title: Identification and cloning of class II and III chitinases from alkaline floral nectar of Rhododendron irroratum, Ericaceae.
abstract: 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.
authors.affiliation: College of Life and Environment Sciences, Huangshan University, Anhui, 245041, China. zhahg@hsu.edu.cn.
authors.forename: Hong-Guang
authors.initials: HG
authors.lastname: Zha
authors.affiliation: Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, EH9 3JH, UK.|Royal Botanic Garden, Edinburgh, EH3 5LR, UK.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Milne
authors.affiliation: College of Life and Environment Sciences, Huangshan University, Anhui, 245041, China.
authors.forename: Hong-Xia
authors.initials: HX
authors.lastname: Zhou
authors.affiliation: College of Life and Environment Sciences, Huangshan University, Anhui, 245041, China.
authors.forename: Xiang-Yang
authors.initials: XY
authors.lastname: Chen
authors.affiliation: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650204, China.
authors.forename: Hang
authors.initials: H
authors.lastname: Sun
mesh_terms: D000468:Alkalies; D000595:Amino Acid Sequence; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D035264:Flowers / Q000201:enzymology / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D057048:Plant Nectar / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029793:Rhododendron / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
chemical_list: D000468:Alkalies; D057048:Plant Nectar; D010940:Plant Proteins; D002688:Chitinases; C110892:chitinase, class III
keywords: Alkaline nectar; Chitinolytic activity; Class II chitinase; Class III chitinase; Glycoside hydrolase; Pathogenesis-related proteins