Genome-Wide Identification, Characterization and Expression Profiling of Potato (Solanum tuberosum) Frataxin (FH) Gene.

Frataxin (FH) plays a crucial role in the biogenesis of mitochondria and the regulation of iron in the cells of various organisms. However, there has been very little research on FH in plants. In this study, the potato FH gene (StFH) was identified and characterized using a genome-wide approach, and its sequence was compared to those of FH genes from Arabidopsis, rice, and maize. The FH genes were found to have a lineage-specific distribution and were more conserved in monocots than in dicots. While multiple copies of FH genes have been reported in some species, including plants, only one isoform of FH was found in potato. The expression of StFH in leaves and roots was analyzed under two different abiotic stress conditions, and the results showed that StFH was upregulated more in leaves and that its expression levels increased with the severity of the stress. This is the first study to examine the expression of an FH gene under abiotic stress conditions.

Mitochondrial iron transporter (MIT) gene in potato (Solanum tuberosum): comparative bioinformatics, physiological and expression analyses in response to drought and salinity.

Mitochondrial iron transporter (MIT) genes are essential for mitochondrial acquisition/import of iron and vital to proper functioning of mitochondria. Unlike other organisms, research on the MITs in plants is limited. The present study provides comparative bioinformatics assays for the potato MIT gene (StMIT) as well as gene expression analyses. The phylogenetic analyses revealed monocots-dicot divergence in MIT proteins and it was also found clade specific motif diversity. In addition, docking analyses indicated that Asp172 and Gly100 residues to be identified as the closest residues binding to ferrous iron. The percentage of structure overlap of the StMIT 3D protein model with Arabidopsis, maize and rice MIT proteins was found between 80.18% and 85.71%. The transcript analyses exhibited that the expression of StMIT was triggered under drought and salinity stresses. The findings of the present study would provide valuable leads for further studies targeting specifically the MIT gene and generally the plant iron metabolism.

Genome-wide identification and characterization of high-affinity nitrate transporter 2 (NRT2) gene family in tomato (Solanum lycopersicum) and their transcriptional responses to drought and salinity stresses.

The high-affinity nitrate transporter 2 (NRT2) proteins play vital roles in both nitrate (NO3-) uptake and translocation in plants. Although the gene families coding the NRT2 proteins have been identified and functionally characterized in many plant species, the systematic identification of NRT2 gene family members has not previously been reported in tomato (Solanum lycopersicum). Moreover, little is known about their expression profiles in response to different environmental stresses. The present study sought to identify the NRT2 gene family members within the tomato genome, and then to characterize them in detail by means of bioinformatics, physiological and expression analyses. Four novel NRT2 genes were identified in the tomato genome, all of which contained the same domain belonging to the major facilitator superfamily (PF07690). The co-expression network of the SlNRT2 genes revealed that they were co-expressed with several other genes in a number of different molecular pathways, including the transport, photosynthesis, fatty acid metabolism and amino acid catabolism pathways. Several phosphorylation sites were predicted in the NRT2 proteins. The SlNRT2 genes interact with many other genes that perform various functions in many crucial pathways within the tomato genome. The sequence variations observed at the gene and protein levels indicate the dynamic regulation of the SlNRT2 gene family members in relation to cell metabolism, particularly with regard to the nitrogen assimilation pathway. The responses of the SlNRT2 genes to drought and salinity stresses are diverse, and they are neither stress- nor tissue-specific. The findings of this study should provide a useful scientific basis for future studies concerning the roles of the NRT2 gene family in plants.

Genes involved in mRNA surveillance are induced in Brachypodium distachyon under cadmium toxicity.

BACKGROUND: Cd accumulation in plant cells results in dramatic problems including oxidative stress and inhibition of vital enzymes. It also affects mineral uptakes by disrupting membrane permeability. Interaction among Cd and other plant nutrient elements changes the nutritional contents of crops and reduces their yield. METHODS AND RESULTS: In the present study, Cd stress in Brachypodium distachyon led to the upregulation of some heavy metal transport genes (influx or efflux) encoding cation-efflux proteins, heavy metal-associated proteins and NRAMP proteins. The Arabidopsis orthologs of the differentially expressed B. distachyon genes (DEGs) under Cd toxicity were identified, which exhibited Bradi4g26905 was an ortholog of AtALY1-2. Detailed co-expression network and gene ontology analyses found the potential involvement of the mRNA surveillance pathway in Cd tolerance in B. distachyon. These genes were shown to be downregulated by sulfur (S) deficiency. CONCLUSIONS: This is the first transcriptomic study investigating the effect of Cd toxicity in B. distachyon, a model plant for genomic studies in Poaceae (Gramineae) species. The results are expected to provide valuable information for more comprehensive research related to heavy metal toxicity in plants.

Genome-wide investigation of proline transporter (ProT) gene family in tomato: Bioinformatics and expression analyses in response to drought stress.

Proline has various functions in plants, such as growth, development and stress response to biotic and abiotic factors. Therefore, proline accumulation and transport are vital for crop production in higher quality and quantity. The present study addresses genome-wide identification and bioinformatics analyses of tomato (Solanum lycopersicum) proline transporter (ProT) genes and their expression profiles under drought stress. The analyses indicated four novel ProT genes (SlProTs) in the tomato genome and their protein lengths ranged from 439 to 452 amino acid residues. All SlProTs contained a PF01490 (transmembrane amino acid transporter protein) domain and seven exons, and they had a basic pI. The phylogeny analysis proved that monocot-dicot divergence was not present and the SlProT proteins were distinct from the ProT proteins in monocots and Arabidopsis. Based on the digital expression analysis, SlProT1 and SlProT2 genes seemed to be more active than the others in response to abiotic stress conditions. However, detected by RT-qPCR, the expression levels of all SlProT genes under drought stress were similar. The promotor analyses of SlProT genes revealed that they contained many transcription factors binding sites in cis-elements, such as MYB, Dof, Hox, bZIP, bHLH, AP2/ERF and WRKY. Finally, our findings could contribute to the understanding of SlProT genes and proline metabolism in plants.

Novel TALEN-generated mCitrine-FANCD2 fusion reporter mouse model for in vivo research of DNA damage response.

Reporter gene mouse lines are routinely used for studies related to functional genomics, proteomics, cell biology or cell-based drug screenings, and represent a crucial platform for in vivo research. In the generation of knock-in reporter lines, new gene targeting methods provide several advantages over the standard transgenic techniques. First of all, specific targeting of the genome allows expression of the reporter gene under controlled conditions, whether in a specific locus in the genome or in a "safe harbor" locus. Historically, the ROSA26 locus is used for gene knock-in strategies by homologous recombination in mouse embryonic stem cells. The other preferred place for integration of the reporter transgene in the mouse genome is the endogenous promoter of a target gene. In this study, we employed TALENs to generate a reporter fusion protein expressed from its native promoter. For monitoring DNA damage response, we generated a mouse line expressing a mCitrine-tagged version of the FANCD2 protein, involved in DNA damage response and repair, and the Fanconi anemia (FA) pathway. This model could be a valuable tool for in vivo investigation of DNA damage.

Pathogenesis related protein-1 (PR-1) genes in tomato (Solanum lycopersicum L.): Bioinformatics analyses and expression profiles in response to drought stress.

The pathogenesis-related protein 1 (PR-1) gene family play important roles in the plant metabolism in response to biotic and abiotic stresses. The present study aimed genome-wide identification and bioinformatics analyses of PR-1 genes in tomato (Solanum lycopersicum L.). The analyses resulted in the identification of 13 novel SlPR-1 genes, each of which produce a protein belonging to the CAP superfamily (PF00188). The KEGG annotation analyses revealed that the SlPR-1 proteins functioned in the environmental information processing (09130). The expression patterns of the PR-1 genes and some stress-related physiological parameters were investigated in Fusarium oxysporum sensitive and tolerant tomato varieties under drought stress. The drought stress leaded upregulation of all SlPR-1 genes, reaching up to 50 folds. The results indicate that the SlPR-1 genes play active roles in response to drought. This is the first study exhibiting the expression profiles of SlPR-1 genes under an abiotic stress, drought, in tomato.

Ammonium transporter 1 (AMT1) gene family in tomato (Solanum lycopersicum L.): Bioinformatics, physiological and expression analyses under drought and salt stresses.

Nitrogen (N) is an essential macronutrient for plants, and mainly taken from the soil as ammonium (NH+4). It is particularly transported into the plants by AMmonium Transporters (AMTs), which are plasma membrane proteins. In the present study, genome-wide identification, physiological and expression analyses of tomato (Solanum lycopersicum L.) ammonium transporters 1 (SlAMT1) genes under drought and salt stresses were performed. Sequence analyses revealed the presence of variations in SlAMT1s at nucleotide and protein levels. While all the SlAMT1s comprise an ammonium transporter domain (PF00909), the numbers of their transmembrane helices were found to be diverse. Digital expression analyses proved that SlAMT1-3 gene had different expression patterns compared to the others, suggesting its functional diversities. The expression analyses revealed that SlAMT1 genes were 0.16 and 5.94 -fold down-regulated under drought and salt stresses, respectively. The results suggested that expression of SlAMT1 genes were adversely affected by abiotic stress conditions.

Whirly (Why) transcription factors in tomato (Solanum lycopersicum L.): genome-wide identification and transcriptional profiling under drought and salt stresses.

Whirly (Why) transcription factor (TFs) constitute one of the important TF families which plays essential roles in plant metabolism to cope with environmental stresses. In the present study, Why genes were identified at genome-wide scale in tomato (Solanum lycopersicum), and bioinformatics analyses were implemented. Validation of Why genes expressions under drought and salt stresses were also performed using RT-qPCR. The analyses revealed the presence of two Why genes in tomato genome, SlWhy1 (Solyc05g007100.2.1) and SlWhy2 (Solyc11g044750.1.1). Both genes contained Whirly transcription factor domain structure (PF08536), and Why proteins were in basic character (pI ≥ 7). While the lengths of the proteins ranged from 268 to 236 amino acid residues for SlWhy1 and SlWhy2 respectively, exon numbers identified in both genes were seven. According to the digital expression data, SlWhy genes are expressed at medium level in different anatomical parts and developmental stages. In the promotor sequence analysis, 13 types of putative TF binding sites were identified, and the highest motif number was 46, found for GATA TF. Gene co-expression analyses revealed that complex networks for SlWhy genes, which are connected with various metabolic pathways. Based on the RT-qPCR data, both SlWhy1 and SlWhy2 genes were up-regulated under salt and drought stresses. 3D structure analyses revealed that SlWhy1 protein had a more diverged structure than SlWhy2 protein, based on their comparisons in Arabidopsis and potato. The results obtained in the present study could be a useful scientific basis for understanding Why genes in tomato and their functions under abiotic stress conditions.

Genome-wide analyses of ATP sulfurylase (ATPS) genes in higher plants and expression profiles in sorghum (Sorghum bicolor) under cadmium and salinity stresses.

ATP sulfurylase (ATPS, EC: 2.7.7.4) is a crucial enzyme for sulfate assimilation pathway in both plastids and cytosol in plants. In this study, genome-wide and comparative analyses of ATPSs in 11 higher plant species, including sequence and structural analyses have been performed. Expression of ATPS genes in sorghum under cadmium (Cd) and salinity (NaCl) stresses were also investigated to provide a model experimental data for the regulation of ATPS genes under stress conditions. Thirty-one ATPS genes from 11 plant species were found. It showed that ATPSs from different species have high sequence divergences, which cause structural differences among them. Phylogenetic analysis has shown that there are two major types of ATPSs evolved in dicots while monocots were evolved to have one type of ATPs. Finally, expression analysis of ATPS genes revealed tissue and stress dependent expression pattern, which indicates expressions of ATPS genes are tightly regulated.

Identification of O-acetylserine(thiol)lyase (OASTL) genes in sorghum (Sorghum bicolor) and gene expression analysis under cadmium stress.

Cysteine (Cys) is the first identified molecule in plant metabolism which includes both sulfur and nitrogen. It can be synthesized in three cellular compartments, containing chloroplast, cytoplasm and mitochondrion. The final step of cysteine biosynthesis is catalyzed by the O-acetylserine(thiol)lyase enzyme (OASTL, E.C. 4.2.99). In the present study, seven members of the OASTL gene family in the sorghum (Sorghum bicolor) genome were identified at a genome-wide scale and comparative bioinformatics analyses were performed between sorghum and Arabidopsis OASTLs. In all OASTL proteins, a pyridoxal-phosphate dependent domain structure (PALP, PF00291) was identified. The gene ontology annotations also revealed that all sorghum OASTL genes have KOG1252 (Cystathionine beta-synthase and related enzyme) and K01738 (cysteine synthase A) activities. In promotor sequences of OASTL genes, diverse cis-acting elements were found, including hormone and light responsiveness, abiotic stress responsiveness, and tissue-specific ones (meristem and endosperm). Sorghum OASTL genes demonstrated medium or high level expressions in anatomical parts and developmental stages based on the digital expression data. Expression of OASTL genes were also analyzed under cadmium (Cd) stress in sorghum by Real Time-quantitative PCR (RT-qPCR). The results exclusively showed that OASTL A1-2 gene was 1.12 fold up-regulated in roots, whereas cysteine synthase 26 was 2.25 fold down-regulated in leaves. The predicted 3D structure of OASTLs indicated some structural diversities as well as variations in the secondary structures.

DREB2 (dehydration-responsive element-binding protein 2) type transcription factor in sorghum (Sorghum bicolor): genome-wide identification, characterization and expression profiles under cadmium and salt stresses.

Biotic and abiotic stresses negatively affect fitness, biomass production, and crop yield in plants. The dehydration-responsive element-binding proteins (DREB) are important transcription factors (TFs), and are induced by abiotic and biotic stresses. In this study, genome-wide identification, in silico sequence, and phylogenetic analyses and expression analyses of DREB2 genes under cadmium (Cd) and salt (NaCl) stresses in sorghum (Sorghum bicolor, Sb) were performed. Six putative SbDREB2 genes were identified in sorghum genome and all contained AP2 domain (PF00847). Nucleotide diversities in SbDREB2 genes were calculated as π: 0.53 and θ: 0.39, respectively. While exon numbers of them were either one or two, length of SbDREB2 proteins ranged from 238 to 388 amino acid residues. Fifty-six cis-acting regulatory elements, which are tissue specific, light, hormone, and stress responsive, were identified in the promotor regions of SbDREB2 genes. Analyses on digital expression data indicated that SbDREB2A and SbDREB2B are more expressed genes than other SbDREB genes in sorghum. Under Cd and NaCl stresses, expressions of SbDREB2 genes were induced at different levels. All SbDREB2 genes in root were up-regulated under salt stress. In case of Cd stress, SbDREB2D gene was particularly up-regulated in leaves and roots. Co-expression analyses revealed four of TFs in co-expression network, indicating that they have roles in transcriptional cascade. Furthermore, five miRNA target regions were identified for four SbDREB2 genes, indicating their roles in post-transcriptional regulation. The predicted 3D structure of SbDREB2 proteins showed some structural divergences and structure overlap between rice and sorghum varied at between 26.58 and 50%. Finally, obtained data could be used in breeding of stress-tolerant plants, particularly genetically engineered DREB2 expressing plants. Findings in this study would also contribute to the understanding of DREB2 genes in plants, especially in sorghum.

Genome-wide identification and cadmium induced expression profiling of sulfate transporter (SULTR) genes in sorghum (Sorghum bicolor L.).

Sulfur is an essential element for all living organisms. Plants can convert inorganic sulfur into organic sulfur compounds by complex enzymatic steps. In this study, we conducted a genome-wide analysis of sulfate transporter genes (SULTRs) in the sorghum (Sorghum bicolor) genome and examined expression profiles of SbSULTR genes under 200 µM cadmium (Cd) exposure. As a result of sorghum genome analysis, 11 SULTR genes were identified, including SbSULTR1;1, SbSULTR1;2, SbSULTR1;3, SbSULTR2;1, SbSULTR2;2, SbSULTR3;1, SbSULTR3;2, SbSULTR3;3, SbSULTR3;4, SbSULTR3;5, and SbSULTR4. Given names are based on phylogeny and chromosomal locations. Except SbSULTR4, all SbSULTR proteins contained Sulfate_transp (PF00916), STAS (PF01740) domains and 12 trans-membrane domains. Phylogenetic analysis revealed that four major groups were identified such as SULTR1, 2, 3, and 4 groups and SULTR4 group was separated to other SULTR groups. In promotor sequences of SbSULTR genes, many diverse cis-acting elements were found mainly related with physiological processes such as light, stress and hormone responsiveness. The expression profiles of SbSULTR genes showed that SULTR1;2, 1;3, 3;3, and 3;5 genes up-regulated in root, while expression level of SULTR4 decreased under 200 µM Cd exposure. The predicted 3D structures of SULTR proteins showed some conformational changes, suggesting functional diversities of SbSULTRs. Finally, results of this study may contribute towards understanding SbSULTR genes and their regulations and roles in Cd stress in sorghum.

Improved FLP recombinase, FLPe, efficiently removes marker gene from transgene locus developed by Cre-lox mediated site-specific gene integration in rice.

Site-specific recombination systems, such as FLP-FRT and Cre-lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP-FRT system for excising marker gene from the transgene locus developed by Cre-lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP-FRT-based biotechnology applications in plants.

Dosage-dependent gene expression from direct repeat locus in rice developed by site-specific gene integration.

In the standard plant transformation practice, transgene copy number is often inversely correlated with transgene expression. As the integration locus generated by standard methods is mostly complex, consisting of both full-length and partial copies arranged in direct or inverted repeat configurations, it is difficult to parse the effect of copy number and locus structure. To clearly study the effect of transgene copy number on gene expression, it is important to control the locus structure and integrate full-length copies. In this study, the effect of transgene copy number on transgene expression in plant cells was determined using rice callus as a model. To generate full-length integrations, Cre-lox-mediated site-specific gene integration method was used. Transgenic rice lines consisting of one to three copies of beta-glucuronidase or green fluorescent protein genes were developed. Site-specific integration lines were characterized and subjected to expression analysis. Lines containing two or three copies of either reporter genes displayed 2-4 times higher expression compared to the single-copy lines. Therefore, dosage-dependent transgene expression can be obtained by integrating full-length copies, and site-specific gene integration approach can serve as an efficient tool for generating precise multi-copy integrations.

Root and Crown Rot Fungi Associated with Spring, Facultative, and Winter Wheat in Turkey.

The objective of this study was to determine the distribution frequency of the fungi associated with wheat (Triticum aestivum) crowns and roots in cereal producing areas of Turkey through a targeted survey of 518 commercial fields over a 2-year period. More than 26% of the fields had one or more of the fungal species commonly reported as part of the dryland root rot complex, Fusarium culmorum (14%) > Bipolaris sorokiniana (10%) > F. pseudograminearum (2%). The fungi considered to be part of the high rainfall root rot complex were found at very low frequencies: 2% for Gaeumannomyces graminis and 3% for Pythium spp. Species of Rhizoctonia were found in 22% of the fields. Several Fusarium species considered to be less or nonpathogenic to cereals were also found in high frequencies at 11% (F. oxysporum, F. chlamydosporum), 10% (F. sporotrichioides), and 8% (F. avenaceum and F. solani). The mostly random distribution of cereal root-rotting species across the survey area suggests the fungi are not distributed in any distinct agroecological relationship. As a result, the relative economic importance of a given species on wheat will be determined by a number of factors, such as their fungal pathogenicity, host susceptibility/tolerance, and the seasonal conditions. Results from this study suggest that there are a wide range of fungal species associated with root and crown tissues of wheat.