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

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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.

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Abbreviations

GH:

Glycosyl hydrolase

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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).

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Authors and Affiliations

  1. College of Life and Environment Sciences, Huangshan University, Anhui, 245041, China

    Hong-Guang Zha, Hong-Xia Zhou & Xiang-Yang Chen

  2. Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, EH9 3JH, UK

    Richard I. Milne

  3. Royal Botanic Garden, Edinburgh, EH3 5LR, UK

    Richard I. Milne

  4. Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650204, China

    Hang Sun

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  1. Hong-Guang Zha
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Correspondence to Hong-Guang Zha.

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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)

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)

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)

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Zha, HG., Milne, R.I., Zhou, HX. et al. Identification and cloning of class II and III chitinases from alkaline floral nectar of Rhododendron irroratum, Ericaceae. Planta 244, 805–818 (2016). https://doi.org/10.1007/s00425-016-2546-y

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