CesA Genes Research Articles

Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cell wall biosynthesis

Higher plants contain a family of cellulose synthase catalytic subunit (CesA) genes that encode components of an enzyme complex embedded in the cell membrane. Recent studies in several higher plant species have demonstrated that two groups of CesA genes exist, associated with either primary or secondary cell wall deposition. We cloned six full-length CesA cDNAs from Eucalyptus grandis W. Hill ex Maiden (EgCesA1 through 6) and determined their expression patterns in a variety of organs from an adult tree. The six EgCesA genes encode predicted proteins of 978 to 1097 amino acid residues, each of which contains all of the key regions and motifs characteristic of functional CESA proteins. The predicted proteins share limited amino acid identity with each other, ranging from 61 to 70%. In contrast, similar CESA proteins from higher plant species exhibit 81 to 90% identity with the six EgCESAs. Gene expression analysis using quantitative reverse-transcription polymerase chain reaction indicated that transcripts of EgCesA1 to 3 were abundant in tissues enriched with cells laying down secondary cell walls (e.g., xylem), but were weakly expressed in tissues undergoing primary growth (e.g., unfolding leaves). Expression of EgCesA4 and EgCesA5 was upregulated in tissues rich in rapidly dividing cells undergoing primary wall synthesis, whereas EgCesA6 was weakly expressed in all tissues analyzed. These results suggest that Eucalyptus, like other higher plants, expresses two contrasting groups of apparently co-regulated CesAs involved in either primary or secondary cell wall biosynthesis.

Biosynthesis of cellulose‐enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis

Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) x tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5'-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified.

The genome sequence of black cottonwood (Populus trichocarpa) reveals 18 conserved cellulose synthase (CesA) genes

The genome sequence of Populus trichocarpa was screened for genes encoding cellulose synthases by using full-length cDNA sequences and ESTs previously identified in the tissue specific cDNA libraries of other poplars. The data obtained revealed 18 distinct CesA gene sequences in P. trichocarpa. The identified genes were grouped in seven gene pairs, one group of three sequences and one single gene. Evidence from gene expression studies of hybrid aspen suggests that both copies of at least one pair, CesA3-1 and CesA3-2, are actively transcribed. No sequences corresponding to the gene pair, CesA6-1 and CesA6-2, were found in Arabidopsis or hybrid aspen, while one homologous gene has been identified in the rice genome and an active transcript in Populus tremuloides. A phylogenetic analysis suggests that the CesA genes previously associated with secondary cell wall synthesis originate from a single ancestor gene and group in three distinct subgroups. The newly identified copies of CesA genes in P. trichocarpa give rise to a number of new questions concerning the mechanism of cellulose synthesis in trees.

Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms

Specific plant cellulose synthases (CesA), encoded by a multigene family, are necessary for secondary wall synthesis in vascular tissues and are critical to wood production. We obtained full-length clones for the three CesAs that are highly expressed in developing xylem and examined their phylogenetic relationships and expression patterns in loblolly pine tissues. Full-length CesA clones were isolated from cDNA of developing loblolly pine (Pinus taeda) xylem and phylogenetic inferences made from plant CesA protein sequences. Expression of the three genes was examined by Northern blot analysis and semiquantitative RT-PCR. Each of three PtCesA genes is orthologous to one of the three angiosperm secondary cell wall CesAs. The PtCesAs are coexpressed in tissues of loblolly pine with tissues undergoing secondary cell wall biosynthesis showing the highest levels of expression. Phylogenetic and expression analyses suggest that functional roles for these loblolly pine CesAs are analogous to those of orthologs in angiosperm taxa. Based upon evidence from this and other studies, we suggest division of seed plant CesA genes into six major paralogous groups, each containing orthologs from various taxa. Available evidence suggests that paralogous CesA genes and their distinct functional roles evolved before the divergence of gymnosperm and angiosperm lineages.

Cellulose synthase (CesA) genes in algae and seedless plants

Microfibril structure is determined largely by the organization of arrays of integral plasma membrane protein particles known as “terminal complexes”, which include cellulose synthase catalytic subunits encoded by CesA genes. Although the CesA genes of plants and bacteria share conserved regions, variations in terminal complex and microfibril structure presumably result from sequence differences. Thus, the CesA domains that influence terminal complex assembly may be revealed by examining the differences between CesA genes from green algae in which terminal complex structure ranges from rosettes (plant-like) to linear (bacteria-like). This report describes a second CesA gene that has been cloned from Mesotaenium caldariorum, a unicellular green alga from the order Zygnematales, which have rosette terminal complexes. Both McCesA1 and McCesA2 are similar to seed plant CesAs in domain structure and intron position. Seed plants have multiple CesAs and CesA-like (Csl) genes, some of which appear to be expressed specifically during cell expansion, secondary cell wall deposition in vascular tissue, or tip growth. Diversification of the CesA and Csl gene families can be explored by comparing these genes in mosses, which lack vascular tissue with secondary cell walls, and early divergent vascular plants such as ferns. Degenerate primers were used to amplify and clone five unique CesA and Csl fragments from genomic DNA isolated from Physcomitrella patens. Probes derived from the cloned fragments were used to isolate several clones from a Physcomitrella genomic library. One Csl fragment was amplified from genomic DNA isolated from the fern Ceratopteris richardii. Phylogenetic analysis supports the presence of CslD genes in both mosses and ferns, but does not support the presence of secondary cell wall specific CesA orthologs in mosses.

Global expression analysis of CESA and CSL genes in Arabidopsis

We have used Affymetrix gene chips to measure the expression of 10 CESA and 29 CSL genes of Arabidopsis in different developmental stages or organs. These measurements reveal that many of the genes exhibit different levels of expression in the various organs. While several CESA genes are highly expressed in all the tissues examined, very few CSL genes approach such high levels of expression. This suggests that the CSL genes either encode enzymes for the synthesis of minor components of cell walls or are expressed only in specific cell types. The expression data also highlights the potential importance of the CESA genes for primary and secondary cell wall formation during different developmental stages and in the different organs examined.

Identification and expression analysis of genes encoding putative cellulose synthases (CesA) in the hybrid aspen, Populus tremula (L.) P. tremuloides (Michx.)

Cellulose is synthesized in plant cell walls by large membrane-bound protein complexes proposed to contain several copies of the catalytic subunit of the cellulose synthase, CesA. Here we report identification of 10 distinct CesA genes within a database of 100,000 ESTs of the hybrid aspen, Populus tremula (L.) × P. tremuloides (Michx.). Expression analyses in normal wood undergoing xylogenesis and in tension wood indicate xylem specific expression of four putative CesA isoenzymes, PttCesA1, PttCesA3-1, PttCesA3-2 and PttCesA9. Both the protein sequences and the expression profiles of PttCesA3-1 and PttCesA3-2 are very similar, and they may thus represent redundant copies of an enzyme with essentially the same function. Further, one of the generally more constitutively expressed CesA genes, PttCesA2, seems to be activated on the opposite side of a tension wood induced stem, while PttCesA6 appears to be more specific for leaf tissues. The rest of the hybrid aspen CesA genes were found to be relatively evenly expressed over the poplar tissues hereby studied.

Cellulose synthesis in maize: isolation and expression analysis of the cellulose synthase (CesA) gene family

Stalk lodging in maize results in significant yield losses. We have determined that cellulose per unit length of the stalk is the primary determinant of internodal strength. An increase in cellulose concentration in the wall might allow simultaneous improvements in stalk strength and harvest index. Cellulose formation in plants can be perturbed by mutations in the genes involved in cellulose synthesis, post-synthetic cellulose alteration or deposition, N-glycosylation, and some other genes with as yet unknown functions. We have isolated 12 members of the cellulose synthase (CesA) gene family from maize. The genes involved in primary wall formation appear to have duplicated relatively independently in dicots and monocots. The deduced amino acid sequences of three of the maize genes, ZmCesA10–12, cluster with the Arabidopsis CesA sequences that have been shown to be involved in secondary wall formation. Based on their expression patterns across multiple tissues, these three genes appear to be coordinately expressed. The remaining genes show overlapping expression to varying degrees with ZmCesA1, 7, and 8 forming one group, ZmCesA3 and 5 a second group, and ZmCesA2 and 6 exhibiting independent expression of any other gene. This suggests that the varying levels of coexpression may just be incidental except in the case of ZmCesA10–12, which may interact with each other to form a functional enzyme complex. Isolation of the expressed CesA genes from maize and their association with primary or secondary wall formation has made it possible to test their respective roles in cellulose synthesis through mutational genetics or transgenic approaches. This information would be useful in improving stalk strength.

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.

Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in aspen trees.

The quality and quantity of cellulose deposited in the primary and secondary cell walls of plants vary in accordance with their biological function. However, the molecular basis of such cellulose heterogeneity has so far remained unclear. Since enrichment of better-quality cellulose, in terms of increased degree of polymerization and crystallinity, is one of the goals of forest biotechnology, our main objective is to decipher the roles of distinct cellulose synthase (CesA) genes in tree development, with special reference to wood production. Here, we report two full-length CesA cDNAs, PtrCesA3 and PtrCesA4, from an economically important tree aspen (Populus tremuloides). PtrCesA3 is orthologous to the Arabidopsis AtCesA4 gene involved in secondary wall formation, whereas PtrCesA4 is orthologous to the Arabidopsis AtCesA1 gene involved in primary cell wall formation. To define the specific cell types expressing these CesA genes, we explored the natural distribution patterns of PtrCesA3 and PtrCesA4 transcripts in a variety of aspen organs, such as leaves, petiole, stem, and roots, using in situ hybridization with hypervariable region-specific antisense riboprobes. Such a side-by-side comparison suggested that PtrCesA3 is exclusively expressed in secondary-wall-forming cells of xylem and phloem fibers, whereas PtrCesA4 is predominantly expressed in primary-wall-forming expanding cells in all aspen organs examined. These findings suggest a functionally distinct role for each of these two types of PtrCesAs during primary and secondary wall biogenesis in aspen trees, and that such functional distinction appears to be conserved between annual herbaceous plants and perennial trees.

Molecular cloning of ten distinct hypervariable regions from the cellulose synthase gene superfamily in aspen trees.

Recent molecular genetic data suggest that cellulose synthase (CesA) genes coding for the enzymes that catalyze cellulose biosynthesis (CESAs) in Arabidopsis and other herbaceous plants belong to a large gene family. Much less is known about CesA genes from forest trees. To isolate new CesA genes from tree species, discriminative but easily obtainable homologous DNA probes are required. Hypervariable regions (HVRII) of CesA genes represent highly divergent DNA sequences that can be used to examine structural, expressional and functional relationships among CesA genes. We used a reverse transcriptase-polymerase chain reaction (RT-PCR)-based technique to identify HVRII regions from eight types of CesA genes and two types of CesA-like D (CslD) genes in quaking aspen (Populus tremuloides Michx.). Comparison of these aspen CESA/CSLD HVRII regions with the predicted proteins from eight full-length CesA/CslD cDNAs available in our laboratory and with searches for aspen CesA/CslD homologs in the recently released Populus trichocarpa Torr. & A. Gray. genome confirmed the utility of this approach in identifying several CesA/CslD gene members from the Populus genome. Phylogenetic analysis of 56 HVRII domains from a variety of plant species suggested that at least six distinct classes of CESAs exist in plants, supporting a previous proposal for renaming HVRII regions as class-specific regions (CSR). This method of CSR cloning could be applied to other crop plants and tree species, especially softwoods, for which the whole genome sequence is unlikely to become available in the near future because of the large size of these genomes.

Differential expression patterns of two new primary cell wall-related cellulose synthase cDNAs, PtrCesA6 and PtrCesA7 from aspen trees

Based on elegant molecular genetic analyses, distinct classes of cellulose synthase ( CesA) genes have been associated with either primary or secondary cell wall development in Arabidopsis. Here, we report on cloning of two new CesA cDNAs, PtrCesA6 and PtrCesA7 involved in the primary cell wall development in aspen ( Populus tremuloides) trees. Both these distinct cDNAs, isolated from a developing xylem cDNA library, share only 60–67% identities with each other as well as with five other previously known aspen CesA cDNAs. Interestingly, PtrCESA6 from aspen, a dicot species, shares maximum identity of 81–84% with three CESA isoforms from maize and rice, two monocot species. On the other hand, PtrCESA7 shares a maximum identity of 86% with AtCESA2, a primary wall-related CesA member from Arabidopsis, a dicot species. Gene expression analyses by reverse transcriptase-polymerase chain reactions (RT-PCRs) suggested that both these genes are expressed at a low level in all aspen tissues examined but PtrCesA7 is expressed at a higher level than PtrCesA6. While corroborating these results, in situ mRNA hybridization studies using three different aspen organs also suggested that PtrCesA6 and PtrCesA7 genes are expressed in all expanding cells depositing primary cell wall but PtrCesA7 is expressed at a higher level than PtrCesA6. These differential gene expression profiles suggest that each of these CesAs may be playing a specific role during primary cell wall development in aspen trees. Isolation of two primary wall related CesA genes from xylem tissues also suggest their importance during xylem development, which is traditionally considered to be enriched in secondary cell wall forming cells of economical significance.

Modulation of the cellulose content of tuber cell walls by antisense expression of different potato ( Solanum tuberosum L.) CesA clones

Four potato cellulose synthase ( CesA) homologs ( StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE- StCesA3) and antisense (AS- StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.

Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall.

Several brittle culm mutations of rice (Oryza sativa) causing fragility of plant tissues have been identified genetically but not characterized at a molecular level. We show here that the genes responsible for three distinct brittle mutations of rice, induced by the insertion of the endogenous retrotransposon Tos17, correspond to CesA (cellulose synthase catalytic subunit) genes, OsCesA4, OsCesA7 and OsCesA9. Three CesA genes were expressed in seedlings, culms, premature panicles, and roots but not in mature leaves, and the expression profiles were almost identical among the three genes. Cellulose contents were dramatically decreased (8.9%-25.5% of the wild-type level) in the culms of null mutants of the three genes, indicating that these genes are not functionally redundant. Consistent with these results, cell walls in the cortical fiber cells were shown to be thinner in all the mutants than in wild-type plants. Based on these observations, the structure of a cellulose-synthesizing complex involved in the synthesis of the secondary cell wall is discussed.

Isolation and characterization of a new, full-length cellulose synthase cDNA, PtrCesA5 from developing xylem of aspen trees.

Cellulose synthases (CesA) represent a group of beta-1,4 glycosyltransferases involved in cellulose biosynthesis. Recent molecular studies confirmed that a large CesA gene family encoding various CesA isoforms exists in plants. The isolation and characterization of a new, full-length CesA cDNA, PtrCesA5 from an economically important tree, quaking aspen (Populus tremuloides) is reported here. The predicted PtrCesA5 protein is highly similar to Arabidopsis AtCesA3 (88%) involved in primary cell wall synthesis. However, gene expression studies here suggest that PtrCesA5 transcripts are greatly enhanced in developing xylem tissues enriched in secondary cell wall synthesis as compared with leaf tissues actively undergoing primary cell wall synthesis. Availability of this new, dual-function CesA from a woody species will assist in a better understanding of the mechanism of cellulose biosynthesis during wood development.

Control of cellulose synthase complex localization in developing xylem.

Cellulose synthesis in the developing xylem vessels of Arabidopsis requires three members of the cellulose synthase (CesA) gene family. In young vessels, these three proteins localize within the cell, whereas in older vessels, all three CesA proteins colocalize with bands of cortical microtubules that mark the sites of secondary cell wall deposition. In the absence of one subunit, however, the remaining two subunits are retained in the cell, demonstrating that all three CesA proteins are required to assemble a functional complex. CesA proteins with altered catalytic activity localize normally, suggesting that cellulose synthase activity is not required for this localization. Cortical microtubule arrays are required continually to maintain normal CesA protein localization. By contrast, actin microfilaments do not colocalize with the CesA proteins and are unlikely to play a direct role in their localization. Green fluorescent protein-tagged CesA reveals a novel process in which the structure and/or local environment of the cellulose synthase complex is altered rapidly.

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.

Recent progress in cellulose biosynthesis.

Cellulose comprises the major polymer of the plant cell wall. It consists of a set of parallel chains composed of glucans and these chains are highly oriented to form a structure known as a microfibril. The orientation of the microfibrils controls the extension of the direction of the plant cell. Extensive studies on the cellulose biosynthesis have been carried out for over three decades, and recently (1996) genes for cellulose biosynthesis in plants ( CesA) were isolated. In the year 2002, a specific primer for cellulose biosynthesis reaction has been discovered and cellulose synthetic activity has been also confirmed by recombinant protein derived from the plant CesA gene. Furthermore, other proteins involved in cellulose biosynthesis besides CesA proteins were also proposed at the same time. One of these proteins, Korrigan cellulase, was suggested to act by removing sitosterol from the primer for biosynthesis reaction of cellulose. A membrane-bound sucrose synthase was also suggested to provide UDP-glucose as a substrate for cellulose biosynthesis. On the basis of these results, a new pathway for cellulose biosynthesis was proposed. Now, the research field of cellulose biosynthesis is facing a major turning point.

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis.

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2 (AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1(-)) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3 were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3 antisense plants. In keeping with the mild phenotype compared with Rsw1(-), reductions in total cellulose levels in antisense CesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, like CesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 in rsw1 but were unable to complement that mutant's defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31 degrees C.

Pollen tubes of Nicotiana alata express two genes from different beta-glucan synthase families.

The walls deposited by growing pollen tubes contain two types of beta-glucan, the (1,3)-beta-glucan callose and the (1,4)-beta-glucan cellulose, as well as various alpha-linked pectic polysaccharides. Pollen tubes of Nicotiana alata Link et Otto, an ornamental tobacco, were therefore used to identify genes potentially encoding catalytic subunits of the callose synthase and cellulose synthase enzymes. Reverse transcriptase-polymerase chain reactions (RT-PCR) with pollen-tube RNA and primers designed to conserved regions of bacterial and plant cellulose synthase (CesA) genes amplified a fragment that corresponded to an abundantly expressed cellulose-synthase-like gene named NaCslD1. A fragment from a true CesA gene (NaCesA1) was also amplified, but corresponding cDNAs could not be identified in a pollen-tube library, consistent with the very low level of expression of the NaCesA1 gene. RT-PCR with pollen-tube RNA and primers designed to regions conserved between the fungal FKS genes [that encode (1,3)-beta-glucan synthases] and their presumed plant homologs (the Gsl or glucan-synthase-like genes) amplified a fragment that corresponded to an abundantly expressed gene named NaGsl1. A second Gsl gene detected by RT-PCR (NaGsl2) was expressed at low levels in immature floral organs. The structure of full-length cDNAs of NaCslD1, NaCesA1, and NaGsl1 are presented. Both NaCslD1 and NaGsl1 are predominantly expressed in the male gametophyte (developing and mature pollen and growing pollen tubes), and we propose that they encode the catalytic subunits of two beta-glucan synthases involved in pollen-tube wall synthesis. Different beta-glucans deposited in one cell type may therefore be synthesized by enzymes from different gene families.