Characterization of inhibitory mechanism and antifungal activity between group-1 and group-2 phytocystatins from taro (Colocasia esculenta).

Tarocystatin from Colocasia esculenta, a group-2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N-terminal region, which is homological to group-1 cystatin, and a repetitive peptide at the C-terminus. The purified recombinant proteins of tarocystatin, such as full-length (FL), N-terminus (Nt) and C-terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K(ia) = 0.098 microM and K(ib) = 0.252 microM) and Nt peptide showed competitive inhibition (K(i) = 0.057 microM), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group-2) and Nt (group-1) peptides on papain activity, an inhibitory mechanism of group-2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase.

Carbohydrate mobilization and gene regulatory profile in the pseudobulb of Oncidium orchid during the flowering process.

The pseudobulb of Oncidium orchid is a storage organ for supplying water, minerals and carbohydrates to the developing inflorescence. Different patterns of mannan, starch and pectin metabolism were observed in the pseudobulb of three developmental stages by histochemical staining and high performance anion exchange chromatographic (HPAEC) analysis. Copious pectin was strongly stained by ruthenium red in young pseudobulbs demonstrating that mannan and pectin were preferentially accumulated in the young pseudobulb sink at inflorescence pre-initiation stage. Concomitant with the emergence of the inflorescence, mannan and pectin decreased gradually and converted to starch. The starch, synthesized at the inflorescence developing stage, was eventually degraded at the floral development stage. A systematic survey on the subtractive EST (expression sequence tag) library of pseudobulb in the inflorescence pre-initiation stage revealed the presence of five groups of gene homologues related to sucrose, mannan, starch, pectin and other carbohydrate metabolism. The transcriptional level of 13 relevant genes related to carbohydrate metabolism was characterized from pseudobulbs of three different developmental stages. The specific activities of the enzymes encoded by these genes were also assayed. The expression profiles of these genes show that the transcriptional levels largely correlated with the enzyme activities, which were associated with the respective carbohydrate pools. These results demonstrated a novel functional profile of polysaccharide mobilization pathway as well as their relevant gene expression in the pseudobulb of Oncidium orchid during the flowering process.