Changes in the activity of ascorbate peroxidase under anaerobiosis in cocoyam (Colocasia esculenta).

This study was conducted to determine the activity of ascorbate peroxidase in the cormels of cocoyam (Colocasia esculenta var. antiquorum) immediately after harvest and in storage under anaerobiosis for one and three weeks, respectively. During stress condition in plants, hydrogen peroxide is released and mechanisms to detoxify it must be maintained. The cocoyam tubers that were neither damaged nor affected by disease were harvested from a local farm in Ugbogui, Ovia North Local Government Area in Edo State, Nigeria. The selected cocoyam tubers were peeled manually, washed with ice cold water and cut into pieces. The root tissues (50 g) were homogenised with 100 mL of ice cold 0.05 M phosphate buffer. The extract obtained was clarified by centrifugation for 15 min at 8000 g at 4 degrees C. Ascorbate-peroxidising activity was assayed using the initial rate of decrease in ascorbate concentration as measured by its absorbance at 290 nm using Milton Roy Spectron 21D. Results showed the weight of the cormels decreased all through during storage. Immediately after harvest the activity of ascorbate peroxidase was 15.49 unit mL(-1) with a significant increase (p < 0.05) after one week to 73.05 U mL(-1). Thereafter there was a significant decrease in activity of the enzyme after three weeks of storage to 33.33 U mL(-1). This increase in activity of ascorbate peroxidase after three weeks of storage may be related to increase in response to various biotic stresses. Therefore, manipulation of the capacity of cocoyam to tolerate anaerobiosis is a function of its ability to modulate the antioxidant enzymes' armory in case of need.

Crystal structure of tarocystatin-papain complex: implications for the inhibition property of group-2 phytocystatins.

Tarocystatin (CeCPI) from taro (Colocasia esculenta cv. Kaohsiung no. 1), a group-2 phytocystatin, shares a conserved N-terminal cystatin domain (NtD) with other phytocystatins but contains a C-terminal cystatin-like extension (CtE). The structure of the tarocystatin-papain complex and the domain interaction between NtD and CtE in tarocystatin have not been determined. We resolved the crystal structure of the phytocystatin-papain complex at resolution 2.03 Å. Surprisingly, the structure of the NtD-papain complex in a stoichiometry of 1:1 could be built, with no CtE observed. Only two remnant residues of CtE could be built in the structure of the CtE-papain complex. Therefore, CtE is easily digested by papain. To further characterize the interaction between NtD and CtE, three segments of tarocystatin, including the full-length (FL), NtD and CtE, were used to analyze the domain-domain interaction and the inhibition ability. The results from glutaraldehyde cross-linking and yeast two-hybrid assay indicated the existence of an intrinsic flexibility in the region linking NtD and CtE for most tarocystatin molecules. In the inhibition activity assay, the glutathione-S-transferase (GST)-fused FL showed the highest inhibition ability without residual peptidase activity, and GST-NtD and FL showed almost the same inhibition ability, which was higher than with NtD alone. On the basis of the structures, the linker flexibility and inhibition activity of tarocystatins, we propose that the overhangs from the cystatin domain may enhance the inhibition ability of the cystatin domain against papain.

Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus.

Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to ß-lactam antibiotics. PBP2A cross-links nascent peptidoglycan when the native TPs are inhibited by ß-lactams. Although mecA expression is essential for ß-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes methicillin-resistant S. aureus (MRSA) strains to ß-lactams even though the ß-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and ß-lactams is noteworthy not simply because strategies to overcome MRSA are desperately needed but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The "synthetic lethality" of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic-resistant bacterial infections.

Antioxidative enzymes and isozymes analysis of taro genotypes and their implications in Phytophthora blight disease resistance.

Assessment of the differential expression of antioxidative enzymes and their isozymes, was done in 30 day-old ex vitro raised plants of three highly resistant (DP-25, Jhankri and Duradim) and one highly susceptible (N-118) genotypes of taro [Colocasia esculenta (L.) Schott]. Antioxidative enzymes were assayed in the ex vitro plants, 7 days after inoculation with the spores (15,000 spores ml(-1) water) of Phytophthora colocasiae Raciborski to induce taro leaf blight disease. Uninoculated ex vitro plants in each genotype were used as control. The activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased under induced blight condition when compared with control. Increase in antioxidative enzymes was more (67-92%) in the resistant genotypes than that (21-29%) of the susceptible genotype. The zymograms of SOD and GPX in the resistant genotypes, with pathogenic infection, showed increased activity for anodal isoform of SOD and increased expression and/or induction of either POX 1 or POX 2 isoforms of GPX. In susceptible genotype, expression of the above isoforms was faint for SOD and nearly absent for GPX under both blight free and induced blight conditions. Induction and/or increased activity of particular isoform of SOD and GPX against infection of Phytophthora colocasiae in the resistant genotypes studied led to the apparent conclusion of linkage of isozyme expression with blight resistance in taro. This might be an important criterion in breeding of taro for Phytophthora leaf blight resistance.

cDNA cloning, expression, and characterization of Taro SSII: a novel member of starch synthase II family.

A novel soluble starch synthase II (SSII) gene was isolated from taro (Colocasia esculenta var. esculenta) tubers. This 2939 bp SSII transcript encodes 804 amino acids with a putative transit peptide of 52 residues. It displays 58-63% identity and 63-69% similarity with SSIIs from other sources. Alignment and phylogenetic analyses showed that taro SSII is more closely related with dicot SSIIs than with the monocot ones, though taro is a monocot. The identification of taro SSII clone as starch synthase was confirmed by the expression of its enzymatic activity in Escherichia coli. Genomic DNA blot analysis revealed a single copy or low number copies of SSII in taro. Expression profile showed that more transcript and protein were accumulated in tubers of 597 +/- 37 g fresh weight, that is, a stage of rapid starch synthesis, than tubers of other stages. By Western blot analysis, SSII was found in both soluble and granule bound portions of tuber extracts, and more SSII protein was found in aged leaves than in leaves of other stages. These results suggest that taro SSII is a novel starch synthase for the synthesis of both transit and storage starch.

Cloning, expression, and characterization of soluble starch synthase I cDNA from taro (Colocasia esculenta Var. esculenta).

Soluble starch synthase I (SSSI) cDNA was isolated from taro (Colocasia esculenta var. esculenta) by RT-PCR and rapid amplification of cDNA ends reaction. The transcript of this single-copy gene is 2340 bp and encodes 642 amino acids protein containing a putative transit peptide of 54 residues. Recombinant SSSI protein displayed both primer-dependent and primer-independent activities of starch synthase. More SSSI transcript was expressed in taro leaves than in tubers, with no evident expression in petioles; and more transcript and protein were found in tubers of 597 +/- 37 g of fresh weight than in smaller or larger ones. Two forms of SSSI, i.e., 72 and 66 kDa, exist in leaves, and only the 66 kDa form was found in tubers. The taro SSSI, proposed as a novel member, was located only in the soluble fraction of tuber extract, while SSSI from other sources exist in both soluble and granule-bound forms.