Abstract
A putative barley (1→3)-β-d-glucan synthase cDNA of 6.1 kb, which is homologous to the yeast FKS gene, was assembled from DNA fragments obtained through screening of barley cDNA and BAC libraries, and by PCR amplification. The corresponding gene, designated HvGSL1, is a member of a family of at least six genes in barley. Gene transcripts are detected at relatively high levels in early developing grain, florets, coleoptiles and roots, but not in leaves infected with a fungal pathogen. A (1→3)-β-d-glucan synthase has been purified more than 60-fold from barley suspension-cultured cells by detergent extraction, CaCl2 treatment, sucrose density gradient centrifugation and non-denaturing gel electrophoresis. The enzyme synthesizes (1→3)-β-d-glucan in vitro and is recognized by antibodies raised against a 17 kDa protein generated by heterologous expression of a fragment of the HvGSL1 cDNA. Furthermore, mass spectrometric analyses show that tryptic peptides produced by in-gel digestion of the active enzyme match peptides predicted from the gene sequence. Thus, the amino acid sequence predicted from the HvGSL1 gene has been linked with the actual amino acid sequence of an active (1→3)-β-d-glucan synthase fraction from barley.
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Brown, R.C., Lemmon, B.E., Stone, B.A. and Olsen, O.-A. 1997. Cell wall (1→3)-and (1→3;1→4)-β-glucans during early grain development in rice endosperm. Planta 202: 414–426.
Bulone, V., Fincher, G.B. and Stone, B.A. 1995. In vitro synthesis of a microfibrillar (1→3)-β-D-glucan by a ryegrass (Lolium multiflorum) endosperm (1→3)-β-D-glucan synthase enriched by product entrapment. Plant J. 8: 213–225.
Cabib, E., Drgonová, J. and Drgon, T. 1998. Role of small G proteins in yeast cell polarization and wall biosynthesis. Annu. Rev. Biochem. 67: 307–333.
Cabib, E., Roh, D-H., Schmidt, M., Crotti, L.B. and Varma, A. 2001. The yeast cell wall and septum as paradigms of cell growth and morphogenesis. J. Biol. Chem. 276: 19679–19682.
Coutinho, P.M. and Henrissat, B. 1999. Carbohydrate-active enzymes: an integrated database approach. In: H.J. Gilbert, G. Davies, B. Henrissat and B. Svensson (Eds.) Recent Advances in Carbohydrate Bioengineering, Royal Society of Chemistry, Cambridge, UK, pp. 3–12.
Cui, X., Heungsop, S., Song, C., Laosinchai, W., Amano, Y., Brown, R.M. Jr. 2001. A putative plant homolog of the yeast β-(1→3)-D-glucan synthase subunit FKS1 from cotton (Gossypium hirsutum L.) fibers. Planta 213: 223–230.
Delmer, D.P., Volokita, M., Solomon, M., Fritz, U., Delphendahl, W. and Herth, W. 1993. A monoclonal antibody recognizes a 65 kDa higher plant membrane polypeptide which undergoes cation-dependent association with callose deposition in vivo. Protoplasma 176: 3–42.
Delmer, D.P., Pear, J.R., Andrawis, A. and Stalker, D.M. 1995. Genes encoding small GTP-binding proteins analogous to mammalian Rac are preferentially expressed in developing cotton fibers. Mol. Gen. Genet. 248: 43–51.
Devereux, J., Haeberli, P. and Smithies, O. 1984. A comprehensive set of sequence analysis programs for the Vax. Nucl. Acids Res. 12: 387–395.
Dhugga, K.S. and Ray, P.M. 1994. Purification of (1→3)-β-Dglucan synthase activity from pea tissue-two polypeptides of 55 kD and 70 kD copurify with enzyme activity. Eur. J. Biochem. 220: 943–953.
Dijkgraaf, G.J., Abe, M., Ohya, Y. and Bussey, H. 2002. Mutations in Fks1p affect the cell wall content of β-(1→3) and β-(1→6)-D-glucan in Saccharomyces cerevisiae. Yeast 19: 671–690.
Doblin, M.S., Melis, L.D., Newbigin, E., Bacic, A. and Read, S.M. 2001. Pollen tubes of Nicotiana alata express two genes from different β-D-glucan synthase families. Plant Physiol. 125: 2040–2052.
Donofrio, N.M. and Delaney, T.P. 2001. Abnormal callose response phenotype and hypersusceptibility to Peronospora parasitica in defence-compromised Arabidopsis nim1-1 and salicylate hydroxylase-expressing plants. Mol. Plant-Microbe Interact. 14: 439–450.
Douglas, C.M., Foor, F., Marrinan, J.A., Morin, N., Nielsen, J.B., Dahl, A.M., Mazur, P., Baginsky, W., Li, W.L., Elsherbeini, M., Clemas, J.A., Mandala, S.M., Frommer, B.R. and Kurtz, M.B. 1994. The Saccharomyces cerevisiae Fks1 (Etg1) gene encodes an integral membrane protein which is a subunit of (1→3)-β-Dglucan synthase. Proc. Natl. Acad. Sci. USA 91: 12907–12911.
Feingold, D.S., Neufeld, E.F. and Hassid, W.Z. 1958. Synthesis of a β-(1→3)-linked glucan by extracts of Phaseolus aureus seedlings. J. Biol. Chem. 233: 783–788.
Gregory, A.C.E., Smith, C., Kerry M.E., Wheatley, E.R. and Bolwell, G.P. 2002. Comparative subcellular immunolocation of polypeptides associated with xylan and callose synthases in French bean (Phaseolus vulgaris) during secondary wall formation. Phytochemistry 59: 249–259.
Him, J.L.K., Pelosi, L., Chanzy, H., Putaux, J.L. and Bulone, V. 2001. Biosynthesis of (1→3)-β-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana. Eur. J. Biochem. 268: 4628–4638.
Hong, Z., Delauney, A.J. and Verma, D.P.S. 2001. A cell platespecific callose synthase and its interaction with phragmoplastin. Plant Cell 13: 755–768.
Jacobs, A.K., Lipka, V., Burton, R.A., Panstruga, R., Strizhov, N., Schulze-Lefert, P. and Fincher, G.B. 2003. An Arabidopsis thaliana callose synthase, GSL5, is required for wound and papillary callose formation. Plant Cell, in press.
Kudlicka, K. and Brown, R.M. 1997. Cellulose and callose biosynthesis in higher plants 1. Solubilization and separation of (1→3)-and (1→4)-β-D-glucan synthase activities from mung bean. Plant Physiol. 115: 643–656.
Kudlicka, K., Brown, R.M., Li, L.K., Lee, J.H., Shin, H. and Kuga, S. 1995. Beta-D-glucan synthesis in the cotton fiber. 4. In vitro assembly of the cellulose I allomorph. Plant Physiol. 107: 111–123.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Mazur, P., Morin, N., Baginsky, W., Elsherbeini, M., Clemas, J.A., Nielsen, J.B. and Foor, F. 1995. Differential expression and function of two homologous subunits of yeast (1→3)-β-D-glucan synthase. Mol. Cell. Biol. 15: 5671–5681.
McCormack, B.A., Gregory, A.C.E., Kerry, M.E., Smith, C. and Bolwell, G.P. 1997. Purification of an elicitor-induced glucan synthase (callose synthase) from suspension cultures of French bean (Phaseolus vulgaris L.): purification and immunolocation of a probable Mr-65000 subunit of the enzyme. Planta 203: 196–203.
Meikle, P.J., Bonig, I., Hoogenraad, N.J., Clarke, A.E. and Stone, B.A. 1991. The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan specific monoclonal antibody. Planta 485: 1–8.
Nishimura, M.T., Stein, M., Hou, B.H., Vogel, J.P., Edwards, H. and Somerville, S.C. 2003. Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science 301: 969–972.
Olsen, O.-A., 2001. Endosperm development: cellularization and cell fate specification. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52: 233–267.
Østergaard, L., Petersen, M., Mattsson, O. and Mundy, J. 2002. An Arabidopsis callose synthase. Plant Mol. Biol. 49: 559–566.
Pear, J.R., Kawagoe, Y., Schreckengost, W.E., Delmer, D.P. and Stalker, D.M. 1996. Higher plants contain homologs of the bacterial CelA genes encoding the catalytic subunit of cellulose synthase. Proc. Natl. Acad. Sci. USA 93: 12637–12642.
Pelosi, L., Imai, T., Chanzy, H., Heux, L., Buhler, E. and Bulone, V. 2003. Structural and morphological diversity of (1→3)-β-D-glucans synthesized in vitro by enzymes from Saprolegnia monoica. Comparison with a corresponding in vitro product from blackberry (Rubus fruticosus). Biochemistry 42: 6264–6274.
Richmond, T.A. and Somerville, C.R. 2000. The cellulose synthase superfamily. Plant Physiol. 124: 495–498.
Ryals, J.A., Neuenschwander, U.H., Willits, M.G., Molina, A., Steiner, H.Y. and Hunt, M.D. 1996. Systemic Acquired Resistance. Plant Cell 8: 1809–1819.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 1.25–1.31.
Samuels, A.L., Giddings, T.H. Jr. and Staehelin, L.A. 1995. Cytokinesis in tobacco BY-2 and root tip cells: a new model of cell plate formation in higher plants. J. Cell Biol. 130: 1345–1357.
Sanger, F., Nicklen, S. and Coulsen, A.R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467.
Saxena, I.M., Brown, R.M. Jr. and Dandekar, T. 2001. Structurefunction characterization of cellulose synthase: relationship to other glycosyltransferases. Phytochemistry 57: 1135–1148.
Schlupmann, H., Bacic, A. and Read, S.M. 1993. A novel callose synthase from pollen tubes of Nicotiana. Planta 191: 470–481.
Slay, R.M., Watada, A.E., Frost, D.J. and Wasserman, B.P. 1992. Characterization of the UDP-glucose:(1,3)-β-glucan (callose) synthase from plasma membranes of celery: polypeptide profiles and photolabeling patterns of enriched fractions suggest callose synthase complexes from various sources share a common structure. Plant Sci. 86: 125–136.
Smith, M.M. and Stone, B.A. 1973. β-Glucan synthesis by cell-free extracts from Lolium multiflorum endsperm. Biochim. Biophys. Acta 313: 72–94.
Stasinopoulos, S.J., Fisher, P.R., Stone, B.A. and Stanisich, V.A. 1999. Detection of two loci involved in (1→3)-β-D-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene. Glycobiology 9: 31–41.
Stone, B.A. and Clarke, A.E. 1992. Chemistry and biology of (1→3)-β-D-glucans. La Trobe University Press, Australia.
Tucker, M.R., Paech, N.A., Willemse, M.T.M. and Koltunow, A.M.G. 2001. Dynamics of callose deposition and (1→3)-β-D225 glucanase expression during reproductive events in sexual and apomictic Hieracium. Planta 212: 487–498.
Turner, A., Bacic, A., Harris, P.J. and Read, S.M. 1998. Membrane fractionation and enrichment of callose synthase from pollen tube of Nicotiana alata Link et Otto. Planta 205: 380–388.
Verma, D.P.S. and Hong, Z. 2001. Plant callose synthase complexes. Plant Mol. Biol. 47: 693–701.
von Heijne, G. 1992. Membrane protein structure prediction: hydrophobicity analysis and the ‘Positive Inside’ rule. J. Mol. Biol. 225: 487–494.
Zhou, F., Kurth, J., Wei, F., Elliott, C., Vale, G., Yahiaoui, N., Keller, B., Somerville, S., Wise, R. and Schulze-Lefert, P. 2001. Cell-autonomous expression of barley Mla1 confers race-specific resistance to the powdery mildew fungus via a Rar1-independent signaling pathway. Plant Cell 13: 337–350.
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Li, J., Burton, R.A., Harvey, A.J. et al. Biochemical evidence linking a putative callose synthase gene with (1→3)-β-d-glucan biosynthesis in barley. Plant Mol Biol 53, 213–225 (2003). https://doi.org/10.1023/B:PLAN.0000009289.50285.52
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DOI: https://doi.org/10.1023/B:PLAN.0000009289.50285.52