Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization

Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.     

Phylogeny and molecular evolution of the Acc1 gene within the StH genome species in Triticeae (Poaceae)

To estimate the phylogeny and molecular evolution of a single-copy gene encoding plastid acetyl-CoA carboxylase (Acc1) within the StH genome species, two Acc1 homoeologous sequences were isolated from nearly all the sampled StH genome species and were analyzed with those from 35 diploid taxa representing 19 basic genomes in Triticeae. Sequence diversity patterns and genealogical analysis suggested that (1) the StH genome species from the same areas or neighboring geographic regions are closely related to each other; (2) the Acc1 gene sequences of the StH genome species from North America and Eurasia are evolutionarily distinct; (3) Dasypyrum has contributed to the nuclear genome of Elymus repens and Elymus mutabilis; (4) the StH genome polyploids have higher levels of sequence diversity in the H genome homoeolog than the St genome homoeolog; and (5) the Acc1 sequence may evolve faster in the polyploid species than in the diploids. Our result provides some insight on evolutionary dynamics of duplicate Acc1 gene, the polyploidy speciation and phylogeny of the StH genome species.     

Antioxidant status of Lobiger serradifalci and Oxynoe olivacea (Opisthobranchia, Mollusca)

Invasion of the Mediterranean Sea by the two world-wide famous exotic algae species, Caulerpa taxifolia and Caulerpa racemosa, is still a problem and has adverse effects on the Mediterranean sublittoral ecosystem. Biological control studies revealed that the two native Sacoglossans, Oxynoe olivacea and Lobiger serradifalci, may have an effect on the expansion of invasive Caulerpa spp. in the Mediterranean. In the framework of this study, antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, as oxidative stress markers in L. serradifalci and O. olivacea were determined at two different temperature conditions (20 and 27 °C). In both species, SOD, CAT and GSH-Px activities were found to be positively correlated with temperature. The SOD activities in L. serradifalci were higher than those in O. olivacea at both temperatures, whereas the CAT and GSH-Px activities were significantly (p<0.05) higher in O. olivacea, compared to L. serradifalci. As expected, both species showed decreased LPO levels at 27 °C compared to 20 °C. GSSG level at 27 °C in O.olivacea was significantly (p<0.05) higher than that of 20 °C. On the other hand, no statistical (p>0.05) difference in L.serradifalci existed between GSSG levels at two temperatures. But, despite the variations in the antioxidant enzyme activities, there was no significant difference in LPO levels between the species, suggesting that the oxidative consequences of a given environmental condition may vary among different species. Inasmuch as the GSSG levels were in accordance with antioxidant enzyme activities, GSH might have acted as a cofactor of GSH-Px and an individual antioxidant in these sea slugs.     

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.     

Ultrastructural characterization and multilocus sequence analysis (MLSA) of ‘Candidatus Rickettsiella isopodorum’, a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda)

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation ‘Candidatus Rickettsiella isopodorum’ is introduced for this new lineage of isopod-associated Rickettsiella bacteria.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.     

Stimulation of chlororespiration by drought under heat and high illumination in Rosa meillandina

Rosa meillandina plants were used to study the effects of water deficit on photosynthesis and chlororespiration. Plants showed high tolerance to heat and high illumination in controlled conditions that ensured that there was no water deficit. However, when heat and high illumination were accompanied by low watering photosynthetic linear electron transport was down regulated, as indicated by the reduced photochemistry efficiency of PS II, which was associated with an increase in the non-photochemical quenching of fluorescence. In addition to the effects on the photosynthetic activity, changes were also observed in the plastidial NDH complex, PTOX and PGR5. In plants exposed to heat and high illumination without water deficit, the activities and amounts of the chlororespiration enzymes, NDH complex and PTOX, remained similar to the control and only increased in response to drought, high light and heat stress, applied together. In contrast, both the PS I activity and the amount of PGR5 polypeptide were higher in plants exposed to heat and high illumination without water deficit than in those with water deficit. The results indicated that in the conditions studied, the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when there is no water deficit, and another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PS II activity is inhibited by drought, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.     

Association of glutathione S-transferase polymorphisms (GSTM1 and GSTT1) with primary open-angle glaucoma: An evidence-based meta-analysis

Studies investigating the associations between glutathione S-transferase (GST) genetic polymorphisms and primary open-angle glaucoma (POAG) have reported controversial results. Therefore, a meta-analysis was performed to clarify the effects of GSTM1 and GSTT1 polymorphisms on POAG risk. Published literatures from PubMed, EMBASE, ISI Web of Science and CBM databases were retrieved. All studies evaluating the association between GSTM1/GSTT1 polymorphisms and POAG were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using fixed- or random-effects model. Eleven studies on GSTM1 (1339 cases and 1412 controls) and seven studies on GSTT1 (958 cases, 1003 controls) were included. Overall analysis showed that the association between GSTM1 and GSTT1 null genotype and POAG risk is not statistically significant. Subgroup analyses showed that the null genotype of GSTM1 increased the risk of POAG in Asians. In GSTM1–GSTT1 interaction analysis, individuals with dual null genotype were associated with a significantly increased risk of POAG when compared with the dual present genotype. In conclusion, the present meta-analysis suggested that GSTM1 null genotypes are associated with increased POAG risk in Asian populations but not in Caucasian and mixed populations. Dual null genotype of GSTM1/GSTT1 is associated with increased risk of POAG. Given the limited sample size, the finding on GST polymorphisms needs further investigation.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.