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Similar to cellulose synthases (CESAs), cellulose synthase-like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens to investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca 2+ , rather than structural differences in the microfibrils they produce.

Assessing the number of glucan chains in cellulose microfibrils (CMFs) is crucial for understanding their structure-property relationships and interactions within plant cell walls. This Review examines the conclusions and limitations of the major experimental techniques that have provided insights into this question. Small-angle X-ray and neutron scattering data predominantly support an 18-chain model, although analysis is complicated by factors such as fibril coalescence and matrix polysaccharide associations. Solid-state nuclear magnetic resonance (NMR) spectroscopy allows the estimation of the CMF width from the ratio of interior to surface glucose residues. However, there is uncertainty in the assignment of NMR spectral peaks to surface or interior chains. Freeze-fracture transmission electron microscopy images show cellulose synthase complexes to be "rosettes" of six lobes each consistent with a trimer of cellulose synthase enzymes, consistent with the synthesis of 18 parallel glucan chains in the CMF. Nevertheless, the number of chains in CMFs remains to be conclusively demonstrated.

Premise: The shape of young cotton ( Gossypium ) fibers varies within and between commercial cotton species, as revealed by previous detailed analyses of one cultivar of G. hirsutum and one of G. barbadense . Both narrow and wide fibers exist in G. hirsutum cv. Deltapine 90, which may impact the quality of our most abundant renewable textile material. More efficient cellular phenotyping methods are needed to empower future research efforts., Methods: We developed semi-automated imaging methods for young cotton fibers and a novel machine learning algorithm for the rapid detection of tapered (narrow) or hemisphere (wide) fibers in homogeneous or mixed populations., Results: The new methods were accurate for diverse accessions of G. hirsutum and G. barbadense and at least eight times more efficient than manual methods. Narrow fibers dominated in the three G. barbadense accessions analyzed, whereas the three G. hirsutum accessions showed a mixture of tapered and hemisphere fibers in varying proportions., Discussion: The use or adaptation of these improved methods will facilitate experiments with higher throughput to understand the biological factors controlling the variable shapes of young cotton fibers or other elongating single cells. This research also enables the exploration of links between early cell shape and mature cotton fiber quality in diverse field-grown cotton accessions., (© 2022 The Authors. Applications in Plant Sciences published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Observable qualitative traits are relatively stable across environments and are commonly used to evaluate crop genetic diversity. Recently, molecular markers have largely superseded describing phenotypes in diversity surveys. However, qualitative descriptors are useful in cataloging germplasm collections and for describing new germplasm in patents, publications, and/or the Plant Variety Protection (PVP) system. This research focused on the comparative analysis of standardized cotton traits as represented within the National Cotton Germplasm Collection (NCGC). The cotton traits are named by 'descriptors' that have non-numerical sub-categories (descriptor states) reflecting the details of how each trait manifests or is absent in the plant. We statistically assessed selected accessions from three major groups of Gossypium as defined by the NCGC curator: (1) "Stoneville accessions (SA)," containing mainly Upland cotton ( Gossypium hirsutum ) cultivars; (2) "Texas accessions (TEX)," containing mainly G. hirsutum landraces; and (3) Gossypium barbadense (Gb), containing cultivars or landraces of Pima cotton ( Gossypium barbadense ). For 33 cotton descriptors we: (a) revealed distributions of character states for each descriptor within each group; (b) analyzed bivariate associations between paired descriptors; and (c) clustered accessions based on their descriptors. The fewest significant associations between descriptors occurred in the SA dataset, likely reflecting extensive breeding for cultivar development. In contrast, the TEX and Gb datasets showed a higher number of significant associations between descriptors, likely correlating with less impact from breeding efforts. Three significant bivariate associations were identified for all three groups, bract nectaries:boll nectaries , leaf hair:stem hair , and lint color:seed fuzz color . Unsupervised clustering analysis recapitulated the species labels for about 97% of the accessions. Unexpected clustering results indicated accessions that may benefit from potential further investigation. In the future, the significant associations between standardized descriptors can be used by curators to determine whether new exotic/unusual accessions most closely resemble Upland or Pima cotton. In addition, the study shows how existing descriptors for large germplasm datasets can be useful to inform downstream goals in breeding and research, such as identifying rare individuals with specific trait combinations and targeting breakdown of remaining trait associations through breeding, thus demonstrating the utility of the analytical methods employed in categorizing germplasm diversity within the collection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Restrepo-Montoya, Hulse-Kemp, Scheffler, Haigler, Hinze, Love, Percy, Jones and Frelichowski.)

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana , to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella ) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript.The Authors did not report any conflict of interest., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

Main Conclusion: Variable cotton fiber diameter is set early in anisotropic elongation by cell-type-specific processes involving the temporal and spatial regulation of microtubules in the apical region. Cotton fibers are single cells that originate from the seed epidermis of Gossypium species. Then, they undergo extreme anisotropic elongation and limited diametric expansion. The details of cellular morphogenesis determine the quality traits that affect fiber uses and value, such as length, strength, and diameter. Lower and more consistent diameter would increase the competitiveness of cotton fiber with synthetic fiber, but we do not know how this trait is controlled. The complexity of the question is indicated by the existence of fibers in two major width classes in the major commercial species: broad and narrow fibers exist in commonly grown G. hirsutum, whereas G. barbadense produces only narrow fiber. To further understand how fiber diameter is controlled, we used ovule cultures, morphology measurements, and microtubule immunofluorescence to observe the effects of microtubule antagonists on fiber morphology, including shape and diameter within 80 µm of the apex. The treatments were applied at either one or two days post-anthesis during different stages of fiber morphogenesis. The results showed that inhibiting the presence and/or dynamic activity of microtubules caused larger diameter tips to form, with greater effects often observed with earlier treatment. The presence and geometry of a microtubule-depleted-zone below the apex were transiently correlated with the apical diameter of the narrow tip types. Similarly, the microtubule antagonists had somewhat different effects between tip types. Overall, the results demonstrate cell-type-specific mechanisms regulating fiber expansion within 80 µm of the apex, with variation in the impact of microtubules between tip types and over developmental time.

Main Conclusion: A novel method for culturing ovules of Gossypium barbadense allowed in vitro comparisons with Gossypium hirsutum and revealed variable roles of microtubules in controlling cotton fiber cell expansion. Cotton fibers undergo extensive elongation and secondary wall thickening as they develop into our most important renewable textile material. These single cells elongate at the apex as well as elongating and expanding in diameter behind the apex. These multiple growth modes represent an interesting difference compared to classical tip-growing cells that needs to be explored further. In vitro ovule culture enables experimental analysis of the controls of cotton fiber development in commonly grown Gossypium hirsutum cotton, but, previously, there was no equivalent system for G. barbadense, which produces higher quality cotton fiber. Here, we describe: (a) how to culture the ovules of G. barbadense successfully, and (b) the results of an in vitro experiment comparing the role of microtubules in controlling cell expansion in different zones near the apex of three types of cotton fiber tips. Adding the common herbicide fluridone, 1-Methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]-4(1H)-pyridinone, to the medium supported G. barbadense ovule culture, with positive impacts on the number of useful ovules and fiber length. The effect is potentially mediated through inhibited synthesis of abscisic acid, which antagonized the positive effects of fluridone. Fiber development was perturbed by adding colchicine, a microtubule antagonist, to ovules of G. barbadense and G. hirsutum cultured 2 days after flowering. The results supported the zonal control of cell expansion in one type of G. hirsutum fiber tip and highlighted differences in the role of microtubules in modulating cell expansion between three types of cotton fiber tips.

Cellulose microfibrils are synthesized by membrane-embedded cellulose synthesis complexes (CSCs), currently modeled as hexamers of cellulose synthase (CESA) trimers. The three paralogous CESAs involved in secondary cell wall (SCW) cellulose biosynthesis in Arabidopsis (CESA4, CESA7, CESA8) are similar, but nonredundant, with all three isoforms required for assembly and function of the CSC. The molecular basis of protein-protein recognition among the isoforms is not well understood. To investigate the locations of the interfaces that are responsible for isoform recognition, we swapped three domains between the Arabidopsis CESAs required for SCW synthesis (CESA4, CESA7, and CESA8): N-terminus, central domain containing the catalytic core, and C-terminus. Chimeric genes with all pairwise permutations of the domains were tested for in vivo functionality within knockout mutant backgrounds of cesa4 , cesa7 , and cesa8 . Immunoblotting with isoform-specific antibodies confirmed the anticipated protein expression in transgenic plants. The percent recovery of stem height and crystalline cellulose content was assayed, as compared to wild type, the mutant background lines, and other controls. Retention of the native central domain was sufficient for CESA8 chimeras to function, with neither its N-terminal nor C-terminal domains required. The C-terminal domain is required for class-specific function of CESA4 and CESA7, and CESA7 also requires its own N-terminus. Across all isoforms, the results indicate that the central domain, as well as the N- and C-terminal regions, contributes to class-specific function variously in Arabidopsis CESA4, CESA7, and CESA8.

Competing Interests: The author declares no conflict of interest.

Cellulose synthases (CESAs) are glycosyltransferases that catalyze formation of cellulose microfibrils in plant cell walls. Seed plant CESA isoforms cluster in six phylogenetic clades, whose non-interchangeable members play distinct roles within cellulose synthesis complexes (CSCs). A 'class specific region' (CSR), with higher sequence similarity within versus between functional CESA classes, has been suggested to contribute to specific activities or interactions of different isoforms. We investigated CESA isoform specificity in the moss, Physcomitrella patens (Hedw.) B. S. G. to gain evolutionary insights into CESA structure/function relationships. Like seed plants, P. patens has oligomeric rosette-type CSCs, but the PpCESAs diverged independently and form a separate CESA clade. We showed that P. patens has two functionally distinct CESAs classes, based on the ability to complement the gametophore-negative phenotype of a ppcesa5 knockout line. Thus, non-interchangeable CESA classes evolved separately in mosses and seed plants. However, testing of chimeric moss CESA genes for complementation demonstrated that functional class-specificity is not determined by the CSR. Sequence analysis and computational modeling showed that the CSR is intrinsically disordered and contains predicted molecular recognition features, consistent with a possible role in CESA oligomerization and explaining the evolution of class-specific sequences without selection for class-specific function., (© 2018 Institute of Botany, Chinese Academy of Sciences.)

Leaf shape varies spectacularly among plants. Leaves are the primary source of photoassimilate in crop plants, and understanding the genetic basis of variation in leaf morphology is critical to improving agricultural productivity. Leaf shape played a unique role in cotton improvement, as breeders have selected for entire and lobed leaf morphs resulting from a single locus, okra (l-D 1 ), which is responsible for the major leaf shapes in cotton. The l-D 1 locus is not only of agricultural importance in cotton, but through pioneering chimeric and morphometric studies, it has contributed to fundamental knowledge about leaf development. Here we show that an HD-Zip transcription factor homologous to the LATE MERISTEM IDENTITY1 (LMI1) gene of Arabidopsis is the causal gene underlying the l-D 1 locus. The classical okra leaf shape allele has a 133-bp tandem duplication in the promoter, correlated with elevated expression, whereas an 8-bp deletion in the third exon of the presumed wild-type normal allele causes a frame-shifted and truncated coding sequence. Our results indicate that subokra is the ancestral leaf shape of tetraploid cotton that gave rise to the okra allele and that normal is a derived mutant allele that came to predominate and define the leaf shape of cultivated cotton. Virus-induced gene silencing (VIGS) of the LMI1-like gene in an okra variety was sufficient to induce normal leaf formation. The developmental changes in leaves conferred by this gene are associated with a photosynthetic transcriptomic signature, substantiating its use by breeders to produce a superior cotton ideotype., Competing Interests: The authors declare no conflict of interest.

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.

Cotton fibers arise through highly anisotropic expansion of a single seed epidermal cell. We obtained evidence that apical cell wall synthesis occurs through examining the tips of young elongating Gossypium hirsutum (Gh) and G. barbadense (Gb) fibers. We characterized two tip types in Gh fiber (hemisphere and tapered), each with distinct apical diameter, central vacuole location, and distribution of cell wall components. The apex of Gh hemisphere tips was enriched in homogalacturonan epitopes, including a relatively high methyl-esterified form associated with cell wall pliability. Other wall components increased behind the apex including cellulose and the α-Fuc-(1,2)-β-Gal epitope predominantly found in xyloglucan. Gb fibers had only one narrow tip type featuring characters found in each Gh tip type. Pulse-labeling of cell wall glucans indicated wall synthesis at the apex of both Gh tip types and in distal zones. Living Gh hemisphere and Gb tips ruptured preferentially at the apex upon treatment with wall degrading enzymes, consistent with newly synthesized wall at the apex. Gh tapered tips ruptured either at the apex or distantly. Overall, the results reveal diverse cotton fiber tip morphologies and support primary wall synthesis occurring at the apex and discrete distal regions of the tip.

Cellulose synthases (CESAs) synthesize the β-1,4-glucan chains that coalesce to form cellulose microfibrils in plant cell walls. In addition to a large cytosolic (catalytic) domain, CESAs have eight predicted transmembrane helices (TMHs). However, analogous to the structure of BcsA, a bacterial CESA, predicted TMH5 in CESA may instead be an interfacial helix. This would place the conserved FxVTxK motif in the plant cell cytosol where it could function as a substrate-gating loop as occurs in BcsA. To define the functional importance of the CESA region containing FxVTxK, we tested five parallel mutations in Arabidopsis thaliana CESA1 and Physcomitrella patens CESA5 in complementation assays of the relevant cesa mutants. In both organisms, the substitution of the valine or lysine residues in FxVTxK severely affected CESA function. In Arabidopsis roots, both changes were correlated with lower cellulose anisotropy, as revealed by Pontamine Fast Scarlet. Analysis of hypocotyl inner cell wall layers by atomic force microscopy showed that two altered versions of Atcesa1 could rescue cell wall phenotypes observed in the mutant background line. Overall, the data show that the FxVTxK motif is functionally important in two phylogenetically distant plant CESAs. The results show that Physcomitrella provides an efficient model for assessing the effects of engineered CESA mutations affecting primary cell wall synthesis and that diverse testing systems can lead to nuanced insights into CESA structure-function relationships. Although CESA membrane topology needs to be experimentally determined, the results support the possibility that the FxVTxK region functions similarly in CESA and BcsA., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A cellulose synthesis complex with a "rosette" shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Background: The morphogenesis of single-celled cotton fiber includes extreme elongation and staged cell wall differentiation. Designing strategies for improving cotton fiber for textiles and other uses relies on uncovering the related regulatory mechanisms. In this research we compared the transcriptomes and metabolomes of two Gossypium genotypes, Gossypium barbadense cv Phytogen 800 and G. hirsutum cv Deltapine 90. When grown in parallel, the two types of fiber developed similarly except for prolonged fiber elongation in the G. barbadense cultivar. The data were collected from isolated fibers between 10 to 28 days post anthesis (DPA) representing: primary wall synthesis to support elongation; transitional cell wall remodeling; and secondary wall cellulose synthesis, which was accompanied by continuing elongation only in G. barbadense fiber., Results: Of 206 identified fiber metabolites, 205 were held in common between the two genotypes. Approximately 38,000 transcripts were expressed in the fiber of each genotype, and these were mapped to the reference set and interpreted by homology to known genes. The developmental changes in the transcriptomes and the metabolomes were compared within and across genotypes with several novel implications. Transitional cell wall remodeling is a distinct stable developmental stage lasting at least four days (18 to 21 DPA). Expression of selected cell wall related transcripts was similar between genotypes, but cellulose synthase gene expression patterns were more complex than expected. Lignification was transcriptionally repressed in both genotypes. Oxidative stress was lower in the fiber of G. barbadense cv Phytogen 800 as compared to G. hirsutum cv Deltapine 90. Correspondingly, the G. barbadense cultivar had enhanced capacity for management of reactive oxygen species during its prolonged elongation period, as indicated by a 138-fold increase in ascorbate concentration at 28 DPA., Conclusions: The parallel data on deep-sequencing transcriptomics and non-targeted metabolomics for two genotypes of single-celled cotton fiber showed that a discrete developmental stage of transitional cell wall remodeling occurs before secondary wall cellulose synthesis begins. The data showed how lignification can be transcriptionally repressed during secondary cell wall synthesis, and they implicated enhanced capacity to manage reactive oxygen species through the ascorbate-glutathione cycle as a positive contributor to fiber length.

Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given.

The β-1,4-glucan chains comprising cellulose are synthesized by cellulose synthases in the plasma membranes of diverse organisms including bacteria and plants. Understanding structure-function relationships in the plant enzymes involved in cellulose synthesis (CESAs) is important because cellulose is the most abundant component in the plant cell wall, a key renewable biomaterial. Here, we explored the structure and function of the region encompassing transmembrane helices (TMHs) 5 and 6 in CESA using computational and genetic tools. Ab initio computational structure prediction revealed novel bi-modal structural conformations of the region between TMH5 and 6 that may affect CESA function. Here we present our computational findings on this region in three CESAs of Arabidopsis thaliana (AtCESA1, 3, and 6), the Atcesa3(ixr1-2) mutant, and a novel missense mutation in AtCESA1. A newly engineered point mutation in AtCESA1 (Atcesa1(F954L) ) that altered the structural conformation in silico resulted in a protein that was not fully functional in the temperature-sensitive Atcesa1(rsw1-1) mutant at the restrictive temperature. The combination of computational and genetic results provides evidence that the ability of the TMH5-6 region to adopt specific structural conformations is important for CESA function., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The remarkable mechanical strength of cellulose reflects the arrangement of multiple β-1,4-linked glucan chains in a para-crystalline fibril. During plant cellulose biosynthesis, a multimeric cellulose synthesis complex (CSC) moves within the plane of the plasma membrane as many glucan chains are synthesized from the same end and in close proximity. Many questions remain about the mechanism of cellulose fibril assembly, for example must multiple catalytic subunits within one CSC polymerize cellulose at the same rate? How does the cellulose fibril bend to align horizontally with the cell wall? Here we used mathematical modeling to investigate the interactions between glucan chains immediately after extrusion on the plasma membrane surface. Molecular dynamics simulations on groups of six glucans, each originating from a position approximating its extrusion site, revealed initial formation of an uncrystallized aggregate of chains from which a protofibril arose spontaneously through a ratchet mechanism involving hydrogen bonds and van der Waals interactions between glucose monomers. Consistent with the predictions from the model, freeze-fracture transmission electron microscopy using improved methods revealed a hemispherical accumulation of material at points of origination of apparent cellulose fibrils on the external surface of the plasma membrane where rosette-type CSCs were also observed. Together the data support the possibility that a zone of uncrystallized chains on the plasma membrane surface buffers the predicted variable rates of cellulose polymerization from multiple catalytic subunits within the CSC and acts as a flexible hinge allowing the horizontal alignment of the crystalline cellulose fibrils relative to the cell wall.

Detailed information about the structure and biochemical mechanisms of cellulose synthase (CelS) proteins remained elusive until a complex containing the catalytic subunit (BcsA) of CelS from Rhodobacter sphaeroides was crystalized. Additionally, a 3D structure of most of the cytosolic domain of a plant CelS (GhCESA1 from cotton, Gossypium hirsutum) was produced by computational modeling. This predicted structure contributes to our understanding of how plant CelS proteins may be similar and different as compared with BcsA. In this review, we highlight how these structures impact our understanding of the synthesis of cellulose and other extracellular polysaccharides. We show how the structures can be used to generate hypotheses for experiments testing mechanisms of glucan synthesis and translocation in plant CelS., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fourier transform infrared (FTIR) spectra of cotton fibers harvested at different stages of development were acquired using Universal Attenuated Total Reflectance FTIR (UATR-FTIR). The main goal of the study was to monitor cell wall changes occurring during different phases of cotton fiber development. Two cultivars of Gossypium hirsutum L. were planted in a greenhouse (Texas Marker-1 and TX55). On the day of flowering, individual flowers were tagged and bolls were harvested. From fibers harvested on numerous days between 10 and 56 dpa, the FTIR spectra were acquired using UATR (ZnSe-Diamond crystal) with no special sample preparation. The changes in the FTIR spectra were used to document the timing of the transition between primary and secondary cell wall synthesis. Changes in cellulose during cotton fiber growth and development were identified through changes in numerous vibrations within the spectra. The intensity of the vibration bands at 667 and 897 cm(-1) correlated with percentage of cellulose analyzed chemically., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose-synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs.

Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition, Gossypium hirsutum (Gh) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in Gh and Gossypium barbadense (Gb), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the Gb fiber had a prolonged elongation period and developed higher quality compared to Gh fiber. The Gb fibers had a CFML, but it was not directly required for fiber elongation because Gb fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of Gb fiber contained lower levels of xyloglucan compared to Gh fiber. Xyloglucan endo-hydrolase activity was also higher in Gb fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which 't' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

Background: We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns., Results: The biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively., Conclusions: These innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

The genome sequence of the moss Physcomitrella patens has stimulated new research examining the cell wall polysaccharides of mosses and the glycosyl transferases that synthesize them as a means to understand fundamental processes of cell wall biosynthesis and plant cell wall evolution. The cell walls of mosses and vascular plants are composed of the same classes of polysaccharides, but with differences in side chain composition and structure. Similarly, the genomes of P. patens and angiosperms encode the same families of cell wall glycosyl transferases, yet, in many cases these families have diversified independently in each lineage. Our understanding of land plant evolution could be enhanced by more complete knowledge of the relationships among glycosyl transferase functional diversification, cell wall structural and biochemical specialization, and the roles of cell walls in plant adaptation. As a foundation for these studies, we review the features of P. patens as an experimental system, analyses of cell wall composition in various moss species, recent studies that elucidate the structure and biosynthesis of cell wall polysaccharides in P. patens, and phylogenetic analysis of P. patens genes potentially involved in cell wall biosynthesis.

Cotton fibers are single-celled extensions of the seed epidermis. They can be isolated in pure form as they undergo staged differentiation including primary cell wall synthesis during elongation and nearly pure cellulose synthesis during secondary wall thickening. This combination of features supports clear interpretation of data about cell walls and cellulose synthesis in the context of high throughput modern experimental technologies. Prior contributions of cotton fiber to building fundamental knowledge about cell walls will be summarized and the dynamic changes in cell wall polymers throughout cotton fiber differentiation will be described. Recent successes in using stable cotton transformation to alter cotton fiber cell wall properties as well as cotton fiber quality will be discussed. Futurec prospects to perform experiments more rapidly through altering cotton fiberwall properties via virus-induced gene silencing will be evaluated.

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Genetic manipulation of cellulose biosynthesis in trees may provide novel insights into the growth and development of trees. To explore this possibility, the overexpression of an aspen secondary wall-associated cellulose synthase (PtdCesA8) gene was attempted in transgenic aspen (Populus tremuloides L.) and unexpectedly resulted in silencing of the transgene as well as its endogenous counterparts. The main axis of the transgenic aspen plants quickly stopped growing, and weak branches adopted a weeping growth habit. Furthermore, transgenic plants initially developed smaller leaves and a less extensive root system. Secondary xylem (wood) of transgenic aspen plants contained as little as 10% cellulose normalized to dry weight compared to 41% cellulose typically found in normal aspen wood. This massive reduction in cellulose was accompanied by proportional increases in lignin (35%) and non-cellulosic polysaccharides (55%) compared to the 22% lignin and 36% non-cellulosic polysaccharides in control plants. The transgenic stems produced typical collapsed or 'irregular' xylem vessels that had altered secondary wall morphology and contained greatly reduced amounts of crystalline cellulose. These results demonstrate the fundamental role of secondary wall cellulose within the secondary xylem in maintaining the strength and structural integrity required to establish the vertical growth habit in trees.

Background: Understanding the evolutionary genetics of modern crop phenotypes has a dual relevance to evolutionary biology and crop improvement. Modern upland cotton (Gossypium hirsutum L.) was developed following thousands of years of artificial selection from a wild form, G. hirsutum var. yucatanense, which bears a shorter, sparser, layer of single-celled, ovular trichomes ('fibre'). In order to gain an insight into the nature of the developmental genetic transformations that accompanied domestication and crop improvement, we studied the transcriptomes of cotton fibres from wild and domesticated accessions over a developmental time course., Results: Fibre cells were harvested between 2 and 25 days post-anthesis and encompassed the primary and secondary wall synthesis stages. Using amplified messenger RNA and a custom microarray platform designed to interrogate expression for 40,430 genes, we determined global patterns of expression during fibre development. The fibre transcriptome of domesticated cotton is far more dynamic than that of wild cotton, with over twice as many genes being differentially expressed during development (12,626 versus 5273). Remarkably, a total of 9465 genes were diagnosed as differentially expressed between wild and domesticated fibres when summed across five key developmental time points. Human selection during the initial domestication and subsequent crop improvement has resulted in a biased upregulation of components of the transcriptional network that are important for agronomically advanced fibre, especially in the early stages of development. About 15% of the differentially expressed genes in wild versus domesticated cotton fibre have no homology to the genes in databases., Conclusions: We show that artificial selection during crop domestication can radically alter the transcriptional developmental network of even a single-celled structure, affecting nearly a quarter of the genes in the genome. Gene expression during fibre development within accessions and expression alteration arising from evolutionary change appears to be 'modular' - complex genic networks have been simultaneously and similarly transformed, in a coordinated fashion, as a consequence of human-mediated selection. These results highlight the complex alteration of the global gene expression machinery that resulted from human selection for a longer, stronger and finer fibre, as well as other aspects of fibre physiology that were not consciously selected. We illustrate how the data can be mined for genes that were unwittingly targeted by aboriginal and/or modern domesticators during crop improvement and/or which potentially control the improved qualities of domesticated cotton fibre.See Commentary: http://www.biomedcentral.com/1741-7007/8/137.

Construction of three-dimensional volumes from a series of two-dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour-keying and manipulation of two-dimensional images in true three-dimensional space allowed us to construct three-dimensional volumes from pixel-based images of stained plant and animal tissue without generating vector information. We present three-dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin-embedded tissues. It is widely applicable, having potential to recover three-dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy., (Published 2010. This article is a US Government work and is in the public domain in the USA.)

Abstract 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, AtCesA1,2,3,5, 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.

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.

Use of a synthetic auxin (naphthalene-1-acetic acid, NAA) to start (Gossypium hirsutum) ovule/fiber cultures hindered fiber secondary wall cellulose synthesis compared with natural auxin (indole-3-acetic acid, IAA). In contrast, NAA promoted fiber elongation and ovule weight gain, which resulted in larger ovule/fiber units. To reach these conclusions, fiber and ovule growth parameters were measured and cell wall characteristics were examined microscopically. The differences in fiber from NAA and IAA culture were underpinned by changes in the expression patterns of marker genes for three fiber developmental stages (elongation, the transition stage, and secondary wall deposition), and these gene expression patterns were also analyzed quantitatively in plant-grown fiber. The results demonstrate that secondary wall cellulose synthesis: (1) is under strong transcriptional control that is influenced by auxin; and (2) must be specifically characterized in the cotton ovule/fiber culture system given the many protocol variables employed in different laboratories.

Cotton (Gossypium hirsutum) provides the world's dominant renewable textile fiber, and cotton fiber is valued as a research model because of its extensive elongation and secondary wall thickening. Previously, it was assumed that fibers elongated as individual cells. In contrast, observation by cryo-field emission-scanning electron microscopy of cotton fibers developing in situ within the boll demonstrated that fibers elongate within tissue-like bundles. These bundles were entrained by twisting fiber tips and consolidated by adhesion of a cotton fiber middle lamella (CFML). The fiber bundles consolidated via the CFML ultimately formed a packet of fiber around each seed, which helps explain how thousands of cotton fibers achieve their great length within a confined space. The cell wall nature of the CFML was characterized using transmission electron microscopy, including polymer epitope labeling. Toward the end of elongation, up-regulation occurred in gene expression and enzyme activities related to cell wall hydrolysis, and targeted breakdown of the CFML restored fiber individuality. At the same time, losses occurred in certain cell wall polymer epitopes (as revealed by comprehensive microarray polymer profiling) and sugars within noncellulosic matrix components (as revealed by gas chromatography-mass spectrometry analysis of derivatized neutral and acidic glycosyl residues). Broadly, these data show that adhesion modulated by an outer layer of the primary wall can coordinate the extensive growth of a large group of cells and illustrate dynamic changes in primary wall structure and composition occurring during the differentiation of one cell type that spends only part of its life as a tissue.

A new procedure has been developed for the isolation of wild-type and mutant Arabidopsis trichomes. The isolated trichomes maintained enzymatic activity and were used for DNA, protein, and RNA isolation. The RNA was used to generate probes suitable for Affymetrix analysis. The validity of the Affymetrix results was confirmed by quantitative PCR analysis on a subset of genes that are preferentially expressed in trichomes or leaves. Sufficient quantities of trichomes were isolated to probe the biochemical nature of trichome cell walls. These analyses provide evidence for the presence of lignin in Arabidopsis trichome cell walls. The monosaccharide analysis and positive staining with ruthenium red indicates that the walls also contain a large portion of pectin. The 2.23-fold ratio of pectin-related sugars compared with potential cellulosic glucose suggests that the polysaccharides of the trichome cell walls are more like those of typical primary walls even though the wall becomes quite thick. Overall, these analyses open the door to using the Arabidopsis trichome cell wall as an excellent model to probe various questions concerning plant cell wall biosynthesis.

Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar 'Deltapine 5415' bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30 degrees C/26 degrees C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22 degrees C/18 degrees C. However, endogenous gene silencing decreased at 30 degrees C/26 degrees C. There was an approximately 7 d delay in the onset of gene silencing at 22 degrees C/18 degrees C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

Members of the large Arabidopsis NAC domain transcription factor family are regulators of meristem development, organ elongation and separation, and deposition of patterned secondary cell walls. XYLEM NAC DOMAIN 1 (XND1) is highly expressed in xylem. Changes observed for XND1 knockout plants compared with wild-type Arabidopsis thaliana included a reduction in both plant height and tracheary element length and an increase in metaxylem relative to protoxylem in roots of plants treated with the proteasome inhibitor MG132. Overexpression of XND1 resulted in extreme dwarfism associated with the absence of xylem vessels and little or no expression of tracheary element marker genes. In contrast, phloem marker-gene expression was not altered and phloem-type cells still formed. Transmission electron microscopy showed that parenchyma-like cells in the incipient xylem zone in hypocotyls of XND1 overexpressors lacked secondary wall thickenings and retained their cytoplasmic content. Considered together, these findings suggest that XND1 affects tracheary element growth through regulation of secondary wall synthesis and programmed cell death.

Prior data indicated that enhanced availability of sucrose, a major product of photosynthesis in source leaves and the carbon source for secondary wall cellulose synthesis in fiber sinks, might improve fiber quality under abiotic stress conditions. To test this hypothesis, a family of transgenic cotton plants (Gossypium hirsutum cv. Coker 312 elite) was produced that over-expressed spinach sucrose-phosphate synthase (SPS) because of its role in regulation of sucrose synthesis in photosynthetic and heterotrophic tissues. A family of 12 independent transgenic lines was characterized in terms of foreign gene insertion, expression of spinach SPS, production of spinach SPS protein, and development of enhanced extractable V (max) SPS activity in leaf and fiber. Lines with the highest V (max) SPS activity were further characterized in terms of carbon partitioning and fiber quality compared to wild-type and transgenic null controls. Leaves of transgenic SPS over-expressing lines showed higher sucrose:starch ratio and partitioning of (14)C to sucrose in preference to starch. In two growth chamber experiments with cool nights, ambient CO(2) concentration, and limited light below the canopy, the transgenic line with the highest SPS activity in leaf and fiber had higher fiber micronaire and maturity ratio associated with greater thickness of the cellulosic secondary wall.

The aim of this study was to characterise the response of CO 2 assimilation (A) of cotton (Gossypium hirsutum L.) to short- and long-term exposures to night chilling. We hypothesised that short-term exposures to night chilling would induce reductions in g s and, therefore, A during the following days, while growth of cotton plants for several weeks in cool night conditions would cause elevated leaf carbohydrate content, leading to the down-regulation of the capacity for A. Transferring warm-grown seedlings of wild type cotton, transgenic cotton with elevated sucrose-phosphate synthase activity (SPS+) that might produce and export more sucrose from the leaf, and a segregating null to cool nights (9°C minimum) for 1 or 2 d caused a small reduction in A (12%) and g s (21-50%) measured at 28°C. Internal CO 2 did not change, suggesting some biochemical restriction of A along with a g s restriction. After 30 d, new leaves that developed in cool nights exhibited acclimation of A and partial acclimation of g s . Despite the elevated leaf carbohydrate content when plants were grown to maturity with night chilling, no reduction in A, g s , carboxylation capacity, electron transport capacity, or triose-phosphate utilisation capacity occurred. Instead, growth in cool nights tended to retard the diminishing of photosynthetic parameters and g s for aging stem and subtending leaves. However, elevated SPS activity did not affect any photosynthetic parameters. Therefore, when cotton that is well fertilised with nitrogen is grown with continuous night chilling, photosynthesis should not be negatively affected. However, an occasional exposure to cool nights could result in a small reduction in A and g s for leaves that have developed in warm night conditions.

The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC50 values during primary and secondary wall synthesis in Z. elegans were 3.91 x 10(-8) M and 3.67 x 10(-9) M, respectively. The IC50 in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67 x 10(-10) M. At maximally inhibitory concentrations, 18-33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 microM 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.

Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 microm thick. Immunolabeling also showed that callose (beta-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers.

Cellulose is an important component of the cell walls of higher plants and the world's most abundant organic compound. As a major sink for carbon on earth, it is of interest to examine possible means by which the quality or quantity of cellulose deposited in various plant parts might be manipulated by metabolic engineering techniques. This review outlines basic knowledge about the genes and proteins that are involved in cellulose biosynthesis and presents a model that summarizes our current thinking on the overall cellulose biosynthesis pathway. Strategies that might be used for altering the flux of carbon into this pathway are discussed.

Based on work with cotton fibers, a particulate form of sucrose (Suc) synthase was proposed to support secondary wall cellulose synthesis by degrading Suc to fructose and UDP-glucose. The model proposed that UDP-glucose was then channeled to cellulose synthase in the plasma membrane, and it implies that Suc availability in cellulose sink cells would affect the rate of cellulose synthesis. Therefore, if cellulose sink cells could synthesize Suc and/or had the capacity to recycle the fructose released by Suc synthase back to Suc, cellulose synthesis might be supported. The capacity of cellulose sink cells to synthesize Suc was tested by analyzing the Suc phosphate synthase (SPS) activity of three heterotrophic systems with cellulose-rich secondary walls. SPS is a primary regulator of the Suc synthesis rate in leaves and some Suc-storing, heterotrophic organs, but its activity has not been previously correlated with cellulose synthesis. Two systems analyzed, cultured mesophyll cells of Zinnia elegans L. var. Envy and etiolated hypocotyls of kidney beans (Phaseolus vulgaris), contained differentiating tracheary elements. Cotton (Gossypium hirsutum L. cv Acala SJ-1) fibers were also analyzed during primary and secondary wall synthesis. SPS activity rose in all three systems during periods of maximum cellulose deposition within secondary walls. The Z. elegans culture system was manipulated to establish a tight linkage between the timing of tracheary element differentiation and rising SPS activity and to show that SPS activity did not depend on the availability of starch for degradation. The significance of these findings in regard to directing metabolic flux toward cellulose will be discussed.

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca2+ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis.