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Planta

, Volume 244, Issue 4, pp 805–818 | Cite as

Identification and cloning of class II and III chitinases from alkaline floral nectar of Rhododendron irroratum, Ericaceae

  • Hong-Guang ZhaEmail author
  • Richard I. Milne
  • Hong-Xia Zhou
  • Xiang-Yang Chen
  • Hang Sun
Original Article

Abstract

Main conclusion

Class II and III chitinases belonging to different glycoside hydrolase families were major nectarins in Rhododendron irroratum floral nectar which showed significant chitinolytic activity.

Previous studies have demonstrated antimicrobial activity in plant floral nectar, but the molecular basis for the mechanism is still poorly understood. Two chitinases, class II (Rhchi2) and III (Rhchi3), were characterized from alkaline Rhododendron irroratum nectar by both SDS-PAGE and mass spectrometry. Rhchi2 (27 kDa) and Rhchi3 (29 kDa) are glycoside hydrolases (family 19 and 18) with theoretical pI of 8.19 and 7.04. The expression patterns of Rhchi2 and Rhchi3 were analyzed by semi-quantitative RT-PCR. Rhchi2 is expressed in flowers (corolla nectar pouches) and leaves while Rhchi3 is expressed in flowers. Chitinase in concentrated protein and fresh nectar samples was visualised by SDS-PAGE and chitinolytic activity in fresh nectar was determined spectrophotometrically via chitin-azure. Full length gene sequences were cloned with Tail-PCR and RACE. The amino acid sequence deduced from the coding region for these proteins showed high identity with known chitinases and predicted to be located in extracellular space. Fresh R. irroratum floral nectar showed significant chitinolytic activity. Our results demonstrate that class III chitinase (GH 18 family) also exists in floral nectar. The functional relationship between class II and III chitinases and the role of these pathogenesis-related proteins in antimicrobial activity in nectar is suggested.

Keywords

Alkaline nectar Chitinolytic activity Class II chitinase Class III chitinase Glycoside hydrolase Pathogenesis-related proteins 

Abbreviation

GH

Glycosyl hydrolase

Notes

Acknowledgments

We thank Prof. Bernard Henrissat (CNRS) for his suggestions and critical reading of the manuscript. This study was supported by National Science Foundation of China (Grant No. 31170216 to HG Zha) and Major Program of National Natural Science Foundation of China (Grant No. 31590823 to Hang Sun).

Compliance with ethical standards

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

Our work complies to the ethical rules applicable for this journal.

Supplementary material

425_2016_2546_MOESM1_ESM.tif (2.2 mb)
Supplementary Fig. S1 MS spectra of identified proteins and fragments. a Rhchi2 MS spectra. b MS/MS spectra of m/z 1451.65, “GFYTYEAFI(L)AAAK” fragment in Rhchi2. c MS/MS spectra of m/z 2642.2, “TAL(I)WFWMTPQSPKPSSHDVITGR” fragment in Rhchi2. d Rhchi3 MS spectra. e MS/MS spectra of m/z 1110.5, “YGGI(L)ML(I)WDR” fragment in Rhchi3. f MS/MS spectra of m/z 1526.77, “I(L)VNL(I)GFL(I)SAFGNFK” fragment in Rhchi3 (TIFF 2255 kb)
425_2016_2546_MOESM2_ESM.doc (106 kb)
Supplementary Fig. S2 Comparison of Rhchi2 amino acid sequence with that of six class II plant chitinase homologues. Amino acids, which are completely conserved are marked with asterisks, and the highly conserved amino acids are marked with dots or double dots. -, gap left to improve alignment. Numbers refer to amino acid residues at the end of the respective lines. Species names are abbreviated at the left and represent with accession number: Zmchi2 (Zea mays, B6SZC6), Gmchi2 (Glycine max, C6TNB0), Ntchi2 (Nicotiana tabacum, Q9ZWS3), Vvchi2 (Vitis vinifera, A5AT00), Qschi2 (Oryza sativa, Q7XCK6), Ghchi2 (Gossypium hirsutum, P931545) (DOC 105 kb)
425_2016_2546_MOESM3_ESM.doc (110 kb)
Supplementary Fig. S3 Comparison of Rhchi3 amino acid sequence with that of six class III plant chitinase homologues. Amino acids, which are completely conserved are marked with asterisks, and the highly conserved amino acids are marked with dots or double dots. -, gap left to improve alignment. Numbers refer to amino acid residues at the end of the respective lines. Species names are abbreviated at the left and represent an accession number: Zmchi3 (Zea mays, B4G1T3), Gmchi3 (Glycine max, C6T8G2), Ntchi3 (Nicotiana tabacum, P29061), Vvchi3 (Vitis vinifera, Q84S31), Qschi3 (Oryza sativa, Q84ZH2), Ghchi3 (Gossypium hirsutum, A2TJX5) (DOC 110 kb)

References

  1. Adrangi S, Faramarzi MA (2013) From bacteria to human: a journey into the world of chitinases. Biotechnol Adv 31:1786–1795CrossRefPubMedGoogle Scholar
  2. Akimoto C, Aoyagi H, Dicosmo F, Tanaka H (2000) Synergistic effect of active oxygen species and alginate on chitinase production by Wasabia japonica cells and its application. J Biosci Bioeng 89:131–137CrossRefPubMedGoogle Scholar
  3. Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92:773–784CrossRefPubMedGoogle Scholar
  4. Baker HG, Baker I (1983) A brief historical review of the chemistry of floral nectar. In: Bentley B, Elias T (eds) The biology of nectaries. Columbia University Press, New York, pp 126–152Google Scholar
  5. Beintema JJ (1994) Structural features of plant chitinases and chitin-binding proteins. FEBS Lett 350:159–163CrossRefPubMedGoogle Scholar
  6. Benhamou N, Joosten MHAJ, de Wit PJGM (1990) Subcellular localization of chitinase and of potential substrate in tomato root tissue infected by Fusarium oxysporum f. sp. radicis-lycopersici. Plant Physiol 92:1108–1120CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Briesemeister S, Rahnenführer J, Kohlbacher O (2010) YLoc–an interpretable web server for predicting subcellular localization. Nucleic Acids Res 38:W497–W502CrossRefPubMedPubMedCentralGoogle Scholar
  9. Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194–1197CrossRefPubMedGoogle Scholar
  10. Brunner F, Stintzi A, Fritig B, Goddard Iii WA (1998) Substrate specificities of tobacco chitinases. Plant J 14:225–234CrossRefPubMedGoogle Scholar
  11. Carter C, Thornburg RW (2000) Tobacco nectarin I: purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defense of floral reproductive tissues. J Biol Chem 275:36726–36733CrossRefPubMedGoogle Scholar
  12. Carter C, Thornburg RW (2004) Is the nectar redox cycle a floral defense against microbial attack? Trends Plant Sci 9:320–324CrossRefPubMedGoogle Scholar
  13. Chamberlain DF (1982) A revision of Rhododendron II. subgenus Hymenanthes. Notes from the Royal Botanic Garden, Edinburgh 39:209–486Google Scholar
  14. Chamnongpol S, Willekens H, Moeder W, Langebartels C, Sandermann H Jr, van Montagu M, Inzé D, van Camp W (1998) Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Proc Natl Acad Sci USA 95:5818–5823CrossRefPubMedPubMedCentralGoogle Scholar
  15. Chang KLB, Tai MC, Cheng FH (2001) Kinetics and products of the degradation of chitosan by hydrogen peroxide. J Agr Food Chem 49:4845–4851CrossRefGoogle Scholar
  16. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3:31–40CrossRefPubMedGoogle Scholar
  17. Dore I, Legrand M, Cornelissen BJC, Bol JF (1991) Subcellular localization of acidic and basic PR proteins in tobacco mosaic virus infected tobacco. Arch Virol 120:97–107CrossRefPubMedGoogle Scholar
  18. Dubois M, Gilles KA, Hamilton JR, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  19. Eilenberg H, Pnini-Cohen S, Schuster S, Movtchan A, Zilberstein A (2006) Isolation and characterization of chitinase genes from pitchers of the carnivorous plant Nepenthes khasiana. J Exp Bot 57:2775–2784CrossRefPubMedGoogle Scholar
  20. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971CrossRefPubMedGoogle Scholar
  21. Esaka M, Teramoto T (1998) cDNA cloning, gene expression and secretion of chitinase in winged bean. Plant Cell Physiol 39:349–356CrossRefPubMedGoogle Scholar
  22. Escalante-Perez M, Jaborsky M, Reinders J, Kurzai O, Hedrich R, Ache P (2012) Poplar extrafloral nectar is protected against plant and human pathogenic fungus. Mol Plant 5:1157–1159CrossRefPubMedGoogle Scholar
  23. Evans JD, Armstrong TN (2006) Antagonistic interactions between honey bee bacterial symbionts and implications for disease. BMC Ecol 6:4. doi: 10.1186/1472-6785-6-4 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Ferrari M, Bjornstad O, Partain J, Antonovics J (2006) A gravity model for the spread of a pollinator-borne plant pathogen. Am Nat 168:294–303CrossRefPubMedGoogle Scholar
  25. Ferre F, Clote P (2006) DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification. Nucleic Acids Res 34(Suppl 2): W182–W185Google Scholar
  26. Flach J, Pilet PE, Jolles P (1992) What’s new in chitinase research? Experientia 48:701–716CrossRefPubMedGoogle Scholar
  27. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols Handbook. Humana Press, Totowa, pp 571–607CrossRefGoogle Scholar
  28. Gonzalez-Teuber M, Eilmus S, Muck A, Svatos A, Heil M (2009) Pathogenesis-related proteins protect extrafloral nectar from microbial infestation. Plant J 58:464–473CrossRefPubMedGoogle Scholar
  29. Gonzalez-Teuber M, Pozo MJ, Muck A, Svatos A, Adame-Alvarez RM, Heil M (2010) Glucanases and chitinases as causal agents in the protection of acacia extrafloral nectar from infestation by phytopathogens. Plant Physiol 152:1705–1715CrossRefPubMedGoogle Scholar
  30. Grover A (2012) Plant chitinases: genetic diversity and physiological roles. CRC Crit Rev Plant Sci 31:57–73CrossRefGoogle Scholar
  31. Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine. Oxford University Press, New YorkGoogle Scholar
  32. Hamel F, Boivin R, Tremblay C, Bellemare G (1997) Structural and evolutionary relationships among chitinases of flowering plants. J Mol Evol 44:614–624CrossRefPubMedGoogle Scholar
  33. Hamid R, Khan MA, Ahmad M et al (2013) Chitinases: an update. J Pharm Bioallied Sci 5:21–29PubMedPubMedCentralGoogle Scholar
  34. Heil M (2011) Nectar: generation, regulation and ecological functions. Trends Plant Sci 16:191–200CrossRefPubMedGoogle Scholar
  35. Heil M (2015) Extrafloral nectar at the plant-insect interface: a spotlight on chemical ecology, phenotypic plasticity, and food webs. Annu Rev Entomol 60:213–232CrossRefPubMedGoogle Scholar
  36. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644CrossRefPubMedGoogle Scholar
  37. Hillwig MS, Kanobe C, Thornburg RW, MacIntosh GC (2011) Identification of S-RNase and peroxidase in petunia nectar. J Plant Physiol 168:734–738CrossRefPubMedGoogle Scholar
  38. Huet J, Rucktooa P, Clantin B, Azarkan M, Looze Y, Villeret V, Wintjens R (2008) X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases. Biochemistry 47:8283–8291CrossRefPubMedGoogle Scholar
  39. Iseli B, Armand S, Boller T, Neuhaus JM, Henrissat B (1996) Plant chitinases use two different hydrolytic mechanisms. FEBS Lett 382:186–188CrossRefPubMedGoogle Scholar
  40. Jaakola L, Pirttilä A, Halonen M, Hohtola A (2001) Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit. Mol Biotechnol 19:201–203CrossRefPubMedGoogle Scholar
  41. Jach G, Goruhardt B, Mundy J, Logemann J, Pinsdorf E, Leah R, Schell J, Maas C (1995) Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8:97–109CrossRefPubMedGoogle Scholar
  42. Kasprzewska A (2003) Plant chitinases-regulation and function. Cell Mol Biol Lett 8:809–824PubMedGoogle Scholar
  43. Kikuchi T, Masuda K (2009) Class II chitinase accumulated in the bark tissue involves with the cold hardiness of shoot stems in highbush blueberry (Vaccinium corymbosum L.). Sci Hortic-Amsterdam 120:230–236CrossRefGoogle Scholar
  44. Kobayashi N, Horikoshi T, Katsuyama H, Handa T, Takayanagi K (1998) A simple and efficient DNA extraction method for plants, especially woody plants. Plant Tissue Culture Biotech 4:72–80Google Scholar
  45. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  46. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  47. Lawton K, Ward E, Payne G, Moyer M, Ryals J (1992) Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco. Plant Mol Biol 19:735–743CrossRefPubMedGoogle Scholar
  48. Lin W, Hu X, Zhang W, Rogers WJ, Cai W (2005) Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice. J Plant Physiol 162:937–944CrossRefPubMedGoogle Scholar
  49. Liu YG, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43:649–656CrossRefPubMedGoogle Scholar
  50. Liu D, Cai J, Xie CC, Liu C, Chen YH (2010) Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri, and its biocontrol potential. Enzyme Microb Tech 46:252–256CrossRefGoogle Scholar
  51. Melchers LS, Ponstein AS, Sela-Buurlage MB, Vloemans SA, Cornelissen BJC (1993) In vitro anti-microbial activities of defense proteins and biotechnology. In: Fritig B, Legrand M (eds) Mechanisms of plant defense. Kluwer Academic Publishers, Dordrecht, pp 401–410CrossRefGoogle Scholar
  52. Minic Z (2008) Physiological roles of plant glycoside hydrolases. Planta 227:723–740CrossRefPubMedGoogle Scholar
  53. Molan PC, Mizrahi A, Lensky Y (1997) Honey as an antimicrobial agent. In: Mizrahi A, Lensky Y (eds) Bee products: properties, applications and apitherapy. Plenum Press, New York, pp 27–37CrossRefGoogle Scholar
  54. Nakatsuka A, Mizuta D, Kii Y, Miyajimac I, Kobayashia N (2008) Isolation and expression analysis of flavonoid biosynthesis genes in evergreen azalea. Sci Hortic-Amsterdam 118:314–320CrossRefGoogle Scholar
  55. Neuhaus JM, Fritig B, Linthorst HJM, Meins F, Mikkelsen J, Ryals J (1996) A revised nomenclature for chitinase genes. Plant Mol Biol Rep 14:102–104CrossRefGoogle Scholar
  56. Nicolson S, Thornburg RW (2007) Nectar chemistry. In: Pacini E, Nepi M, Nicolson S (eds) Nectary and nectar: a modern treatise. Springer, Amsterdam, pp 215–263CrossRefGoogle Scholar
  57. Nocentini D, Guarnieri M, Soligo C (2015) Nectar defense and hydrogen peroxide in floral nectar of Cucurbita pepo. Acta Agrobotanica 68:187–193CrossRefGoogle Scholar
  58. Oldach KH, Becker D, Lorz H (2001) Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat. Mol Plant Microbe Interact 14:832–838CrossRefPubMedGoogle Scholar
  59. Pacini E, Nicolson SW (2007) Introduction. In: Nicolson SW, Nepi M, Pacini E (eds) Nectaries and nectar. Springer, Dordrecht, pp 1–18CrossRefGoogle Scholar
  60. Park CH, Kim S, Park JY, Ahn IP, Jwa NS, Im KH, Lee YH (2004) Molecular characterization of a pathogenesis-related protein 8 gene encoding a class III chitinase in rice. Mol Cells 17:144–150PubMedGoogle Scholar
  61. Peng Y, Arora R, Li GW, Wang X, Fessehaie A (2008) Rhododendron catawbiense plasma membrane intrinsic proteins are aquaporins, and their over-expression compromises constitutive freezing tolerance and cold acclimation ability of transgenic Arabidopsis plants. Plant, Cell Environ 31:1275–1289CrossRefGoogle Scholar
  62. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786CrossRefPubMedGoogle Scholar
  63. Primack RB (1985) Longevity of individual flowers. Annu Rev Ecol Syst 16:15–37CrossRefGoogle Scholar
  64. Prŷs-Jones OE, Willmer PG (1992) The biology of alkaline nectar in the purple toothwort (Lathraea clandestina): ground level defences. Biol J Linn Soc 45:373–388CrossRefGoogle Scholar
  65. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379CrossRefPubMedGoogle Scholar
  66. Schlumbaum A, Mauch F, Vögeli U, Boller T (1986) Plant chitinases are potent inhibitors of fungal growth. Nature 324:365–367CrossRefGoogle Scholar
  67. Seo PJ, Wielsch N, Kessler D, Svatos A, Park CM, Baldwin IT, Kim SG (2013) Natural variation in floral nectar proteins of two Nicotiana attenuata accessions. BMC Plant Biol 13:101CrossRefPubMedPubMedCentralGoogle Scholar
  68. Singh A, Isaac Kirubakaran S, Sakthivel N (2007) Heterologous expression of new antifungal chitinase from wheat. Protein Expres Purif 56:100–109CrossRefGoogle Scholar
  69. Takenaka Y, Nakano S, Tamoi M, Sakuda S, Fukamizo T (2009) Chitinase gene expression in response to environmental stresses in Arabidopsis thaliana: chitinase inhibitor allosamidin enhances stress tolerance. Biosci Biotech Biochem 73:1066–1071CrossRefGoogle Scholar
  70. Trudel J, Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178:362–366CrossRefPubMedGoogle Scholar
  71. Udaya Prakash NA, Jayanthi M, Sabarinathan R, Sabarinathan R, Kangueane P, Mathew L, Sekar K (2010) Evolution, homology conservation, and identification of unique sequence signatures in GH19 family chitinases. J Mol Evol 70:466–478CrossRefPubMedGoogle Scholar
  72. Vitale A, Chrispeels MJ (1992) Sorting of proteins to the vacuoles of plant cells. BioEssays 14:151–160CrossRefPubMedGoogle Scholar
  73. Wagner R, Mugnaini S, Sniezko R, Hardie D, Poulis B, Nepi M, Pacini E, von Aderkas P (2007) Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions. Sex Plant Reprod 20:181–189CrossRefGoogle Scholar
  74. Weston RJ (2000) The contribution of catalase and other natural products to the antibacterial activity of honey: a review. Food Chem 71:235–239CrossRefGoogle Scholar
  75. Zha HG, Milne RI, Sun H (2010) Asymmetric hybridization in Rhododendron agastum: a hybrid taxon comprising mainly F1 s in Yunnan, China. Ann Bot 105:89–100CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Hong-Guang Zha
    • 1
    Email author
  • Richard I. Milne
    • 2
    • 3
  • Hong-Xia Zhou
    • 1
  • Xiang-Yang Chen
    • 1
  • Hang Sun
    • 4
  1. 1.College of Life and Environment SciencesHuangshan UniversityAnhuiChina
  2. 2.Institute of Molecular Plant SciencesUniversity of EdinburghEdinburghUK
  3. 3.Royal Botanic GardenEdinburghUK
  4. 4.Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of BotanyChinese Academy of SciencesKunmingChina

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