Abstract
A barley aleurone cDNA library was screened using 32P-labeled cDNA prepared by reverse transcription of mRNA from aleurone layers treated in the presence or absence of gibberellic acid (GA). Besides α-amylase cDNA clones, another set of clones representing an abundant mRNA in aleurone cells was identified. Messenger RNA hybrid-selected by a prototype clone of this group (clone 10) was translated in vitro to yield a 36 kDa protein. Analysis of the DNA sequence and the predicted amino acid sequence of the protein product of this clone indicates that this gene codes for a protein with homology to endochitinases from tobacco and bean. In addition, the predicted amino acid sequence includes a stretch that is closely related to a cyanogen bromide cleavage fragment from an endochitinase isolated from barley endosperm. The structural genes for endochitinase are present as multiple copies on barley chromosome 1. mRNA detectable by this clone increases in abundance in barley aleurone cells incubated in the absence and in the presence of GA. Western blot analysis of proteins from aleurone and endosperm tissues indicates the presence of multiple endochitinases differing in molecular size. Possible mechanisms for the occurrence of multiple forms of endochitinases and their biological significance are discussed.
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References
Boller T: Induction of hydrolases as a defense reaction against pathogens. UCLA Symp Mol Cell Biol 22: 247–262 (1985).
Broglie KE, Gaynor JJ, Broglie RM: Ethylene-regulated gene expression: Molecular cloning of the genes encoding an encochitinase from Phaseolus vulgaris. Proc Natl Acad Sci USA 83: 6820–6824 (1986).
Chandler PM, Zwar JA, Jacobsen JV, Higgins TJV, Inglis AS: The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in aleurone layers studies using an αamylase cDNA lone. Plant Mol Biol 3: 407–418 (1984).
Deikman J, Jones RL: Control of α-amylase mRNA accumulation by gibberellic acid and calcium in barley aleurone layers. Plant Physiol 78: 192–198 (1985).
Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Fincher GB, Lock PA, Morgan MM, Lingelbach K, Wettenhall REH, Mercer JFB, Brandt A, Thomsen KK: Primary structure of the β-1,3–1,4-glucan 4-glucanohydrolase from barley aleurones. Proc Natl Acad Sci USA 83: 2081–2085 (1986).
Gubler U, Hoffman BJ: A simple and very efficient method for generating cDNA libraries. Gene 25: 263–269 (1983).
Huang JK, Swegle M, Dandekar A, Muthukrishnan S: Expression and regulation of α-amylase gene family in barley aleurones. J. Mol Appl Genet 2: 579–588 (1984).
Huijsduijnen RAMH, Kauffmann S, Brederode FT, Cornelissen BJC, Legrand M, Fritting B, Bol JF: Homology between chitinases that are induced by TMV infection of tobacco. Plant Mol Biol 9: 411–420 (1987).
Islam AKMR, Shepherd KW, Sparrow HB: Isolation and characterization of euplasmic wheat-barley chromosome addition lines. Heredity 46: 161–174 (1981).
Knox CAP, Sonthayanon B, Chandra GR, Muthukrishnan S: Structure and organization of two divergent α-amylase genes from barley. Plant Mol Biol 9: 3–17 (1987).
Leah R, Mikkelsen JD, Mundy J, Svendsen I: Identification of a 28 000 dalton endochitinase in barley endosperm. Carlsberg Res Commun 52: 31–37 (1987).
Legrand M, Kauffmann S, Geoffroy P, Frittig B: Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chitinases. Proc Natl Acad Sci USA 84: 6750–6754 (1987).
Lipman DJ, Pearson WR: Rapid and sensitive protein similarity searches. Science 227: 1435–1441 (1985).
Loi L, Ahluwalia B, Fincher GB: Chromosomal location of genes encoding barley (1–3,1–4)-β-glucan 4-glucanohydrolases. Plant Physiol 87: 300–302 (1988).
Mauch F, Hadwiger LA, Boller T: Antifungal hydrolases in pea tissue. Plant Physiol 87: 325–333 (1988).
Miller JS, Paterson BM, Ricciardi RP, Cohen L, Roberts BE: Methods utilizing cell-free protein synthesizing systems for the identification of recombinant DNA molecules. Methods in Enzymol 101: 650–674 (1983).
Molano J, Polacheck I, Duran A, Cabib E: An endochitinase from wheat germ. J. Biol Chem 254: 4901–4907 (1979).
Muthukrishnan S, Chandra GR: Regulation of the expression of hydrolase genes in cereal seeds. Adv Cereal Sci Technol 9: 129–159 (1988).
Muthukrishnan S, Chandra GR, Maxwell ES: Hormone-induced increase in levels of functional mRNA and α-amylase mRNA in barley aleurones. Proc Natl Acad Sci USA 76: 6181–6186 (1979).
Muthukrishnan S, Chandra GR, Maxwell ES: Hormonal control of α-amylase gene expression in barley — Studies using a cloned cDNA probe. J. Biol Chem 258: 2370–2375 (1983).
Muthukrishnan S, Gill BS, Swegle M, Chandra GR: Structural genes for α-amylases are located on barley chromosomes 1 and 6. J Biol Chem 259: 13637–13639 (1984).
Nolan RC, Lin L-S, Ho T-HD: The effect of abscisic acid on the differential expression of α-amylase isozymes in barley aleurone layers. Plant Mol Biol 8: 13–22 (1986).
Rogers JC, Milliman C: Isolation and sequence analysis of a barley α-amylase cDNA clone. J Biol Chem 258: 8169–8174 (1983).
Rogers JC, Dean D, Heck GR: Aleurain: A barley thiol protease closely related to mammalian cathepsin H. Proc Natl Acad Sci USA 82: 6512–6516 (1985).
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 (1977).
Shinshi H, Mohnen D, Meins F: Regulation of a plant pathogenesisrelated enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci USA 84: 89–93 (1987).
Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77: 5201–5205 (1980).
Whittier RF, Dean DA, Rogers JC: Nucleotide sequence of alpha-amylase and thiol protease that are hormonally regulated in barley aleurone cells. Nucleic Acids Res 15: 2515–2535 (1987).
Wilbur WJ, Lipman DJ: Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci USA 80: 726–730 (1983).
Winston S, Fuller S, Hurrel J: Western blotting. In: Ausubel F, Brent R, Kingston R, Moore D, Smith, Seidman J, Struhl K (eds) Current Protocols in Molecular Biology, pp. 10.8.1–10.8.6. John Wiley, New York (1987).
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Swegle, M., Huang, JK., Lee, G. et al. Identification of an endochitinase cDNA clone from barley aleurone cells. Plant Mol Biol 12, 403–412 (1989). https://doi.org/10.1007/BF00017580
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DOI: https://doi.org/10.1007/BF00017580