Comparative de novo transcriptome analysis and random UV mutagenesis: application in high biomass and astaxanthin production enhancement for Haematococcus pluvialis.

BACKGROUND: Astaxanthin is a natural carotenoid with strong antioxidant capacity. The high demand on astaxanthin by cosmetic, food, pharmaceutical and aquaculture industries promote its value in the biotechnological research. Haematococcus pluvialis Flotow 1844 has been characterized as one of the most promising species for natural astaxanthin biosynthesis. Even though H. pluvialis as an advantage in producing astaxanthin, its slow grow-yield limits usage of the species for large-scale production. METHODS AND RESULTS: In this study we generated mutated H. pluvialis strain by using one-step random UV mutagenesis approach for higher biomass production in the green flagellated period and in turn higher astaxanthin accumulation in red stage per unit algae harvest. Isolated mutant strains were tested for the astaxanthin accumulation and yield of biomass. Among tested strains only mutant strain designated as only MT-3-7-2 showed a consistent and higher growth pattern, the rest had shown a fluctuated and then decreased growth rate than wild type. To demonstrate the phenotypical changes in MT-3-7-2 is associated with transcriptome, we carried out comparative analysis of transcriptome profiles between MT-3-7-2 and the wild type strains. De novo assembly was carried out to obtain the transcripts. Differential expression levels for the transcripts were evaluated by functional annotation analysis. CONCLUSIONS: Data showed that increased biomass for the MT-3-7-2 strain was different from wild type with expression of transcripts upregulated in carbohydrate metabolism and downregulated in lipid metabolisms. Our data suggests a switching mechanism is enrolled between carbohydrate and lipid metabolism to regulate cell proliferation and stress responses.

Bacteria isolated from Triticum monococcum ssp. monococcum roots can improve wheat hologenome in agriculture.

BACKGROUND: Triticum monococcum ssp. monococcum is an ancestral wheat species originated from Karacadag Mountain of Turkey more than ten thousand years ago. Because of environmental and anthropogenic effects, food supply and demand are not balanced. Agricultural activities such as breeding, and fertilization are important to sustain the balance. Conventional breeding and fertilization applications usually neglect contribution of plant related hologenomes in agricultural yield. The disruption of plant growth promoting microorganisms results in intensive usage of chemical fertilizers. The harmony between plant and plant-associated microorganisms is important for sustainability. In this study, isolation, biochemical characterization, and impact on plant growth parameters of natural bacteria associated with Triticum monococcum ssp. monococcum hologenome were aimed. METHODS AND RESULTS: The collection of root samples and isolations of the root-associated bacterial species were carried out from local wheat lands. According to interpretation of three identification methods (MALDI-TOF, 16S rDNA, 16S-23S rDNA) eight isolates are Arthrobacter spp. ESU164, Arthrobacter spp. ESU193, Pseudomonas spp. ESU131, Pseudomonas spp. ESU141, Pseudomonas poae strain ESU182, Pseudomonas thivervalensis strain ESU192, Pseudomonas spp. ESU1531, Bacillus subtilis strain ESU181. For each isolate we investigated biochemical properties especially nitrogen fixation, phosphate solubilization, and indole-3-acetic acid production abilities. The results show that all isolates are nitrogen fixers and the best phosphate solubilizer have been reported as Pseudomonas spp. ESU131 with 2.805 ± 0.439. CONCLUSIONS: All isolates are indole-3-acetic acid productors. 2 isolates affected the coleoptile lengths, 7 bacterial isolates showed statistically positive effect on root number, and 5 isolates promote the root lengths and the root fresh weights.

Sequencing, de novo assembly and annotation of Digitalis ferruginea subsp. schischkinii transcriptome.

There is an increasing demand for elucidating the biosynthetic pathway of medicinal plants, which are capable of producing several metabolites with great potentials for industrial drug production. Digitalis species are important medicinal plants for the production of cardenolide compounds. Advancement on culture techniques is strictly related to our understanding of the genomic background of species. There are a limited number of genomic studies on Digitalis species. The goal of this study is to contribute to the genomic data of Digitalis ferruginea subsp. schischkinii by presenting transcriptome annotation. Digitalis ferruginea subsp. schischkinii has a limited distribution in Turkey and Transcaucasia, and has a high level of lanatoside C, an important cardenolide. In the study, we sequenced the cDNA library prepared from RNA pools of D. ferruginea subsp. schischkinii tissues treated with various stress conditions. Comprehensive bioinformatics approaches were used for de novo assembly and functional annotation of D. ferruginea subsp. schischkinii transcriptome sequence data along with TF families predictions and phylogenetic analysis. In the study, 58,369 unigenes were predicted and unigenes were annotated by analyzing the sequence data in the non-redundant (NR) protein database, the non-redundant nucleotide (NT) database, Gene Orthology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), SwissProt, and InterPro databases. This study is the first transcriptome data for D. ferruginea subsp. schischkinii.

Characterization of fungal RTG2 genes in retrograde signaling of Saccharomyces cerevisiae.

Changes in the functional status of mitochondria result in the transcriptional activation of a subset of nuclear-encoded genes in a process referred to as retrograde signaling. In Saccharomyces cerevisiae, this molecular link between mitochondria and the nuclear genome is controlled by three key signaling proteins: Rtg1p, Rtg2p, and Rtg3p. Although the retrograde signaling response has been well characterized in S. cerevisiae, very little is known about this pathway in other fungi. In this study, we selected four species having uncharacterized open reading frames (ORFs) with more than 66% amino acid identity to Rtg2p for further analysis. To determine whether these putative RTG2 ORFs encoded bona fide regulators of retrograde signaling, we tested their ability to complement the defects associated with the S. cerevisiae rtg2Δ mutant. Specifically, we tested for complementation of citrate synthase (CIT2) and aconitase (ACO1) at the transcript and protein levels, glutamate auxotrophy, and changes in the interaction between Rtg2p and the negative regulator Mks1p. Our findings show that all four Rtg2p homologs are functional upon activation of retrograde signaling, although their degree of complementation varied. In addition, all Rtg2p homologs showed a marked reduction in Mks1p binding, which may contribute to their altered responses to retrograde signaling.

A combined de novo assembly approach increases the quality of prokaryotic draft genomes.

Next-generation sequencing methods provide comprehensive data for the analysis of structural and functional analysis of the genome. The draft genomes with low contig number and high N50 value can give insight into the structure of the genome as well as provide information on the annotation of the genome. In this study, we designed a pipeline that can be used to assemble prokaryotic draft genomes with low number of contigs and high N50 value. We aimed to use combination of two de novo assembly tools (SPAdes and IDBA-Hybrid) and evaluate the impact of this approach on the quality metrics of the assemblies. The followed pipeline was tested with the raw sequence data with short reads (< 300) for a total of 10 species from four different genera. To obtain the final draft genomes, we firstly assembled the sequences using SPAdes to find closely related organism using the extracted 16 s rRNA from it. IDBA-Hybrid assembler was used to obtain the second assembly data using the closely related organism genome. SPAdes assembler tool was implemented using the second assembly, produced by IDBA-hybrid as a hint. The results were evaluated using QUAST and BUSCO. The pipeline was successful for the reduction of the contig numbers and increasing the N50 statistical values in the draft genome assemblies while preserving the coverage of the draft genomes.

Effects of moderate high temperature and UV-B on accumulation of withanolides and relative expression of the squalene synthase gene in Physalis peruviana.

Physalis peruviana L. (Cape gooseberry) is a source for a variety of phytocompounds such as withanolides, withanone, withaferin A, and withanolide A. These withanolides are high-value drug candidates due to their various pharmacological properties. To meet the increasing demands for these compounds, plant cell technology offers a reliable alternative. Exogenous addition of elicitors is considered the most effective strategy for enhanced production of secondary metabolites. In this study, we investigated changes in withanolide accumulation and characterized the gene expression level changes of squalene synthase enzyme in P. peruviana shoot cultures exposed to mild nonlethal heat stress (45°C for 2 and 5 h) and UV-B radiation (313 nm for 15 min and 3 h). We demonstrated significant changes in withanolide content with 7.86- and 12.5-fold increases for 2- and 5-hmild high-temperature exposure times, respectively. Exposure to UV-B also changed the withanolide content by 7.22- and 7-fold increases for 15 min and 3 h exposure times, respectively. The relative expression level of squalene synthase gene showed consistent results with1.80- and 10.13-fold increases in withanolide for 2- and 5-h mild high-temperature exposure times, and 1.34- and 2.01-fold increases with 15 min and 3 h UV-B exposure times, respectively.

Identification of alternative oxidase encoding genes in Caulerpa cylindracea by de novo RNA-Seq assembly analysis.

Alternative oxidases (AOX) are defined in plants, fungi and algae. The main function of AOX proteins has been described for electron flow through electron transport chain and regulation of mitochondrial retrograde signaling pathway. The roles of AOX proteins have been characterized in reproduction and resistance against oxidative stress, cold stress, starvation, and biotic attacks. Caulerpa cylindracea is an invasive marine green alga. Although the natural habitats of the species are Australia coasts, the impact of the invasion has been monitored through the Mediterranean Sea and the Aegean Sea. C. cylindracea species have advantages against others by showing higher resistance to stress conditions such as cold, starvation, pathogen attacks and by their capability of sexual and vegetative reproduction. Comparing the advantages of C. cylindracea over the niche and defined functional roles of mitochondrial AOX proteins, it is evident that AOX proteins are likely involved in developing those advantageous skills in C. cylindracea. However, there is limited data about biochemical and molecular mechanisms that take part in stress resistance and invasion characteristics. We aimed to identify mitochondrial alternative oxidase encoding genes in C. cylindracea while annotating whole transcriptome data for the species. Samples were collected from Seferihisar/Izmir. Transcriptome analysis from pooled RNA samples revealed 47,400 assembled contigs represented by 33,340 unigenes. Using standalone Blast analysis, we were able to identify two alternative oxidase encoding genes.

Identification of conserved miRNA molecules in einkorn wheat (Triticum monococcum subsp. monococcum) by using small RNA sequencing analysis.

Triticum monococcum subsp. monococcum as a first cultivated diploid wheat species possesses desirable agronomic and quality characteristics. Drought and salinity are the most dramatic environmental stress factors that have serious impact on yield and quality of crops; however, plants can use alternative defense mechanisms against these stresses. The posttranscriptional alteration of gene expression by microRNAs (miRNAs) is one of the most conserved mechanisms. In plant species including wheat genomes, miRNAs have been implicated in the management of salt and drought stress; however, studies on einkorn wheat (Triticum monococcum subsp. monococcum) are not yet available. In this study, we aimed to identify conserved miRNAs in einkorn wheat using next generation sequencing technology and bioinformatics analysis. In order to include a larger set of miRNAs, small RNA molecules from pooled plant samples grown under normal, drought, and salinity conditions were used for the library preparation and sequence analysis. After bioinformatics analysis, we identified 167 putative mature miRNA sequences belonging to 140 distinct miRNA families. We also presented a comparative analysis to propose that miRNAs and their target genes were involved in salt and drought stress control in addition to a comprehensive analysis of the scanned target genes in the T. aestivum genome.

Effects of deleting mitochondrial antioxidant genes on life span.

Reactive oxygen species (ROS) damage biomolecules, accelerate aging, and shorten life span, whereas antioxidant enzymes mitigate these effects. Because mitochondria are a primary site of ROS generation and also a primary target of ROS attack, they have become a major focus area of aging studies. Here, we employed yeast genetics to identify mitochondrial antioxidant genes that are important for replicative life span. In our studies, it was found that among the known mitochondrial antioxidant genes (TTR1, CCD1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, PRX1), deletion of only three genes, SOD1 (Cu, Zn superoxide dismutase), SOD2 (Manganese-containing superoxide dismutase), and CCS1 (Copper chaperone), shortened the life span enormously. The life span decreased 40% for Deltasod1 mutant, 72% for Deltasod2 mutant, and 50% for Deltaccs1 mutant. Deletion of the other genes had little or no effect on life span.

Small RNA Sequencing Based Identification of MiRNAs in Daphnia magna.

Small RNA molecules are short, non-coding RNAs identified for their crucial role in post-transcriptional regulation. A well-studied example includes miRNAs (microRNAs) which have been identified in several model organisms including the freshwater flea and planktonic crustacean Daphnia. A model for epigenetic-based studies with an available genome database, the identification of miRNAs and their potential role in regulating Daphnia gene expression has only recently garnered interest. Computational-based work using Daphnia pulex, has indicated the existence of 45 miRNAs, 14 of which have been experimentally verified. To extend this study, we took a sequencing approach towards identifying miRNAs present in a small RNA library isolated from Daphnia magna. Using Perl codes designed for comparative genomic analysis, 815,699 reads were obtained from 4 million raw reads and run against a database file of known miRNA sequences. Using this approach, we have identified 205 putative mature miRNA sequences belonging to 188 distinct miRNA families. Data from this study provides critical information necessary to begin an investigation into a role for these transcripts in the epigenetic regulation of Daphnia magna.

Comparison of autogenous bone graft donor site haemostatic agents used in spinal surgery.

OBJECTIVE: The aim of our study was to investigate the effects of haemostatic agents used at the autograft donor sites in spinal fusion. METHODS: The study included 66 patients (26 men, 40 women; mean age: 42.9 years) who underwent spinal fusion surgery between March 1999 and October 2002. Patients were randomly assigned to 4 different groups according to the haemostatic agents used during surgery. In Group 1, bone wax was used on the graft donor site. In Group 2, spongostan was used. In Group 3, spongostan was applied to the donor site and removed after 10 minutes. Group 4 was the control group and no haemostatic agent was applied. Age, sex, diagnosis and incision shape were not taken into consideration during the selection of patient groups. Closed suction drainage systems were used for the evaluation of drainage amount. The drainage system was removed after 48 hours in patients with a daily drainage of less than 30 cc. RESULTS: In Group 1, there was significantly less drainage than the other groups. Group 2 and Group 3 had less drainage than the control group. When a separate incision was used for graft harvesting, keeping the spongostan at the application site (Group 2) was more effective than its removal (Group 3). CONCLUSION: The application of bone wax and spongostan to bleeding cancellous bone surfaces at the donor site is a safe and effective method to reduce bleeding and hematoma. Bone wax is more effective than spongostan for haemostasis.