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Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress

The WRKY superfamily of transcription factors was shown to be involved in biotic and abiotic stress responses in plants such as wheat (Triticum aestivum L.), one of the major crops largely cultivated and consumed all over the world. Drought is an important abiotic stress resulting in a considerable amount of loss in agronomical yield. Therefore, identification of drought responsive WRKY members in wheat has a profound significance. Here, a total of 160 TaWRKY proteins were characterized according to sequence similarity, motif varieties, and their phylogenetic relationships. The conserved sequences of the TaWRKYs were aligned and classified into three main groups and five subgroups. A novel motif in wheat, WRKYGQR, was identified. To putatively determine the drought responsive TaWRKY members, publicly available RNA-Seq data were analyzed for the first time in this study. Through in silico searches, 35 transcripts were detected having an identity to ten known TaWRKY genes. Furthermore, relative expression levels of TaWRKY16/TaWRKY16-A, TaWRKY17, TaWRKY19-C, TaWRKY24, TaWRKY59, TaWRKY61, and TaWRKY82 were measured in root and leaf tissues of drought-tolerant Sivas 111/33 and susceptible Atay 85 cultivars. All of the quantified TaWRKY transcripts were found to be up-regulated in root tissue of Sivas 111/33. Differential expression of TaWRKY16, TaWRKY24, TaWRKY59, TaWRKY61 and TaWRKY82 genes was discovered for the first time upon drought stress in wheat. These comprehensive analyses bestow a better understanding about the WRKY TFs in bread wheat under water deficit, and increased number of drought responsive WRKYs would contribute to the molecular breeding of tolerant wheat cultivars.

Boron Stress Responsive MicroRNAs and Their Targets in Barley

Boron stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. In this study, miRNAs associated with boron stress were identified and characterized in barley. miRNA profiles were also comparatively analyzed between root and leave samples. A total of 31 known and 3 new miRNAs were identified in barley; 25 of them were found to respond to boron treatment. Several miRNAs were expressed in a tissue specific manner; for example, miR156d, miR171a, miR397, and miR444a were only detected in leaves. Additionally, a total of 934 barley transcripts were found to be specifically targeted and degraded by miRNAs. In silico analysis of miRNA target genes demonstrated that many miRNA targets are conserved transcription factors such as Squamosa promoter-binding protein, Auxin response factor (ARF), and the MYB transcription factor family. A majority of these targets were responsible for plant growth and response to environmental changes. We also propose that some of the miRNAs in barley such as miRNA408 might play critical roles against boron exposure. In conclusion, barley may use several pathways and cellular processes targeted by miRNAs to cope with boron stress.

Nutrition Metabolism Plays an Important Role in the Alternate Bearing of the Olive Tree (Olea europaea L.)

The olive tree (Olea europaea L.) is widely known for its strong tendency for alternate bearing, which severely affects the fruit yield from year to year. Microarray based gene expression analysis using RNA from olive samples (on-off years leaves and ripe-unripe fruits) are particularly useful to understand the molecular mechanisms influencing the periodicity in the olive tree. Thus, we carried out genome wide transcriptome analyses involving different organs and temporal stages of the olive tree using the NimbleGen Array containing 136,628 oligonucleotide probe sets. Cluster analyses of the genes showed that cDNAs originated from different organs could be sorted into separate groups. The nutritional control had a particularly remarkable impact on the alternate bearing of olive, as shown by the differential expression of transcripts under different temporal phases and organs. Additionally, hormonal control and flowering processes also played important roles in this phenomenon. Our analyses provide further insights into the transcript changes between ”on year” and “off year” leaves along with the changes from unrpipe to ripe fruits, which shed light on the molecular mechanisms underlying the olive tree alternate bearing. These findings have important implications for the breeding and agriculture of the olive tree and other crops showing periodicity. To our knowledge, this is the first study reporting the development and use of an olive array to document the gene expression profiling associated with the alternate bearing in olive tree.

Comparative genome analysis of five Pasteurella multocida strains to decipher the diversification in pathogenicity and host specialization.

Pasteurella multocida is a Gram-negative bacterial pathogen causing economically important diseases in distinct animal species. Complete genome sequences of five P. multocida strains (Pm70, HB03, HN06, 3480, and 36950) isolated from poultry, swine or bovine, were retrieved from the GenBank database and compared with each other, for the first time. The missense mutations generating a dissimilar amino acid in the peptide chain, nonsense mutations, and insertion/deletions in the nucleotide sequence were identified due to the potential change in the protein function. A total of 500 putative mutant proteins were identified, and categorized into 10 groups including cellular compartments such as outer membrane, capsule and fimbria, and processes such as carbohydrate, energy, nucleic acid and amino acid metabolisms, transport, and drug resistance. The majority of the mutant proteins were associated with the outer compartments of the bacterial cell. Various mutations were also detected in the genes related with biosynthetic pathways. The highest and the lowest numbers of mutant proteins belonged to 36950 vs. HN06 and Pm70 vs. HB03 comparisons, respectively. The major impact on the diversification of P. multocida strains was observed to be conferred by the mutations related with pathogenicity. To exhibit the outcomes of the mutations in the peptide chains, three sample amino acid sequences belonging to AfuA, MetB, and d,d-heptose 1,7-bisphosphate phosphatase were aligned, and their phylogenetic relationships were shown. These comprehensive analyses improve the understanding of molecular pathogenicity and host specialization of P. multocida, and would have a contribution to the recombinant vaccine development against this pathogen.

Global transcriptome analysis of Halolamina sp. to decipher the salt tolerance in extremely halophilic archaea.

Extremely halophilic archaea survive in the hypersaline environments such as salt lakes or salt mines. Therefore, these microorganisms are good sources to investigate the molecular mechanisms underlying the tolerance to high salt concentrations. In this study, a global transcriptome analysis was conducted in an extremely halophilic archaeon, Halolamina sp. YKT1, isolated from a salt mine in Turkey. A comparative RNA-seq analysis was performed using YKT1 isolate grown either at 2.7M NaCl or 5.5M NaCl concentrations. A total of 2149 genes were predicted to be up-regulated and 1638 genes were down-regulated in the presence of 5.5M NaCl. The salt tolerance of Halolamina sp. YKT1 involves the up-regulation of genes related with membrane transporters, CRISPR-Cas systems, osmoprotectant solutes, oxidative stress proteins, and iron metabolism. On the other hand, the genes encoding the proteins involved in DNA replication, transcription, translation, mismatch and nucleotide excision repair were down-regulated. The RNA-seq data were verified for seven up-regulated genes as well as six down-regulated genes via qRT-PCR analysis. This comprehensive transcriptome analysis showed that the halophilic archaeon canalizes its energy towards keeping the intracellular osmotic balance minimizing the production of nucleic acids and peptides.

Functional Characterization of 4'OMT and 7OMT Genes in BIA Biosynthesis.

Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4′OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4′OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4′OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4′OMT, and 7OMT) were observed upon manipulation of 4′OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.

Identification of Gene Families Using Genomics and/or Transcriptomics Data

Thousands of putative open reading frames (ORFs) are identified via annotation of sequenced plant genomes. Classification of these ORFs into gene families has a crucial importance to understand the evolution, function, and structure of the encoded proteins such as transcription factors, and the non-coding RNAs such as microRNAs (miRNAs). Thus, molecular mechanisms underlying the metabolic processes in plants are uncovered as well. Some members of the gene families are species-specific being more dynamic during evolution whereas others are more conserved, phylogenetically sharing common features. The latter are especially important for the annotation of putative ORFs by revealing known counterparts with high sequence identity via sequence alignment to discover conserved motifs. Various bioinformatic tools are available to find out gene families in plants. The BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) is widely used for identification of homologous sequences. Phytozome (http://www.phytozome.net) or GreenPhyl (http://www.greenphyl.org) are the web resources utilized for the functional and comparative genomics in plants to analyze gene families. TRAPID (http://bioinformatics.psb.ugent.be/webtools/trapid) offers a free of charge web source for functional and comparative analyses of transcriptome data sets for identification of gene families, alignment of multiple sequences and phylogenetic tree construction. Some of the databases store specific type of gene families such as plant transcription factor databases PlantTFDB (http://planttfdb.cbi.pku.edu.cn) and PlnTFDB (http://plntfdb.bio.uni-potsdam.de/v3.0), or miRBase (http://www.mirbase.org) for miRNAs. Molecular Evolutionary Genetics Analysis (MEGA) software is an integrated tool for the analyses such as alignment of sequence, construction of phylogenetic trees, and access to online databases. In this chapter, the bioinformatic tools for analyses of genomics and/or transcriptomics data sets to discover gene families as well as sample researches are discussed.

Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics

The increasing resistance of bacteria to antibacterial therapy poses an enormous health problem, it renders the development of new antibacterial agents with novel mechanism of action an urgent need. Peptide deformylase, a metalloenzyme which catalytically removes N‐formyl group from N‐terminal methionine of newly synthesized polypeptides, is an important target in antibacterial drug discovery. In this study, we report the structure‐based virtual screening of ZINC database in order to discover potential hits as bacterial peptide deformylase enzyme inhibitors with more affinity as compared to GSK1322322, previously known inhibitor. After virtual screening, fifteen compounds of the top hits predicted were purchased and evaluated in vitro for their antibacterial activities against one Gram positive (Staphylococcus aureus) and three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella. pneumoniae) bacteria in different concentrations by disc diffusion method. Out of these, three compounds, ZINC00039650, ZINC03872971 and ZINC00126407, exhibited significant zone of inhibition. The results obtained were confirmed using the dilution method. Thus, these proposed compounds may aid the development of more efficient antibacterial agents.

Highly active and stable protease production by an extreme halophilic archaeon Haloarcula sp. TG1 isolated from Lake Tuz, Turkey

Abstract Objective: Isolation of halophilic microorganisms from Çankırı salt mine and Lake Tuz in Turkey to explore versatile protease producers for industry and characterization of protease enzyme from the best protease producer among the isolated strains. Methods: Extreme halophiles were isolated from salt samples of Çankırı salt mine and Lake Tuz. Their protease activities were determined. The isolate with the highest protease activity was characterized. Its protease activity was evaluated in different NaCl concentrations, temperature and pH ranges, and in the presence of different inhibitors and metals. Thermostability and pH stability were also determined. Results: The highest protease producer strain was identified as Haloarcula sp. on the basis of 16S rRNA analysis. The isolate namely, Haloarcula sp. TG1, was found to be 99% identical to Haloarcula salaria strain HST01-2R. The TG1 protease was found to possess very high activity and stability over a broad pH and temperature ranges. Its maximum activity was recorded at pH: 4.0, 50°C and 4 M NaCl. Among inhibitors tested, dimethyl sulfoxide (DMSO) and ethanol caused the highest decrease (ca. 25%) in its activity. Conclusion: Due to the high activity and stability over a wide range of extreme conditions, Haloarcula sp. TG1 protease reported here is a promising candidate in biotechnology.