Induction, purification and characterization of chitinases in cucumber (Cucumis sativus L.) and carrot (Daucus carota L.)

The profiles of chitinases (EC 3.2.1.14) in cucumber (Cucumis sativus L.) and carrot (Daucus carota L.) were studied. Chitinase isoform banding patterns were visualized using a polyacrylamide gel electrophoresis/overlay gel technique. In cucumber cotyledons, chitinase isoforms were induced by treatment with chitosan, salicylic acid, wounding, fungal pathogen or non-pathogen inoculation. The induction of chitinase isoforms was not specific to the inducing agents. Chitinase isoform patterns in true leaves and roots were similar to those in cotyledons, and no tissue-specific isoforms were observed. The uniform induction pattern in cotyledons, true leaves and roots suggested that the induction was systemic. Similar isoform patterns were observed in four cucumber cultivars and were not correlated with their resistance to powdery mildew (Sphaerotheca fuliginea). A time-course study showed that the overall increase in activity after induction could be attributed to the enhanced expression of four constitutive isoforms and the induction of three additional isoforms. The expression of the different isoforms was classified into three groups (low constitutive, enhanced constitutive and newly induced). The pIs for these isoforms ranged from pH 4 to 6, and the molecular weight was estimated to be around 25,600. Chitinase-containing extracts from cucumber tissues were shown to have antifungal activity in vitro against Trichoderma and Thielaviopsis.; In mature carrot roots (cv. Eagle), multiple chitinase isoforms (8-10) were produced. Some of these isoforms were shown to have differential cross-reactivity to antisera raised against chitinases from classes I, II, and III. The molecular weight of carrot chitinases was estimated to range from 20,000 to 40,000. One major chitinase, which did not react with any of the antisera tested, was purified and found to be an acidic protein with pI at 4.3 and a molecular weight of 39,500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25{dollar}rmspcirc C.{dollar} The enzyme was stable at pH values below 8 and temperatures below 60{dollar}rmspcirc C.{dollar} The protein did not have a chitin-binding domain, but showed similarity to tobacco class I chitinase in its amino acid composition. The N-terminal amino acid sequence did not resemble any of the described classes of chitinases. The chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.