Genome-wide characterization, molecular evolution and mexpression profiling of the metacaspases in potato (Solanum tuberosum L.)

Metacaspases are distant relatives of animal caspases found in plants, protozoa and fungi. Some recent studies have demonstrated that metacaspases are involved in regulating the developmental and environmentally induced programmed cell death in plants. In this study, we identified metacaspase gene family in potato (Solanum tuberosum L.) and analyzed their expression pattern in various developmental tissues and stress responses of plants. There were eight metacaspase genes identified in the Peptidase (Cysteine protease) C14 family and based upon sequence alignment and phylogenetic analysis, a systematic nomenclature of potato metacaspases (SotubMCs) has been proposed. Three of the eight candidate genes showing homology with Arabidopsis thaliana type I metacaspase, AtMC1 were given name SotubMC1, SotubMC2 and SotubMC3 as per the degree of relatedness. Similarly, the next three being homologous to A. thaliana type I metacaspase, AtMC3 were named SotubMC4, SotubMC5, and SotubMC6. The remaining two were named SotubMC7 and SotubMC8, showing significant similarity with type II metacaspases of A. thaliana, AtMC4 and AtMC9, respectively. Evolutionary divergence analysis of SotubMCs from its orthologs in seven other members of Solanaceae family as well as with A. thaliana, Vitis vinifera and Oryza sativa was also carried out. The dN/dS ratios of the orthologous pairs suggested the SotubMCs were under purifying (negative) selection in course of plant evolution. Splicing patterns of potato metacaspases were also analyzed. Amongst all SotubMCs, SotubMC2, SotubMC4, SotubMC6 and SotubMC7 genes appeared to produce multiple alternative spliced variants of different lengths. Furthermore using protein modeling tools, we have predicted the protein structure of identified metacaspases. The cis-regulatory elements analysis was also performed exhibiting the presence of development, stress and hormones related cis-elements in the promoter regions of the SotubMCs. This indicates that potato metacaspases might be playing important roles in the development, stress and hormone responsive pathways. Moreover, relative expression analysis of identified genes was carried out using qRT-PCR in various developmental tissues that also include stolons and tubers. The eight metacaspases showed differential expression in different tissues. Some of the tissues such as leaf undergoing senescence among different leaf developmental stages (immature, mature and senescent) displayed higher relative expression of some of the metacaspases, implying their involvement in leaf senescence. The expression pattern of SotubMCs under various abiotic, biotic and hormonal stresses was also analysed. The results showed that many members of the potato metacaspase gene family displayed differential expression patterns under various stress conditions. Taken together, the study could provide crucial resources for further investigations to understand the functional roles of the identified metacaspases in potato

Genome-wide characterization, molecular evolution and mexpression profiling of the metacaspases in potato (Solanum tuberosum L.)

Metacaspases are distant relatives of animal caspases found in plants, protozoa and fungi. Some recent studies have demonstrated that metacaspases are involved in regulating the developmental and environmentally induced programmed cell death in plants. In this study, we identified metacaspase gene family in potato (Solanum tuberosum L.) and analyzed their expression pattern in various developmental tissues and stress responses of plants. There were eight metacaspase genes identified in the Peptidase (Cysteine protease) C14 family and based upon sequence alignment and phylogenetic analysis, a systematic nomenclature of potato metacaspases (SotubMCs) has been proposed. Three of the eight candidate genes showing homology with Arabidopsis thaliana type I metacaspase, AtMC1 were given name SotubMC1, SotubMC2 and SotubMC3 as per the degree of relatedness. Similarly, the next three being homologous to A. thaliana type I metacaspase, AtMC3 were named SotubMC4, SotubMC5, and SotubMC6. The remaining two were named SotubMC7 and SotubMC8, showing significant similarity with type II metacaspases of A. thaliana, AtMC4 and AtMC9, respectively. Evolutionary divergence analysis of SotubMCs from its orthologs in seven other members of Solanaceae family as well as with A. thaliana, Vitis vinifera and Oryza sativa was also carried out. The dN/dS ratios of the orthologous pairs suggested the SotubMCs were under purifying (negative) selection in course of plant evolution. Splicing patterns of potato metacaspases were also analyzed. Amongst all SotubMCs, SotubMC2, SotubMC4, SotubMC6 and SotubMC7 genes appeared to produce multiple alternative spliced variants of different lengths. Furthermore using protein modeling tools, we have predicted the protein structure of identified metacaspases. The cis-regulatory elements analysis was also performed exhibiting the presence of development, stress and hormones related cis-elements in the promoter regions of the SotubMCs. This indicates that potato metacaspases might be playing important roles in the development, stress and hormone responsive pathways. Moreover, relative expression analysis of identified genes was carried out using qRT-PCR in various developmental tissues that also include stolons and tubers. The eight metacaspases showed differential expression in different tissues. Some of the tissues such as leaf undergoing senescence among different leaf developmental stages (immature, mature and senescent) displayed higher relative expression of some of the metacaspases, implying their involvement in leaf senescence. The expression pattern of SotubMCs under various abiotic, biotic and hormonal stresses was also analysed. The results showed that many members of the potato metacaspase gene family displayed differential expression patterns under various stress conditions. Taken together, the study could provide crucial resources for further investigations to understand the functional roles of the identified metacaspases in potato

Carrier-Mediated Partitioning of Artemisinin into Plasmodium falciparum-Infected Erythrocytes

The purpose of the present study was to characterize the partitioning of artemisinin into both uninfected and Plasmodium falciparum-infected red blood cells (RBCs). The partitioning of [(14)C](+)-artemisinin into RBCs was studied at four different hematocrit levels and eight time periods. At the optimum time of 2 h, the partitioning process was investigated with eight different drug concentrations ranging from 0.88 to 3.52 μM at 37 and 4°C. The effect of the presence of unlabeled artemisinin on the partitioning of the same concentration of [(14)C]artemisinin was studied. About 35 to 40% of the drug was seen to partition into uninfected RBCs at a hematocrit of 33%, irrespective of the incubation period or the drug concentration used. In contrast, infected RBCs showed an increase in partitioning of the drug with time until saturation was achieved at 1 h. While the partitioning of artemisinin into parasitized RBCs at 37°C was found to be significantly higher than that in nonparasitized RBCs, at 4°C both parasitized and nonparasitized RBCs showed identical partitioning of the drug. The partitioning of [(14)C]artemisinin into parasitized RBCs was completely inhibited in the presence of the same concentration of unlabeled artemisinin. However, no such effect was observed in nonparasitized cells, and no evidence suggesting that binding of the drug in parasitized RBCs is reversible was found. The partitioning of artemisinin into parasitized RBCs was found to be rapid, saturable, temperature dependent, irreversible, and subject to competitive inhibition with unlabeled artemisinin. The results obtained suggest the involvement of carrier mediation in the partitioning of artemisinin across the parasitized RBC membrane. In contrast, simple passive diffusion of artemisinin was seen in nonparasitized RBCs