Carbon partitioning to cellulose synthesis.

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.

Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants.

In many higher plants, cellulose synthesis is inhibited by isoxaben and thiazolidinone herbicides such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone. Semidominant mutations at the IXR1 and IXR2 loci of Arabidopsis confer isoxaben and thiazolidinone resistance. Isolation of the IXR1 gene by map-based cloning revealed that it encodes the AtCESA3 isoform of cellulose synthase. The two known mutant alleles contain point mutations that replace glycine 998 with aspartic acid, and threonine 942 with isoleucine, respectively. The mutations occur in a highly conserved region of the enzyme near the carboxyl terminus that is well separated from the proposed active site. Although the IXR1 gene is expressed in the same cells as the structurally related RSW1 (AtCESA1) cellulose synthase gene, these two CESA genes are not functionally redundant.

The experimental herbicide CGA 325'615 inhibits synthesis of crystalline cellulose and causes accumulation of non-crystalline beta-1,4-glucan associated with CesA protein.

Developing cotton (Gossypium hirsutum) fibers, cultured in vitro with their associated ovules, were used to compare the effects of two herbicides that inhibit cellulose synthesis: 2,6-dichlorobenzonitrile (DCB) and an experimental thiatriazine-based herbicide, CGA 325'615. CGA 325'615 in nanomolar concentrations or DCB in micromolar concentrations causes inhibition of synthesis of crystalline cellulose. Unlike DCB, CGA 325'615 also causes concomitant accumulation of non-crystalline beta-1,4-glucan that can be at least partially solubilized from fiber walls with ammonium oxalate. The unusual solubility of this accumulated glucan may be explained by its strong association with protein. Treatment of the glucan fraction with protease changes its size distribution and leads to precipitation of the glucan. Treatment of the glucan fraction with cellulase digests the glucan and also releases protein that has been characterized as GhCesA-1 and GhCesA-2--proteins that are believed to represent the catalytic subunit of cellulose synthase. The fact that cellulase treatment is required to release this protein indicates an extremely tight association of the glucan with the CesA proteins. In addition, CGA 325'615, but not DCB, also causes accumulation of CesA protein and a membrane-associated cellulase in the membrane fraction of fibers. In addition to the effects of CGA 325'615 on levels of both of these proteins, the level of both also shows coordinate regulation during fiber development, further suggesting they are both important for cellulose synthesis. The accumulation of non-crystalline glucan caused by CGA 325'615 mimics the phenotype of the cellulose-deficient rsw1 mutant of Arabidopsis that also accumulates an apparently similar glucan (T. Arioli, L. Peng, A.S. Betzner, J. Burn, W. Wittke, W. Herth, C. Camilleri, H. Hofte, J. Plazinski, R. Birch et al. [1998] Science 279: 717).

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.

A comparative analysis of the plant cellulose synthase (CesA) gene family.

CesA genes are believed to encode the catalytic subunit of cellulose synthase. Identification of nine distinct CesA cDNAs from maize (Zea mays) has allowed us to initiate comparative studies with homologs from Arabidopsis and other plant species. Mapping studies show that closely related CesA genes are not clustered but are found at different chromosomal locations in both Arabidopsis and maize. Furthermore, sequence comparisons among the CesA-deduced proteins show that these cluster in groups wherein orthologs are often more similar than paralogs, indicating that different subclasses evolved prior to the divergence of the monocot and dicot lineages. Studies using reverse transcriptase polymerase chain reaction with gene-specific primers for six of the nine maize genes indicate that all genes are expressed to at least some level in all of the organs examined. However, when expression patterns for a few selected genes from maize and Arabidopsis were analyzed in more detail, they were found to be expressed in unique cell types engaged in either primary or secondary wall synthesis. These studies also indicate that amino acid sequence comparisons, at least in some cases, may have value for prediction of such patterns of gene expression. Such analyses begin to provide insights useful for future genetic engineering of cellulose deposition, in that identification of close orthologs across species may prove useful for prediction of patterns of gene expression and may also aid in prediction of mutant combinations that may be necessary to generate severe phenotypes.

Identification of a novel triterpenoid saponin from Pisum sativum as a specific inhibitor of the diguanylate cyclase of Acetobacter xylinum.

A specific and highly potent inhibitor of diguanylate cyclase, the key regulatory enzyme of the cellulose synthesizing apparatus in the bacterium Acetobacter xylinum, was isolated from extracts of etiolated pea shoots (Pisum sativum). The inhibitor has been purified by a multistep procedure, and sufficient amounts of highly purified compound (3-8 mg) for spectral analysis were obtained. The structure of this compound was established as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1--> 2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside. The structure was elucidated on the basis of susceptibility to various enzymes, chemical and spectral methods, such as GC-MS, FAB-MS, and the following types of 2D-NMR: COSY, ROESY, TOCSEY, HMQC, HMBC analyses. An identical or a very similar compound with identical biological activity was also isolated from A. xylinum, strongly suggesting that at least certain aspects of cellulose synthesis in the bacteria and in higher plants may be regulated in a similar manner. The content of this saponin in etiolated plants was about 0.04 mumol (g fresh tissue)-1.

A microtiter-based fluorescence assay for (1,3)-beta-glucan synthases.

A high-throughput assay for UDP-Glc:(1,3)-beta-glucan synthase(EC 2.4.1.34, UDP-glucose:1,3-beta-D-glucan, 3-beta-glucosyltransferase) from fungi and higher plants is described. The assay is performed in microtiter plates and is extremely inexpensive compared to other standard assays for these enzymes. The reduction in price is achieved by replacing the conventional substrate UDP-[14C]Glc with its nonradioactive counterpart, and the nonradioactive glucan produced is quantified as a fluorescent complex following specific interaction with the fluorochrome present in commercial aniline blue. In addition to a > 100-fold reduction in cost, the assay is highly reproducible and nearly as sensitive as radioactive assays and has the additional advantages of increased safety and avoidance of the need for filtration and washing steps to collect the glucan product. As such, the assay is highly suitable for high-throughput screening for inhibitors of these enzymes.

Structures and functions of annexins in plants.

The first evidence that higher plants contain annexins was presented in 1989. Since that time, annexins have been purfied and characterized from a variety of plant sources. Analyses of the deduced proteins encoded by annexin cDNAs indicate that the majority of these plant annexins possess the characteristic four repeats of 70 to 75 amino acids and possess motifs proposed to be involved in Ca2+ binding. Like animal annexins, plant annexins bind Ca2+ and phospholipids and are abundant proteins, but there are indications that the number of distinct plant annexin genes may be considerably fewer than that found in animals. Regarding function, a number of studies show that various members of the annexin family of plants may play roles in secretion and/or fruit ripening, show interaction with the enzyme callose (1.3-beta-glucan) synthase, possess intrinsic nucleotide phosphodiesterase activity, bind to F-actin, and/or have peroxidase activity.

The Differential Expression of Sucrose Synthase in Relation to Diverse Patterns of Carbon Partitioning in Developing Cotton Seed.

Developing cotton (Gossypium hirsutum L.) seed exhibits complex patterns of carbon allocation in which incoming sucrose (Suc) is partitioned to three major sinks: the fibers, seed coat, and cotyledons, which synthesize cellulose, starch, and storage proteins or oils, respectively. In this study we investigated the role of Suc synthase (SuSy) in the mobilization of Suc into such sinks. Assessments of SuSy gene expression at various levels led to the surprising conclusion that, in contrast to that found for other plants, SuSy does not appear to play a role in starch synthesis in the cotton seed. However, our demonstration of functional symplastic connections between the phloem-unloading area and the fiber cells, as well as the SuSy expression pattern in fibers, indicates a major role of SuSy in partitioning carbon to fiber cellulose synthesis. SuSy expression is also high in transfer cells of the seed coat facing the cotyledons. Such high levels of SuSy could contribute to the synthesis of the thickened cell walls and to the energy generation for Suc efflux to the seed apoplast. The expression of SuSy in cotyledons also suggests a role in protein and lipid synthesis. In summary, the developing cotton seed provides an excellent example of the diverse roles played by SuSy in carbon metabolism.

In Vitro Prenylation of the Small GTPase Rac13 of Cotton.

Previous work (D.P. Delmer, J. Pear, A. Andrawis, D. Stalker [1995] Mol Gen Genet 248: 43-51) has identified a gene in cotton (Gossypium hirsutum), Rac13, that encodes a small, signal-transducing GTPase and shows high expression in the fiber at the time of transition from primary to secondary wall synthesis. Since Rac13 may be important in signal transduction pathway(s), regulating the onset of fiber secondary wall synthesis, we continue to characterize Rac13 by determining its ability to undergo posttranslational modification. In animals Rac proteins contain the C-terminal consensus sequence CaaL (where "a" can be any aliphatic residue), which is a site for geranylgeranylation (B.T. Kinsella, R.A. Erdman, W.A. Maltese [1994] J Biol Chem 266: 9786-9794). We have identified activities in developing cotton fibers that resemble in specificity the geranylgeranyl- and farnesyltransferases of animals and yeast. In addition, using prenyltransferases from rabbit reticulocytes, we show that Rac13, having a C-terminal sequence of CAFL, can serve as an in vitro substrate for geranylgeranylation but not farnesylation. However, the presence of the uncommon penultimate F residue appears to slow the rate of prenylation considerably compared with other acceptors.

Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase.

In spite of much effort, no one has succeeded in isolating and characterizing the enzyme(s) responsible for synthesis of cellulose, the major cell wall polymer of plants. We have characterized two cotton (Gossypium hirsutum) cDNA clones and identified one rice (Oryza sativa) cDNA that are homologs of the bacterial celA genes that encode the catalytic subunit of cellulose synthase. Three regions in the deduced amino acid sequences of the plant celA gene products are conserved with respect to the proteins encoded by bacterial celA genes. Within these conserved regions, there are four highly conserved subdomains previously suggested to be critical for catalysis and/or binding of the substrate UDP-glucose (UDP-Glc). An overexpressed DNA segment of the cotton celA1 gene encodes a polypeptide fragment that spans these domains and binds UDP-Glc, while a similar fragment having one of these domains deleted does not. The plant celA genes show little homology at the N- and C-terminal regions and also contain two internal insertions of sequence, one conserved and one hypervariable, that are not found in the bacterial gene sequences. Cotton celA1 and celA2 genes are expressed at high levels during active secondary wall cellulose synthesis in developing cotton fibers. Genomic Southern blot analyses in cotton demonstrate that celA forms a small gene family.

A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants.

Sucrose synthase (SuSy; EC 2.4.1.13; sucrose + UDP reversible UDPglucose + fructose) has always been studied as a cytoplasmic enzyme in plant cells where it serves to degrade sucrose and provide carbon for respiration and synthesis of cell wall polysaccharides and starch. We report here that at least half of the total SuSy of developing cotton fibers (Gossypium hirsutum) is tightly associated with the plasma membrane. Therefore, this form of SuSy might serve to channel carbon directly from sucrose to cellulose and/or callose synthases in the plasma membrane. By using detached and permeabilized cotton fibers, we show that carbon from sucrose can be converted at high rates to both cellulose and callose. Synthesis of cellulose or callose is favored by addition of EGTA or calcium and cellobiose, respectively. These findings contrast with the traditional observation that when UDPglucose is used as substrate in vitro, callose is the major product synthesized. Immunolocalization studies show that SuSy can be localized at the fiber surface in patterns consistent with the deposition of cellulose or callose. Thus, these results support a model in which SuSy exists in a complex with the beta-glucan synthases and serves to channel carbon from sucrose to glucan.

Genes encoding small GTP-binding proteins analogous to mammalian rac are preferentially expressed in developing cotton fibers.

In animals, the small GTP-binding proteins, Rac and Rho, of the ras superfamily participate in the signal transduction pathway that regulates the organization of the actin cytoskeleton. We report here on the characterization of two distinct cDNA clones isolated from a cotton fiber cDNA library that code for homologs of animal Rac proteins. Using gene-specific probes, we have determined that amphidiploid cotton contains two genes that code for each of the two Rac proteins, designated Rac13 and Rac9, respectively. The gene for Rac13 shows highly enhanced expression in developing cotton fibers, with maximal expression occurring at the time of transition between primary and secondary wall synthesis. This is also the time at which reorganization of the cytoskeleton occurs, and thus the pattern of expression of Rac13 is consistent with its possible role, analogous to animal Rac, in the signal transduction pathway that controls cytoskeletal organization.

Cotton fiber annexins: a potential role in the regulation of callose synthase.

Cotton fibers contain a characteristic set of proteins which interact with plasma membranes in a Ca(2+)-dependent manner. The association of these proteins with the membrane is correlated with a reduced level of UDP-glucose: (1-->3)-beta-glucan (callose) synthase activity. Analysis of the proteins released from membranes by EDTA treatment shows that the most abundant proteins comprise a family of at least three polypeptides (p34) which resemble annexins. This resemblance includes similarity in size (about 34 kDa), sequence homology, Ca(2+)-dependent precipitation or interaction with the plasma membrane, and ability to serve as a substrate for phosphorylation by endogenous protein kinase(s) which also bind to the membranes in a Ca(2+)-dependent manner. A purified fraction of these annexins binds to, and inhibits, the activity of a partially purified cotton fiber callose synthase. These findings suggest that one possible function of annexin(s) in plants is to modulate the activity and/or localization of callose synthase.

Stimulation of Callose Synthesis in Vivo Correlates with Changes in Intracellular Distribution of the Callose Synthase Activator [beta]-Furfuryl-[beta]-Glucoside.

[beta]-Furfuryl-[beta]-glucoside (FG) has been shown to be a specific endogenous activator of higher plant callose synthase (P. Ohana, D.P. Delmer, G. Volman, J.C. Steffens, D.E. Matthews, M. Benziman [1992] Plant Physiol 98: 708-715). Because glycosides such as FG are usually sequestered in vacuoles, we have proposed that activation of callose synthesis in vivo may involve a change in the compartmentation of FG and Ca2+, resulting in a synergistic activation of callose synthase. The use of suspension-cultured barley (Hordeum bulbosum L.) cells provides evidence that FG is largely sequestered in the vacuole. Furthermore, conditions that lead to induction of callose synthesis in vivo correspondingly lead to elevation of the cytoplasmic concentration of FG. These conditions include the lowering of cytoplasmic pH or elevation of cytoplasmic Ca2+. Oligogalacturonide elicitors have also been reported to cause similar changes in cytoplasmic pH and Ca2+ concentration (Y. Mathieu, A. Kurkdjian, H. Xia, J. Guern, A. Koller, M.D. Spiro, M. O'Neill, P. Albersheim, A. Darvill [1991] The Plant Journal 1: 333-343), and such an elicitor also causes an elevation in cytoplasmic FG coupled with stimulation of callose synthesis. These results support the concept that a relative redistribution of FG between cytoplasm and vacuole may be one of the components of the signal transduction pathway for elicitation of callose synthase in vivo.

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile : A Comparison between Two Dicotyledonous Plants and a Graminaceous Monocot.

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca(2+)-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane.

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis.

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of beta-furfuryl-beta-glucoside (FG), an endogenous activator of plant UDP-glucose:(1-->3)-beta-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized alpha-furfuryl-beta-glucoside also stimulates callose synthase, exhibiting a slightly higher K(a) of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using beta-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca(2+), is proposed.