Changes in physiological responses of OsCaM1-1 overexpression in the transgenic rice under dehydration stress.

Calmodulin, a primary calcium sensor in eukaryotes, binds calcium and regulates the activity of effector proteins in response to calcium signals that evoked in response to abiotic and biotic stress. To identify physiological responses associated with improved tolerance under dehydration stress that may be regulated by calmodulin in rice, the transgenic rice overexpressing OsCaM1-1, the control, and the wild-type KDML105 differing in their dehydration tolerance were compared 24 h after exposure to dehydration stress. The results demonstrated a greater increase in relative water content, relative growth rate, abscisic acid, photosynthetic pigment and proline contents, and antioxidant activities in the transgenic rice plants, whereas Na/K and Na/Ca ratio, lipid peroxidation, and electrolytic leakage decreased. The OsCaM1-1 gene overexpression in the transgenic rice showed greater tolerance to dehydration stress than non-transgenic rice, suggesting that OsCaM1-1 might play an important role in mitigating dehydration stress.

Cloning and characterization of catalases from rice, Oryza sativa L.

Catalase is the major H(2)O(2)-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)(6)-tagged proteins in Escherichia coli. The recombinant (His)(6)-polypeptides were enriched to apparent homogeneity and characterized. With H(2)O(2) as substrate, the highest catalase k(cat) value (20±1.71×10(-3) min(-1)) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, ß-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.

Biophysical characterization of calmodulin and calmodulin-like proteins from rice, Oryza sativa L.

Calmodulin (CaM) transduces the increase in cytosolic Ca(2+) concentrations by binding to and altering the activities of target proteins, thereby affecting the physiological responses to the vast array of stimuli. Here, we examined the purified recombinant proteins encoded by three Cam and eight Cam-like (CML) genes from rice. With the exception of one OsCML, all recombinant proteins could be purified by Ca(2+)-dependent hydrophobic chromatography and exhibited an electrophoretic mobility shift when incubated with Ca(2+). The three CaMs all bound CaM kinase II peptide, but none of the eight CMLs did, suggesting a possible differential target binding between the CaM and CML proteins. In addition, their conformational changes upon Ca(2+)-binding were evaluated by circular dichroism spectroscopy and fluorescence spectroscopy using 8-Anilino-1-naphthalene-sulfonic acid. Taken together, OsCMLs were found exhibiting a spectrum of both structural and functional characteristics that ranged from typical to atypical of CaMs. From structural comparison, the OsCMLs have overall main-chain conformation nearly identical to OsCaMs, but with distinct distribution of some charged and hydrophobic amino acids on their target-binding site. These results suggest that genetic polymorphism has promoted the functional diversity of the OsCML family, whose members possess modes of actions probably different from, though maybe overlapping with, those of OsCaMs.

Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme.

Plasmodium falciparum, the causative agent of human malaria, is totally dependent on de novo pyrimidine biosynthetic pathway. A gene encoding P. falciparum dihydroorotase (pfDHOase) was cloned and expressed in Escherichia coli as monofunctional enzyme. PfDHOase revealed a molecular mass of 42kDa. In gel filtration chromatography, the major enzyme activity eluted at 40kDa, indicating that it functions in a monomeric form. This was similarly observed using the native enzyme purified from P. falciparum. Interestingly, kinetic parameters of the enzyme and inhibitory effect by orotate and its 5-substituted derivatives parallel that found in mammalian type I DHOase. Thus, the malarial enzyme shares characteristics of both type I and type II DHOases. This study provides the monofunctional property of the parasite DHOase lending further insights into its differences from the human enzyme which forms part of a multifunctional protein.

Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na+/H+ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH.

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.