Summary
The fungicidal class I chitinases are believed to be important in the induced defense response of plants. We isolated and partially characterized genomic clones representing two members, CHN14 and CHN50, of the gene subfamily encoding these enzymes in Nicotiana tabacum L. cv. Havana 425. The coding sequences of genes CHN14, CHN50, and CHN48, which was cloned earlier, are identical at 79–95% of the positions. Tobacco is an amphidiploid species derived from ancestors most closely related to the present-day species N. sylvestris and N. tomentosiformis. Southern analysis of genomic DNA, comparison of deduced amino acid sequences, and partial sequencing of the purified enzymes suggest that the gene pairs CHN48/CHN50 and CHN14/CHN14′ are homeologues. Gene CHN48, which encodes chitinase A (Mr ca. 34 kDa), and gene CHN14 are derived from N. tomentosiformis; whereas gene CHN50, which encodes chitinase B (Mr ca. 32 kDa), and gene CHN14′ are derived from N. sylvestris. Class I chitinases are induced in leaves of plants treated with ethylene or infected with the fungal pathogen Cercospora nicotianae and in cultured cells transferred to medium without added auxin and cytokinin. RNase protection assays show that under these conditions transcripts encoded by the homeologues CHN48 and CHN50 account for > 90% of the total chitinase mRNA. The less abundant transcript, CHN48, consistently showed a greater degree of induction than CHN50. Expression of the homeologues CHN14 and CHN14′ represented < 10% of the total chitinase mRNA. They showed a pattern of hormonal regulation similar to CHN48 and CHN50, but transcripts of these genes were not detected in leaves infected with C. nicotianae. Therefore the two sets of homeologues are regulated in the same way by hormones and respond differently to infection by a pathogen.
Similar content being viewed by others
References
Antoniw JF, Ooms G, White RF, Wullems GJ (1983) Pathogenesisrelated proteins in plants and tissues of Nicotiana tabacum transformed by Agrobacterium tumefaciens. Plant Mol Biol 2:317–320
Boller T (1988) Ethylene and the regulation of antifungal hydrolases in plants. Oxf Surveys Plant Mol Cell Biol 5:145–174
Boller T, Vögeli U (1984) Vacuolar localization of ethylene-induced chitinase in bean leaves. Plant Physiol 74:442–444
Boller T, Gehri A, Mauch F, Vögeli U (1983) Chitinase in bean leaves induction by ethylene, purification, properties, and possible function. Planta 157:22–31
Broglie KE, Biddle P, Cressman R, Broglie R (1989) Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco. Plant Cell 1:599–607
Corbin DR, Sauer N, Lamb C (1987) Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants. Mol Cell Biol 7:337–4344
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
Durand-Tardif M (1986) Étude de l'induction, par l'ethephon, de l'expression du gène codant pour la chitinase chez la tomate et analyse de la structure de ce gène. Doctoral Dissertation, Université de Paris Sud, Paris, France
Eichholz R, Harper J, Felix G, Meins F Jr (1983) Evidence for an abundant 33,00-dalton polypeptide regulated by cytokinins in cultured tobacco tissues. Planta 158:410–415
Felix G, Meins F Jr (1986) Developmental and hormonal regulation of β-1,2-glucanase in tobacco. Planta 167:206–211
Felix G, Meins F Jr (1987) Ethylene regulation of β-1,3-glucanase in tobacco. Planta 172:386–392
Fukuda Y, Ohme M, Shinshi H (1991) Gene structure and expression of a tobacco endochitinase gene in suspension cultured tobacco cells. Plant Mol Biol 16:1–10
Gerstel DU (1966) Evolutionary problems in some polyploid crop plants. Hereditas Suppl 2:481–504
Gerstel DU (1976) Tobacco. In: Simmonds NW (ed) Evolution of Crop Plants, Longman, London, pp 273–277
Goodall G, Wiebauer K, Filipowicz W (1990) Analysis of premRNA processing in transfected plant protoplasts. Methods Enzymol 181:148–160
Gray JC, Kung SD, Wildman SG, Sheen SJ (1974) Origin of Nicotiana tabacum L. detected by polypeptide composition of fraction I protein. Nature 252:226–227
Hahlbrock K, Schell D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369
Herget T, Schell J, Schreier PH (1990) Elicitor-specific induction of one member of the chitinase gene family in Arachnis hypogaea. Mol Gen Genet 224:469–476
Hermann A, Schulz W, Hahlbrock K (1988) Two alleles of the single-copy chalcone synthase gene in parsley differ by a transposon-like element. Mol Gen Genet 212:93–98
Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ (1981) A gasliquid solid phase peptide and protein sequenator. J Biol Chem 256:7990–7997
Hooft van Huijsduijnen RAM, van Loon LC, Bol JF (1986) cDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products. EMBO J 5:2057–2061
Hunkapiller MW, Hood LE (1983) Analysis of phenylthiohydantoins by ultrasensitive gradient of high-performance liquid chromatography. Methods Enzymol 9:486–494
Joshi CP (1987a) An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res 15:6643–6653
Joshi CP (1987b) Putative polyadenylation signals in nuclear genes of higher plants compilation and analysis. Nucleic Acids Res 15:9627–9640
Keefe D, Hinz U, Meins F Jr (1990) The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves. Planta 182:43–51
Linsmaier B, Skoog F (1965) Organic growth factor requirements of tobacco tissue culture. Physiol Plant 18:100–127
Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Anal Biochem 163:21–26
Lotan T, Ori N, Fluhr R (1989) Pathogenesis-related proteins are developmentally regulated in tobacco flowers. Plant Cell 1:881–887
Maniatis T, Fritsch, EF Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Mauch F, Staehelin LA (1989) Functional implications of the subcellular localization of ethylene-induced chitinase and β-1,3-glucanase in bean leaves. Plant Cell 1: 447–457
Mauch F, Mauch-Mani B, Boller T (1988) Antifungal hydrolases in pea tissue II. Inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiol 88:936–942
Meins F Jr, Ahl P (1989) Induction of chitinase and β-1,3-glucanase in tobacco leaves infected with Pseudomonas tabaci and Phytophthora parasitica var. nicotianae. Plant Sci 61:155–161
Meins F Jr, Neuhaus J-M, Sperisen C, Ryals J (1992) The primary structure of plant pathogenesis-related glucanohydrolases and their genes. In: Boller T, Meins F Jr (eds) Genes involved in plant defense. Springer Verlag, Vienna/New York, in press
Métraux J-P, Burkhart W, Moyer M, Dincher S, Middlesteadt W, Williams S, Payne G, Carnes M, Ryals J (1989) Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proc Natl Acad Sci USA 86:896–900
Meyer AD (1990) Vacuolar localization of β-1,3-glucanase and chitinase in tobacco. Diploma in Naturwissenschaften, Universität Basel, Basel
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Nader WF, Edling TD, Huettermann A, Sauer HW (1985) Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host. Proc Natl Acad Sci USA 82:2698–2702
Natt E, Scherer G (1986) EMBL 12, a new lambda replacement vector with sites for SalIl, XbaI, BamHI, SstI and EcoRI. Nucleic Acids Res 14:7128–7128
Neale AD, Wahleithner JA., Lund M, Bennett HT, Kelly A, MeeksWagner DR, Peacock WJ, Dennis ES (1990) Chitinase, β-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation. Plant Cell 2:673–684
Neuhaus J-M, Ahl-Goy P, Hinz U, Flores S, Meins F Jr (1991a) High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol Biol 16:141–151
Neuhaus J-M, Sticher L, Meins F Jr, Boller T (1991b) A short C-terminal sequence necessary and sufficient for the targeting of chitinases to the plant vacuole. Proc Natl Acad Sci USA 88:10362–10366
Payne G, Ahl P, Moyer M, Harper A, Beck J, Meins F Jr, Ryals J (1990) Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco. Proc Natl Acad Sci USA 87:98–102
Pichersky E, Bernatzky R, Tanksley SD, Cashmore AR (1986) Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 83:3880–3884
Roby D, Broglie K, Cressman R, Biddle P, Chet I, Broglie R (1990) Activation of a bean chitinase promoter in transgenic tobacco plants by phytopathogenic fungi. Plant Cell 2:999–1007
Ryder TB, Hedrick SA, Bell JN, Liang X, Clouse SD, Lamb CJ (1987) Organisation and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris. Mol Gen Genet 210:219–233
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schlumbaum A, Mauch F, Vögeli U, Boller T (1986) Plant chitinases are potent inhibitors of fungal growth. Nature 324:365–367
Shinshi H, Mohnen D, Meins F Jr (1987) Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci USA 84:89–93
Shinshi H, Neuhaus J-M, Ryals J, Meins F Jr (1990) Structure of a tobacco endochitinase gene: evidence that different chitinases genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Mol Biol 14:357–368
Sperisen C, Ryals J, Meins F Jr (1991) Comparison of cloned genes provides evidence for intergenomic exchange of DNA in the evolution of a tobacco glucan endo-1,3-β-glucosidase gene family. Proc Natl Acad Sci USA 88:1820–1824
Strobaek S, Gibbons GC, Haslett B, Boulter D, Wildman SG (1976) On the nature of the polymorphism of the small subunit of ribulose-1,5-diphosphate carboxylase (EC 4.1.1.39) in the amphidiploid Nicotiana tabacum. Carlsberg Res Commun 41:335–343
Vaucheret H, Kronenberger J, Rouzé P, Caboche M (1989) Complete nucleotide sequence of the two homeologous tobacco nitrate reductase genes. Plant Mol Biol 12:597–600
Vögeli-Lange R, Hansen-Gehri A, Boller T, Meins F Jr (1988) Induction of the defense-related glucanohydrolases, β-1,3-glucanase and chitinase, by tobacco mosaic virus infection of tobacco leaves. Plant Sci 54:171–176
Wingender R, Rbhrig H, Höricke C, Wing D, Schell J (1991) Differential regulation of soybean chalcone synthase genes in plant defence, symbiosis and upon environmental stimuli. Mol Gen Genet 218:315–322
Author information
Authors and Affiliations
Additional information
Communicated by J. Schell
Rights and permissions
About this article
Cite this article
van Buuren, M., Neuhaus, JM., Shinshi, H. et al. The structure and regulation of homeologous tobacco endochitinase genes of Nicotiana sylvestris and N. tomentosiformis origin. Molec. Gen. Genet. 232, 460–469 (1992). https://doi.org/10.1007/BF00266251
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00266251