The cellulose synthase gene PrCESA10 is involved in cellulose biosynthesis in developing tracheids of the gymnosperm Pinus radiata

Abstract

One full length (PrCESA10) and seven other cDNA clones (PrCESA2, 3, 5–8, 11) encoding cellulose synthases (CESAs) were isolated from the coniferous gymnosperm Pinus radiata. PrCESA10 encodes a protein predicted to contain the same domains and regions as angiosperm CESA genes: a zinc finger domain, a hypervariable region 1 (HVR1), a plant-conserved region (CR-P), a class-specific region or hypervariable region 2 (HVR2), in addition to the four conserved domains U1–U4 that are characteristic of the family 2 processive β-glycosyltransferases. The P. radiata protein is also predicted to contain eight transmembrane domains. The zinc finger domain, the CR-P and the C-terminal portion of the proteins, are highly conserved between P. radiata and the nearest angiosperm CESA protein from Solanum tuberosum. Reverse transcriptase-PCR showed that all the P. radiata genes were expressed in all organs tested, although to different extents. In situ hybridization studies with PrCESA10 in stems of 2- and 12-month-old seedlings showed that it was expressed in the secondary xylem in the two-to-three most recently developed tracheids, which were laying down secondary cell walls.

Introduction

Cellulose, a (1→4)-β-d-glucan, is the most abundant polysaccharide in the biosphere. It is a major component of both primary and secondary cell walls of seed plants (angiosperms and gymnosperms), where it is present in a crystalline form as microfibrils (Bacic et al., 1988). All other embryophytes (land plants), many algae, some protozoa and bacteria, including Acetobacter xylinum, and one group of invertebrate animals, the tunicates, also produce it (Stone, 2001).

Despite the abundance of cellulose in seed plants, attempts to purify and characterize cellulose synthases from these organisms using biochemical methodology have been unsuccessful (Delmer, 1999, Doblin et al., 2002). In contrast, this approach was successfully used to isolate and characterize cellulose synthase from the bacterium A. xylinum. This study led to the subsequent cloning of the first cellulose synthase genes from bacteria, including the CESA gene that encodes the enzyme containing the catalytic site of cellulose synthase (EC 2.4.1.12) (Saxena et al., 1990). With the nucleotide sequence of this gene, Pear et al. (1996) used a bioinformatic approach to identify the first two CESA genes from seed plants in the angiosperm Gossypium hirsutum.

Definitive genetic evidence for the functionality of cellulose synthase genes in angiosperms was first demonstrated using a CESA mutant of Arabidopsis thaliana, rsw1, that exhibits radial swelling of its roots at restrictive temperatures due to a defect in the production of crystalline cellulose; the wild type RSW1 (AtCESA1) gene restored the wild type phenotype (Arioli et al., 1998). Since then, full length CESA genes have been isolated from a range of other angiosperm species (Doblin et al., 2002; http://cellwall.stanford.edu), including hardwood trees such as aspen (Populus tremuloides) (Wang and Loopstra, 1998, Wu et al., 2000, Samuga and Joshi, 2002, Samuga and Joshi, 2004, Kalluri and Joshi, 2003) and monocotyledons such as Zea mays (Holland et al., 2000).

CESA proteins from bacteria and angiosperms are characterized by having four conserved domains U1–U4: U1 to U3 each contain a conserved aspartic acid residue (D) and U4 contains a conserved QXXRW motif. The three D residues and QXXRW motifs are characteristic of family 2 processive β-glycosyltransferases (Saxena et al., 1995). In cellulose synthases they bind the substrate, UDP-glucose and the cofactor Mn²⁺, and catalyse the formation of cellulose (Karnezis et al., 2000, Doblin et al., 2002). They form part of the cytoplasmic domain of the protein that is between the two groups of predicted transmembrane domains, two near the N-terminus and six near the C-terminus (Delmer, 1999). Compared with the bacterial CESA proteins, angiosperm CESA proteins have an extended N-terminal region that includes a zinc finger domain and an adjacent hypervariable region (HVR1), a plant-conserved region (CR-P) between domains U1 and U2, and a class-specific region (CSR) between U2 and U3. The CSR is also known as the hypervariable region 2 (HVR2) and has a limited homology among different angiosperm CESA proteins (Vergara and Carpita, 2001). Recent evidence indicates that the zinc finger domains are involved in the interactions between individual CESA proteins (Kurek et al., 2002). In angiosperms, the CESA proteins are located in rosette structures in the plasma membrane (Kimura et al., 1999). Similar rosettes occur in all embryophytes and charophycean algae, but not in bacteria (Roberts et al., 2002).

Mutant, antisense and expression analyses of the 10 A. thaliana CESA genes suggest that at least three of them (AtCESA1, 3 and 6) encode proteins that function to synthesize cellulose in cell types with only primary cell walls, whereas at least three genes (AtCESA4, 7 and 8) encode proteins that synthesize cellulose in cell types with secondary walls such as xylem tracheary elements (Doblin et al., 2002, Williamson et al., 2002, Gardiner et al., 2003, Taylor et al., 2003). Moreover, pull-down assays indicate that the proteins encoded by AtCESA4, 7 and 8 all interact, suggesting that they may be in the same rosette (Taylor et al., 2003). Expression analyses of CESA genes in other angiosperm species also associate specific genes with the synthesis of cellulose in cells that are either depositing a primary or a secondary cell wall (Doblin et al., 2002). For example, in situ hybridization analysis showed that PtrCESA6 and PtrCESA7 were expressed in all expanding cells depositing primary cell walls (Samuga and Joshi, 2004), whereas PtrCESA1 was expressed in xylem tracheary elements during secondary cell wall deposition in P. tremuloides (Wu et al., 2000). Mutant and expression analyses have also implicated three rice (Oryza sativa) CESA genes, OsCESA4, 7 and 9, in the synthesis of cellulose in cells with lignified secondary walls (Tanaka et al., 2003).

Despite the importance of coniferous gymnosperms as sources of timber and pulp, as far as we are aware, no expression studies of their CESA genes have been reported. In the present study, we report the isolation, characterization and expression of eight CESA genes from the coniferous gymnosperm Pinus radiata. Our in situ hybridisations show that PrCESA10, encoded by a full length cDNA clone, is expressed in recently developed tracheids, which are laying down secondary cell walls.