dependent regulation of cellulose synthase activity. Hence, we have uncovered a mechanism for how BR signaling can modulate cellulose synthesis in plants.

Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex.

A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In summary, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.

Structural Studies of Plant CESA Support Eighteen CESAs in the Plant CSC

The cellulose synthesis in plants is carried by a large multi-subunit transmembrane protein complex known as cellulose synthesis complex (CSC). The structural information on plant cellulose synthase (CESA) proteins that constitute CSC has remained challenging over years due to inherent complexities in the CSC leading to speculations and debates on the composition of CSC and the number of cellulose chains in a microfibril. Here, we report our findings on the structural properties of catalytic domain of Arabidopsis thaliana CESA1 (ATCESA1CatD) analyzed using small-angle scattering and computational modeling techniques. Our main findings include low resolution structures of ATCESA1CatD in monomeric and trimeric complex forms that provide the first experimental evidence supporting the self-assembly of CESAs into stable trimeric complexes. This is of immense importance in the context of CSC formation by plant CESAs and addresses a long standing question in plant biology - how many CESAs in the plant cellulose synthesis complex? Further, the scattering data in combination with computational modeling provided insight into the potential arrangement the monomers in the catalytic trimer and the relative arrangement of P-CR and CSR regions that are unique in the plants. Comparison of the size of the trimer complex with the dimensions of CSCs from TEM images provides compelling evidence that each lobe of a CSC contains three CESAs rather than the long-standing model of six CESAs within each lobe of a rosette CSC. To our knowledge, these studies are the first experimental evidence that CESA trimers form the lobes of rosette CSCs providing strong support for the hexamer of trimers model that synthesizes an 18-chain cellulose microfibril as the fundamental product of cellulose synthesis in plants.

A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice.

Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth.

The cellobiose sensor CebR is the gatekeeper of Streptomyces scabies pathogenicity.

ABSTRACTA relatively small number of species in the large genus Streptomyces are pathogenic; the best characterized of these is Streptomyces scabies. The pathogenicity of S. scabies strains is dependent on the production of the nitrated diketopiperazine thaxtomin A, which is a potent plant cellulose synthesis inhibitor. Much is known about the genetic loci associated with plant virulence; however, the molecular mechanisms by which S. scabies triggers expression of thaxtomin biosynthetic genes, beyond the pathway-specific activator TxtR, are not well understood. In this study, we demonstrate that binding sites for the cellulose utilization repressor CebR occur and function within the thaxtomin biosynthetic cluster. This was an unexpected result, as CebR is devoted to primary metabolism and nutritive functions in nonpathogenic streptomycetes. In S. scabies, cellobiose and cellotriose inhibit the DNA-binding ability of CebR, leading to an increased expression of the thaxtomin biosynthetic and regulatory genes txtA, txtB, and txtR. Deletion of cebR results in constitutive thaxtomin A production and hypervirulence of S. scabies. The pathogenicity of S. scabies is thus under dual direct positive and negative transcriptional control where CebR is the cellobiose-sensing key that locks the expression of txtR, the key necessary to unlock the production of the phytotoxin. Interestingly, CebR-binding sites also lie upstream of and within the thaxtomin biosynthetic clusters in Streptomyces turgidiscabies and Streptomyces acidiscabies, suggesting that CebR is most likely an important regulator of virulence in these plant-pathogenic species as well.

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex.

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-β-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-β-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.

Turnover time of the non‐structural carbohydrate pool influences δ18O of leaf cellulose

Certainty regarding the degree to which organic molecules exchange oxygen with local water during plant cellulose synthesis (p(ex)) is necessary for cellulose oxygen isotope (δ(18)O(cell))-based applications in environmental and ecological studies. However, the currently accepted notion that p(ex) is a constant of ca. 0.42 appears inconsistent with biochemical theory, which predicts that marked variation may be present in p(ex), in relation to variation in the turnover time (τ) of the carbohydrate pool available for cellulose synthesis. The above prediction was tested in the present study with the analysis of data collected from leaves of Ricinus communis grown in controlled environmental conditions that varied in light intensity and vapour pressure deficit. The results revealed the existence of considerable variation in both p(ex) and τ across plants in the various growth environments. Moreover, despite uncertainties in estimates of the proportion of source water in the synthesis water (p(x)) and of the biochemical fractionation factor (ε(o)), our experiment yielded strong evidence that p(ex) exhibits a significant, positive relationship with τ, consistent with biochemical theory. The observed variation in p(ex) in association with τ has important implications for the interpretation of δ(18)O(cell) data in environmental/ecological studies.

Sucrose Synthase is an Integral Component of the Cellulose Synthesis Machinery

Cellulose synthesis in plants is believed to be carried out by the plasma membrane-associated rosette structure which can be observed by electron microscopy. Despite decade-long speculation, it had not been demonstrated whether the rosette is the site of catalytic activity of cellulose synthesis. To determine the relationship between this structure and cellulose synthesis, we successfully isolated detergent-insoluble rosettes from the plasma membrane of bean epicotyls. However, the purified rosettes did not possess cellulose synthesis activity in vitro. Conversely, detergent-soluble granular particles of approximately 9.5-10 nm diameter were also isolated and exhibited UDP-glucose binding activity and possessed beta-1,4-glucan (cellulose) synthesis activity in vitro. The particle, referred to as the catalytic unit of cellulose synthesis, was enriched with a 78 kDa polypeptide which was verified as sucrose synthase like by mass spectrometry and immunoblotting. The catalytic units were able to bind to the rosettes and retained the cellulose synthesis activity in the presence of UDP-glucose or sucrose plus UDP when supplemented with magnesium. The incorporation of the catalytic unit into the rosette structure was confirmed by immunogold labeling with anti-sucrose synthase antibodies under an electron microscope. Our results suggest that the plasma membrane-associated rosette anchors the catalytic unit of cellulose synthesis to form the functional cellulose synthesis machinery.