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Purification, characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

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Abstract

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one β-1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The β-1,3-glucanase (Mr = 36 kDa) and one of the chitinases (Mr = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (Mr = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic β-1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of β-1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.

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Abbreviations

AC:

acidic chitinase

BC:

basic chitinase

BG =:

basic β-1,3-glucanase

CM-Chitin-RBV:

carboxymethylated-chitin-remazol brilliant violet

2,4-D:

2,4-dichlorophenoxyacetic acid

ILC:

international legume chickpea

Mr :

relative molecular mass

pI:

isoelectric point

SDS-PAGE:

sodium dodecyl sulfatepolyacrylamide gel electrophoresis

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Authors and Affiliations

  1. Westfälische Wilhelms-Universität, Institut für Biochemie und Biotechnologie der Pflanzen, Hindenburgplatz 55, W-4400, Münster, Germany

    Ralph Vogelsang & Wolfgang Barz

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  1. Ralph Vogelsang
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  2. Wolfgang Barz
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We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.

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Vogelsang, R., Barz, W. Purification, characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.). Planta 189, 60–69 (1993). https://doi.org/10.1007/BF00201344

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