Modulation of proline metabolic gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> under water-stressed conditions by a drought-mitigating <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ ¹ -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ ¹ -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ ¹ -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.     

Glutathione improves survival of cryopreserved embryogenic calli of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis>

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H₂O₂, OH^·, and O ₂ ^·? ) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in  OH^· generation activity and the contents of H₂O₂ and MDA, as well as enhancement in the inhibition of O ₂ ^·? generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.     

Toxicity and Detoxification Mechanism of Black Pepper and Its Major Constituent in Controlling <Emphasis Type="Italic">Rhynchophorus ferrugineus</Emphasis> Olivier (Curculionidae: Coleoptera)

The survival, feeding response, and detoxification mechanism of Rhynchophorus ferrugineus Olivier, a key pest responsible for destruction of date palm, was examined with different extracts of Piper nigrum and its major constituent (piperine) identified by GC-MS. In the present study, toxicity of different extracts of black pepper was evaluated by incorporating diffferent doses of extracts into the artificial diet of red palm weevil larvae. All extracts showed dose-dependent insecticidal activity to the tested eighth-instar red palm weevil larvae. Among all the extracts, maximum larvicidal activity was exhibited by chloroform (LD₅₀ = 342.62 mg/l), followed by dichloromethane (LD₅₀ = 357.78 mg/l), acetone (LD₅₀ = 372.57 mg/l), and ethanol (LD₅₀ = 408.88 mg/l). However, piperine, a major constituent of all black pepper extracts identified by GC-MS in the present work, was found to be the most potent treatment exhibiting the least LD₅₀ (219.88 mg/l). In addition, nutritional indices evaluated by calculating the efficiency of the conversion of ingested food (ECI) and digested food (ECD) at the same dose (219.88 mg/l) showed that there was maximum reduction in the ECI (49.90%) and ECD (62.21%) index of larvae fed diets incorporated with piperine. Larvae that were fed diets incorporated with different black pepper extracts experienced increases in the expression of detoxification genes (glutathione S-transferase and cytochrome P450), and this upregulation in detoxification genes (glutathione S-transferase, cytochrome P450 and esterase) was tremendously high in larvae fed diets incorporated with piperine. Results suggest that piperine is a promising bio-pesticide agent for the control of R. ferrugineus Olivier.     

Nano-silver modifies the vase life of cut herbaceous peony (<Emphasis Type="Italic">Paeonia lactiflora</Emphasis> Pall.) flowers

Herbaceous peony (Paeonia lactiflora Pall.) is a popular high-grade cut flower because of higher ornamental value. However, its short flowering time severely restricts the production and application of cut P. lactiflora flowers. In this study, nano-silver (NS) was applied to prolong the vase life of cut P. lactiflora flowers. Under the NS treatment, related physiological indices including relative electrical conductivity (REC), malondialdehyde (MDA), superoxide anion free radical (O₂^·?), hydrogen peroxide (H₂O₂) and free proline contents, and protective enzyme activities including superoxide dismutase (SOD), peroxidase (POD) and ascorbic acid peroxidase (APX) all increased in cut P. lactiflora flowers except soluble protein. Meanwhile, NS treatment increased relative water uptake (RWU) and Ag⁺ distribution. Moreover, the observation of microstructures indicated that the stem-ends without NS treatment were blocked by microbes which were identified as Alternaria sp. and Phoma sp., and NS effectively inhibited their growth by antibacterial efficacy observation. Additionally, three aquaporin genes (AQPs) including two plasma membrane intrinsic protein genes (PlPIP1;2, PlPIP2;1) and one NOD26-like intrinsic protein gene (PlNIP) were isolated, PlPIP1;2, and PlPIP2;1 that were induced by NS treatment took common effects on maintaining the water balance of cut P. lactiflora flowers. Consequently, the vase life of cut P. lactiflora flowers was prolonged and flower fresh weight together with flower diameter was well kept because of these above factors. These results would provide a theoretical basis for prolonging the vase life and improving the ornamental quality of cut P. lactiflora flowers with NS application.     

Genes regulating the development and functioning of the nervous system determine life span in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Earlier, it has been shown that genes responsible for differences in longevity between wild-type Drosophila melanogaster lines 2b and Oregon are localized in region 7A6-B2, 36E4-37B9, 37B9-D2, and 64C-65C. Quantitative complementation tests were conducted between the gene mutations localized in these regions and involved in catecholamine biosynthesis (iav (inactive), Catsup (Catecholamines up), amd (alpha metil dopa-resistant), Dox-A2 (Diphenol oxidase A2), ple (pale)) and neuron development control (Fas3 (Fasciclin 3), tup (tail up), Lim3), on the one hand, and two different normal alleles of these genes in lines 2b and Oregon, on the other. Complementation was found for genes iav, Fas3, amd, and ple. The remaining genes (Catsup, Dox-A2, tup, and Lim3) are candidate genes for controlling differences in longevity between lines 2b and Oregon.     

Effects of drought stress on the antioxidant system,osmolytes and secondary metabolites of <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis> seedlings

Saposhnikovia divaricata (Turcz.) Schischk is a traditional herb of East Asia. Bioactive chromones and volatile components in its roots are known to exhibit pharmacological functions. However, limited information is available on the drought resistance of this herb. In this study, potted Saposhnikovia divaricata seedlings were subjected to a progressive drought stress of 20 days by withholding water followed by twice rehydration, which resulted in some physiological, biochemical and secondary metabolite responses as well as drought acclimatization. A decline in leaf water content but increase in electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), glutathione (GSH), proline, soluble sugar, prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol content was observed. After rehydration, some of the indices recovered except proline, soluble sugar, prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol content. Moreover, mild (day 8), moderate (days 12–16) and severe (day 20) drought phases were identified. A total of 18 volatile components were identified by GC–MS under different drought phases, of which aromatic alcohols (42.02%) and sesquiterpenes (37.35%) were the major components. The characteristic component named falcarinol was decreased by severe drought stress. This study demonstrated that Saposhnikovia divaricata had strong drought acclimatization, and resisted drought by activating the antioxidant system and accumulating osmolytes. In addition, moderate and severe drought stress promoted bioactive secondary metabolites prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol accumulation. Severe drought stress reduced falcarinol relative content, which provided an insight for improving the quantity of Saposhnikovia divaricata bioactive components.     

Glutathione reductase gene expression depends on chloroplast signals in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Glutathione reductase (EC 1.6.4.2) is one of the main antioxidant enzymes of the plant cell. In Arabidopsis thaliana, glutathione reductase is encoded by two genes: the gr1 gene encodes the cytosolic-peroxisomal form, and the gr2 gene encodes the chloroplast-mitochondrial form. Little is known about the regulation of expression of plant glutathione reductase genes. In the present work, we have demonstrated that gr2 (but not gr1) gene expression in Arabidopsis leaves changes depending on changes in redox state of the photosynthetic electron transport chain. Expression of both the gr1 and gr2 genes was induced by reactive oxygen species. In heterotrophic suspension cell culture of Arabidopsis, expression of both studied genes did not depend on H₂O₂ level or on changes in the redox state of the mitochondrial electron transport chain. Our data indicate that chloroplasts are involved in the regulation of the glutathione reductase gene expression in Arabidopsis.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

Comparison of L-lactate dehydrogenases of different origins produced in the cells of yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The expression of L-lactate dehydrogenase genes ldh1 (Bos taurus), ldhA (Homo sapiens), ldhA (Rhizopus oryzae), ldh1 (Lactobacillus plantarum), and ldh1 (Lactobacillus pentosus) in the cells of yeast Schizosaccharomyces pombe VKPM U-3106 has been investigated. The catalytic characteristics of the enzymes encoded by these genes have been compared, and the intensity of lactic acid synthesis by the recombinant strains obtained has been evaluated. The enzymatic activity of L-lactate dehydrogenases from L. plantarum and L. pentosus was the highest (approximately 2 to 2.5 times higher than that of the mammalian enzymes), and these enzymes therefore appear to have the highest potential for the development of lactic-acid producing strains of yeast S. pombe.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.