Characterization of chitinase activity and gene expression in muskmelon seeds

Chitinase is often produced in higher plants as a general defence response after wounding or pathogenic attack. Since germinating seeds are exposed to soil pathogens, the activity and expression of chitinase in muskmelon (Cucumis melo L.) seeds was investigated. One acidic and three basic chitinase isoforms were detected, beginning 40 d after anthesis in developing and fully mature seeds. Both acidic and basic chitinase isoforms were found in endosperm tissue during imbibition and after radicle emergence. Basic chitinase isoforms, but not acidic isoforms, were detected in the embryonic axes of imbibed seeds and in seeds before germination, indicating that chitinases are developmentally regulated in specific seed tissues. Two complete cDNAs, Cmchi1 and Cmchi2, were cloned from germinated muskmelon seeds and are predicted to encode chitinases that show 95% identity to a class III chitinase from cucumber (Cucumis sativus L.) and 61% identity to a class II chitinase from soybean (Glycine max L.), respectively. Southern blotting indicated that Cmchi2 was present only once in the muskmelon genome, while Cmchi1 may be present in one or two copies. Cmchi1 and Cmchi2 mRNAs were only detected in radicles of germinating seeds and in roots of mature plants, so additional genes other than Cmchi1 and Cmchi2 must be responsible for the chitinase activity in developing seeds. Salicylic acid and benzothiadiazole stimulated the expression of Cmchi1, but not Cmchi2, after radicle emergence. A putative role for chitinase in muskmelon seeds is defence against fungal pathogens.

Pathogen Challenge, Salicylic Acid, and Jasmonic Acid Regulate Expression of Chitinase Gene Homologs in Pine

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable β-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.