Secondary Wall Cellulose Research Articles

A synthetic auxin (NAA) suppresses secondary wall cellulose synthesis and enhances elongation in cultured cotton fiber

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.

A Cellulose Synthase-Containing Compartment Moves Rapidly Beneath Sites of Secondary Wall Synthesis

The woody secondary walls of plants represent the major sites of cellulose deposition. The polymerization of cellulose occurs at the plasma membrane by the secondary wall cellulose synthase complex (CSC). In the long, cylindrical cells that make up the xylem, secondary wall deposition is confined to discrete regions of the cell, and yellow fluorescent protein (YFP)-labeled CSCs are also localized to these regions. Using fluorescence loss in photobleaching (FLIP) of complete hoops containing YFP-CSCs, we demonstrate movement of the complexes beneath the nascent secondary wall in developing xylem vessels. We have devised a method for determining particle velocities for particles moving around a cylindrical object using data from FLIP. By applying this method to the hoops of YFP-CSCs of the developing vessels, we have obtained the first estimates of speed of these complexes. These speeds are calculated to be in excess of 7 microm s(-1) and are far higher than those speeds previously reported for the primary wall complex. These high speeds are unlikely to be consistent with CSC movement being attributed to cellulose synthesis alone, and suggest the existence of a highly motile compartment beneath the nascent secondary wall.

Elucidating the Mechanisms of Assembly and Subunit Interaction of the Cellulose Synthase Complex of Arabidopsis Secondary Cell Walls

Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.

Carbohydrate-related genes and cell wall biosynthesis in vascular tissues of loblolly pine (Pinus taeda)

Loblolly pine (Pinus taeda L.), the most widely planted tree species in the United States, is an important source of wood and wood fibers for a multitude of consumer products. Wood fibers are primarily composed of secondary cell walls, and cellulose, hemicelluloses and lignin are major components of wood. Fiber morphology and cell wall composition are important determinants of wood properties. We used comparative genomics to identify putative genes for cellulose and hemicellulose synthesis in loblolly pine that are homologous to genes implicated in cell wall synthesis in angiosperms. Sequences encoding putative secondary cell wall cellulose synthase genes, cellulose synthase-like genes, a membrane-bound endoglucanase gene, a sucrose synthase gene, a UDP-glucose pyrophosphorylase gene and GDP-mannose pyrophosphorylase genes were identified in expressed sequence tag (EST) collections from loblolly pine. Full-length coding sequences were obtained from cDNA clones isolated from a library constructed from developing xylem. Phylogenetic relationships between the genes from loblolly pine and angiosperm taxa were examined and transcriptional profiling in vascular tissues was conducted by real-time quantitative, reverse transcriptase-polymerase chain reaction. The putative cell wall synthesis genes were expressed at high levels in vascular tissues and a subset was differentially regulated in xylem and phloem tissues. Inferred phylogenetic relationships and expression patterns for the genes from loblolly pine were consistent with roles in synthesis of complex carbohydrates of the cell wall. These studies suggest functional conservation of homologous wood formation genes in gymnosperm and angiosperm taxa.

Morphological and structural study of seed pericarp of Opuntia ficus-indica prickly pear fruits

The morphological study of pericarp of Opuntia ficus-indica (OFI) seeds showed that the cells were mainly made up of spindle-shaped sclerenchyma fibers. The chemical composition of the pericarp revealed a significant amount of polysaccharides, with cellulose (35%) and xylan (27%). The structure of xylan and cellulose, both in isolated form and as a component of seed pericarp of OFI were studied by X-ray and CP/MAS 13C NMR spectroscopy. The supramolecular structure of xylan is very sensitive to the surrounding environment, in particular to the presence of water and of cellulose fibers. The cellulose fibers presented X-ray diagrams typical of secondary wall cellulose but they were sensitive toward NaOH since they started to be converted into cellulose II at a NaOH concentration as low as 8%. In seed pericarp, cellulose fibers interact with xylan polymers, causing these to adopt a conformation different to the one observed for xylan both in dry or hydrated form, suggesting that xylans were probably present as composites with cellulose fibers.

Identification of cellulose synthase AtCesA7 (IRX3) in vivo phosphorylation sites—a potential role in regulating protein degradation

Cellulose is central to plant development and is synthesised at the plasma membrane by an organised protein complex that contains three different cellulose synthase proteins. The ordered assembly of these three catalytic subunits is essential for normal cellulose synthesis. The way in which the relative levels of these three proteins are regulated within the cell is currently unknown. In this work it is shown that one of the cellulose synthases essential for secondary cell wall cellulose synthesis in Arabidopsis thaliana, AtCesA7, is phosphorylated in vivo. Analysis of in vivo phosphorylation sites by mass spectrometry reveals that two serine residues are phosphorylated. These residues occur in a region of hyper-variability between the cellulose synthase catalytic subunits. The region of the protein containing these phosphorylation sites can be phosphorylated by a plant extract in vitro. Incubation of this region with plant extracts results in its degradation via a proteasome dependant pathway. Full length endogenous CesA7 is also degraded via a proteasome dependant pathway in whole plant extracts. This data suggests that phosphorylation of the catalytic subunits may target them for degradation via a proteasome dependant pathway. This is a possible mechanism by which plants regulate the relative levels of the three proteins whose specific interaction are required to form an active cellulose synthase complex.

Transgenic cotton over-producing spinach sucrose phosphate synthase showed enhanced leaf sucrose synthesis and improved fiber quality under controlled environmental conditions

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.

Rapid cell expansion and cellulose synthesis regulated by plasmodesmata and sugar: insights from the single-celled cotton fibre.

Higher plants comprise mixtures of some 40 different cell types, and this often complicates the interpretation of data obtained at the tissue level. Studies for a given cell type may provide novel insights into the mechanisms underlying defined cellular and developmental processes. In this regard, the cotton fibre represents an excellent single-cell model to study the control of rapid cell elongation and cellulose synthesis. These single cells, initiated from the ovule epidermis at anthesis, typically elongate to ~3-5 cm in the tetraploid species before they switch to intensive secondary cell wall cellulose synthesis. By maturity, more than 94% of fibre weight is cellulose. To unravel the mechanisms of fibre elongation and cellulose synthesis, two hypotheses have been examined: (a) that sucrose degradation and utilisation mediated by sucrose synthase (Sus) may play roles in fibre development and (b) that symplastic isolation of the fibre cells may be required for their rapid elongation. Reverse genetic and biochemical analyses have revealed the critical role that Sus plays in fibre initiation and early elongation. Late in development, plasma-membrane and cell wall association of Sus protein seems to be involved in rapid cellulose synthesis. Cell biology and gene expression studies showed a temporary closure of fibre plasmodesmata (PD), probably due to the deposition of callose, at the rapid phase of elongation. The duration of the PD closure correlates positively with the final fibre length attained. These data support the view that PD closure may be required for fibres to achieve extended elongation. The branching of PD towards the secondary cell wall stage is postulated to function as a molecule sieve for tight control of macromolecule trafficking into fibres to sustain intensive cellulose synthesis.

Recent advances in understanding cotton fibre and seed development

The unique feature of the seed of tetraploid cotton (Gossypium hirsutumandGossypium barbadense) is that about 30% of the seed coat epidermal cells develop into cellulose-enriched fibres, while the embryos synthesize oils and proteins. Hence, both the maternal and filial tissues of the cotton seed are of significant economic value. After initiation from the ovule epidermis at or just before anthesis, the single-celled fibres elongate to 2.5–6.0 cm long in the tetraploid species before they switch to intensive secondary cell wall cellulose synthesis. Thus, apart from its agronomic importance, the cotton fibre represents a model single-cell system to study the control of cell differentiation and elongation, carbon partitioning to cellulose synthesis and also the interaction between maternal (fibre) and embryonic tissues in seeds. Over the past decade or so, significant effort has been made to understand the cellular and molecular basis of cotton fibre development and oil biosynthesis in the embryo. Metabolic engineering of the oil biosynthetic pathway in cotton seed has successfully produced healthier and stable oils. A number of candidate genes and cellular processes that potentially regulate various aspects of fibre development have been identified. Further elucidation of thein vivofunctions of those candidate genes could significantly deepen our understanding of fibre development and offer potential for improvement of fibre quality through genetic engineering or marker-assisted breeding approaches.

Biotechnological improvement of cotton fibre maturity

This mini‐review focuses on the prospects and tools for controlling cotton fibre secondary wall thickness. Cotton fibre secondary walls are composed of almost 100% cellulose, and are responsible for fibre maturity and a large component of fibre yield. Improved fibre yield and maturity would result from the ability to control secondary wall cellulose deposition quantitatively, including making the process less sensitive to environmental stress. Both genetic engineering and marker‐assisted breeding are possible avenues for effecting such improvements, but first key genes that participate in the regulation and control of secondary wall cellulose biogenesis must be identified. Recent advances towards understanding and manipulating cotton fibre secondary wall deposition that are discussed here include: (i) experimental approaches to identify metabolic participants in cellulose biogenesis; (ii) isolation and characterization of promoters to drive foreign gene expression preferentially during secondary wall deposition; and (iii) a novel set of cDNA sequences representing genes that are differentially expressed during cotton fibre secondary wall deposition compared with primary wall deposition.

Cellulose synthesis in the Arabidopsis secondary cell wall

The identification of genes responsible for cellulose synthesis has led to a significant advance in our understanding of the synthesis of this important polymer. The identification of these genes has arisen from the identification of cellulose deficient mutants. The irregular xylem (irx) mutants of Arabidopsis are caused by a severe reduction in cellulose synthesis in the secondary cell wall. Three irx mutants deficient in secondary cell wall cellulose are the result of mutations in three different members of the cellulose synthase catalytic subunit (CesA) gene family. The three proteins encoded by these genes all associate within the same membrane bound complex, and the presence of all three, but not their activity, is required for correct assembly and targeting of this complex. The knowledge that these three proteins associate provides a good opportunity to purify the cellulose synthase complex, and recent results working towards this goal are discussed.

Cool temperature hinders flux from glucose to sucrose during cellulose synthesis in secondary wall stage cotton fibers

Current knowledge about the integration of cellulose synthesis into cellular carbon metabolism and the cool temperature sensitivity of cellulose synthesis is reviewed briefly. Roles for sucrose synthase (to channel UDP-glucose to the cellulose synthase) and sucrose phosphate synthase (to recycle the fructose released by sucrose synthase to more sucrose) in secondary wall cellulose synthesis are described. Data are presented that implicate sucrose synthesis within cotton fibers as a particularly cool temperature-sensitive step in the partitioning of carbon to cellulose. Sugar metabolism during fiber secondary wall deposition was analyzed in in vitro cultures of ovules from two cultivars of Gossypium hirsutum L. (cv. Acala SJ-1 and cv. Paymaster HS 200), which had different levels of cool temperature sensitivity. The sizes of the sucrose, glucose, and fructose pools within fibers at 4 and 7 h after a temperature shift to 15 or 34 °C did not change in either cultivar. Feeding exogenous U-14C-glucose in pulse and pulse/chase experiments showed that uptake of glucose and transport through the ovule into fibers occurred at the same rate at 34 and 15 °C. In contrast, the flux from glucose to sucrose within fibers was greatly hindered at 15 °C in both cultivars. Since sucrose is the preferred donor of UDP-Glc to the cellulose synthase during secondary wall deposition, this sensitivity in sucrose synthesis is likely to at least partially explain the cool temperature sensitivity of cotton fiber cellulose synthesis that is observed in the field.

Xylem-Specific and Tension Stress-Responsive Expression of Cellulose Synthase Genes from Aspen Trees

Genetic improvement of cellulose biosynthesis in woody trees is one of the major goals of tree biotechnology research. Yet, progress in this field has been slow owing to (1) unavailability of key genes from tree genomes, (2) the inability to isolate active and intact cellulose synthase complexes and, (3) the limited understanding of the mechanistic processes involved in the wood cellulose development. Here I report on the recent advances in molecular genetics of cellulose synthases (CesA) from aspen trees. Two different types of cellulose synthases appear to be involved in cellulose deposition in primary and secondary walls in aspen xylem. The three distinct secondary CesAs from aspen- PtrCesA1, PtrCesA2, and PtrCesA3-appear to be aspen homologs of Arabidopsis secondary CesAs AtCesA8, AtCesA7, and AtCesA4, respectively, based on their high identity/similarity (>80%). These aspen CesA proteins share the transmembrane domain (TMD) structure that is typical of all known "true" CesA proteins: two TMDs toward the N-terminal and six TMDs toward the C-terminal. The putative catalytic domain is present between TMDs 2 and 3. All signature motifs of processive glycosyltransferases are also present in this catalytic domain. In a phylogenetic tree based on various predicted CesA proteins from Arabidopsis and aspen, aspen CesAs fall into families similar to those seen with Arabidopsis CesAs, suggesting their functional similarity. The coordinate expression of three aspen secondary CesAs in xylem and phloem fibers, along with their simultaneous tension stress-responsive upregulation, suggests that these three CesAs may play a pivotal role in biosynthesis of better-quality cellulose in secondary cell walls of plants. These results are likely to have a direct impact on genetic manipulation of trees in the future.

Cellulose biosynthesis in plants: from genes to rosettes.

Modern techniques of gene cloning have identified the CesA genes as encoding the probable catalytic subunits of the plant CelS, the cellulose synthase enzyme complex visualized in the plasma membrane as rosettes. At least 10 CesA isoforms exist in Arabidopsis and have been shown by mutant analyses to play distinct role/s in the cellulose synthesis process. Functional specialization within this family includes differences in gene expression, regulation and, possibly, catalytic function. Current data points towards some CesA isoforms potentially being responsible for initiation or elongation of the recently identified sterol beta-glucoside primer within different cell types, e.g. those undergoing either primary or secondary wall cellulose synthesis. Different CesA isoforms may also play distinct roles within the rosette, and there is some circumstantial evidence that CesA genes may encode the catalytic subunit of the mixed linkage glucan synthase or callose synthase. Various other proteins such as the Korrigan endocellulase, sucrose synthase, cytoskeletal components, Rac13, redox proteins and a lipid transfer protein have been implicated to be involved in synthesizing cellulose but, apart from CesAs, only Korrigan has been definitively linked with cellulose synthesis. These proteins should prove valuable in identifying additional CelS components.

Sucrose Phosphate Synthase Activity Rises in Correlation with High-Rate Cellulose Synthesis in Three Heterotrophic Systems

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.

Sucrose synthase localizes to cellulose synthesis sites in tracheary elements

The synthesis of crystalline cellulose microfibrils in plants is a highly coordinated process that occurs at the interface of the cortex, plasma membrane, and cell wall. There is evidence that cellulose biogenesis is facilitated by the interaction of several proteins, but the details are just beginning to be understood. In particular, sucrose synthase, microtubules, and actin have been proposed to possibly associate with cellulose synthases (microfibril terminal complexes) in the plasma membrane. Differentiating tracheary elements of Zinnia elegans L. were used as a model system to determine the localization of sucrose synthase and actin in relation to the plasma membrane and its underlying microtubules during the deposition of patterned, cellulose-rich secondary walls. Cortical actin occurs with similar density both between and under secondary wall thickenings. In contrast, sucrose synthase is highly enriched near the plasma membrane and the microtubules under the secondary wall thickenings. Both actin and sucrose synthase lie closer to the plasma membrane than the microtubules. These results show that the preferential localization of sucrose synthase at sites of high-rate cellulose synthesis can be generalized beyond cotton fibers, and they establish a spatial context for further work on a multi-protein complex that may facilitate secondary wall cellulose synthesis.

Mutations of the secondary cell wall.

It has not been possible to isolate a number of crucial enzymes involved in plant cell wall synthesis. Recent progress in identifying some of these steps has been overcome by the isolation of mutants defective in various aspects of cell wall synthesis and the use of these mutants to identify the corresponding genes. Secondary cell walls offer numerous advantages for genetic analysis of plant cell walls. It is possible to recover very severe mutants since the plants remain viable. In addition, although variation in secondary cell wall composition occurs between different species and between different cell types, the composition of the walls is relatively simple compared to primary cell walls. Despite these advantages, relatively few secondary cell wall mutations have been described to date. The only secondary cell wall mutations characterised to date, in which the basis of the abnormality is known, have defects in either the control of secondary cell wall deposition or secondary cell wall cellulose or lignin biosynthesis. These mutants have, however, provided essential information on secondary cell wall biosynthesis.

Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis.

The irregular xylem 1 (irx1) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.

Quantitative analysis for the cellulose I α crystalline phase in developing wood cell walls

FT-IR and X-ray analyses were employed to determine the relative ratio of cellulose I α and I β crystalline phases present in each developmental stage of coniferous tracheid cell wall formation. The IR spectra showed that initially the I α phase occupies 50% of the crystalline regions in the primary cell wall cellulose and this value drops to 20% after ceasing of the cell enlarging growth for the formation of the secondary wall cellulose (the remaining regions are composed of the I β phase). Although it is reasonable that the content for I β , which is stress-reduced crystalline form, was higher in the secondary wall formation (Kataoka Y, and Kondo T. Macromolecules 1996;29:6356–6358) it is more interesting that during the crystallization of stress-induced I α cellulose for the primary wall the stress-reduced I β is also possible to be crystallized in an alternative way. This means that throughout the period the I α -causing stress may not be necessarily kept loaded. In light of our previously reported hypothesis (Kataoka Y. and Kondo T. Macromolecules 1998;31:760–764) for the formation of I α phase due to cellular growing stresses in the primary wall cellulose, such an alternating on–off stress effect to account for the occurrence of both I α and I β phases might be related to a biological growth system in coniferous wood cells.

FT-IR Microscopic Analysis of Changing Cellulose Crystalline Structure during Wood Cell Wall Formation

A Fourier transform infrared (FT-IR) spectrometer equipped with a microscopic accessory was used to monitor the cellulose crystalline structure at each developmental stage of coniferous tracheid cell wall formation. The spectra showed that the cellulose in the primary cell wall was rich in the metastable Iα crystalline form and interestingly had a higher crystallinity than the secondary wall cellulose composed mainly of the stable Iβ crystalline phase. These results indicate the presence of an in vivo physical force at the cell surface which may stress the primary wall cellulose during the crystallization. Moreover, the primary wall cellulose was oriented parallel to the enlarging direction of growing cells. Thus, we consider that the cellular growing stress generated between the plasma membrane and the primary wall may elongate just-biosynthesized β-glucan chains and cause them to crystallize with a higher crystallinity of the metastable Iα phase, as is the case for crystallization occurring during the d...