Ertugrul Filiz

Identification and Comparative Analysis of H2O2-Scavenging Enzymes (Ascorbate Peroxidase and Glutathione Peroxidase) in Selected Plants Employing Bioinformatics Approaches.

Among major reactive oxygen species (ROS), hydrogen peroxide (H2O2) exhibits dual roles in plant metabolism. Low levels of H2O2 modulate many biological/physiological processes in plants; whereas, its high level can cause damage to cell structures, having severe consequences. Thus, steady-state level of cellular H2O2 must be tightly regulated. Glutathione peroxidases (GPX) and ascorbate peroxidase (APX) are two major ROS-scavenging enzymes which catalyze the reduction of H2O2 in order to prevent potential H2O2-derived cellular damage. Employing bioinformatics approaches, this study presents a comparative evaluation of both GPX and APX in 18 different plant species, and provides valuable insights into the nature and complex regulation of these enzymes. Herein, (a) potential GPX and APX genes/proteins from 18 different plant species were identified, (b) their exon/intron organization were analyzed, (c) detailed information about their physicochemical properties were provided, (d) conserved motif signatures of GPX and APX were identified, (e) their phylogenetic trees and 3D models were constructed, (f) protein-protein interaction networks were generated, and finally (g) GPX and APX gene expression profiles were analyzed. Study outcomes enlightened GPX and APX as major H2O2-scavenging enzymes at their structural and functional levels, which could be used in future studies in the current direction.

Assessment of miRNA expression profile and differential expression pattern of target genes in cold-tolerant and cold-sensitive tomato cultivars

MircroRNAs (miRNAs) are small non-coding RNAs about 21 nt in length. These short transcripts regulate developmental and stress responses in plants. Cold stress is one of the most restraining abiotic factors adversely affecting the plant yield. In the present study, some cold stress-related miRNAs (miR167, miR169, miR172, miR393 and miR397) in tomato (Solanum lycopersicum) were assessed at early time points (0, 1, 4 and 16 h) of cold exposure. Relative expression of miRNAs was measured by stem–loop quantitative reverse transcription polymerase chain reaction. The results showed that miR167, miR169, miR172 and miR393 were activated in the early time points of cold treatment. Especially, miR172 was found to have highest expression level. Furthermore, target genes of selected miRNAs were identified and their expression profiles were assessed between cold-sensitive and cold-tolerant cultivars of tomato. It was found that inferred expression patterns of target genes were differentiated between the cultivars. Analysis of cis-acting elements showed that miRNAs had stress-responsive elements. Meanwhile, since no miR393 sequence is available, putative miR393 sequence and its secondary structure were predicted in tomato. These results may provide a framework for further analysis in terms of understanding the response of miRNAs against cold stress in tomato.

Genome-wide exploration of metal tolerance protein (MTP) genes in common wheat (Triticum aestivum): insights into metal homeostasis and biofortification

Metal transport process in plants is a determinant of quality and quantity of the harvest. Although it is among the most important of staple crops, knowledge about genes that encode for membrane-bound metal transporters is scarce in wheat. Metal tolerance proteins (MTPs) are involved in trace metal homeostasis at the sub-cellular level, usually by providing metal efflux out of the cytosol. Here, by using various bioinformatics approaches, genes that encode for MTPs in the hexaploid wheat genome (Triticum aestivum, abbreviated as Ta) were identified and characterized. Based on the comparison with known rice MTPs, the wheat genome contained 20 MTP sequences; named as TaMTP1–8A, B and D. All TaMTPs contained a cation diffusion facilitator (CDF) family domain and most members harbored a zinc transporter dimerization domain. Based on motif, phylogeny and alignment analysis, A, B and D genomes of TaMTP3–7 sequences demonstrated higher homology compared to TaMTP1, 2 and 8. With reference to their rice orthologs, TaMTP1s and TaMTP8s belonged to Zn-CDFs, TaMTP2s to Fe/Zn-CDFs and TaMTP3–7s to Mn-CDFs. Upstream regions of TaMTP genes included diverse cis-regulatory motifs, indicating regulation by developmental stage, tissue type and stresses. A scan of the coding sequences of 20 TaMTPs against published miRNAs predicted a total of 14 potential miRNAs, mainly targeting the members of most diverged groups. Expression analysis showed that several TaMTPs were temporally and spatially regulated during the developmental time-course. In grains, MTPs were preferentially expressed in the aleurone layer, which is known as a reservoir for high concentrations of iron and zinc. The work identified and characterized metal tolerance proteins in common wheat and revealed a potential involvement of MTPs in providing a sink for trace element storage in wheat grains.

Isolation of a transcription factor DREB1A gene from Phaseolus vulgaris and computational insights into its characterization: protein modeling, docking and mutagenesis

A transcription factor DREB1A (dehydration-responsive element-binding 1A) gene was amplified and sequenced from the common bean (Phaseolus vulgaris). PvDREB1A had a 777 base pairs (bp) open reading frame encoding a protein of 225 residues. The protein sequence contained a conserved DNA-binding AP2 domain of about 60 residues and a nuclear localization signal (NLS) at N-terminus site. PvDREB1A demonstrated high homology with other DREB1 members only in AP2 domain and NLS site. The phylogenetic distribution of different DREB members showed three main groups as DREB1–3 and PvDREB1A was a member of DREB1 group. Homology modeling and secondary structure analyses revealed that PvDREB1A AP2 domain was packed into the three-stranded antiparallel beta sheets (β1–3) and an alpha helix (α1) almost parallel to these beta sheets. Moreover, DNA-binding AP2 domain of PvDREB1A and GCC-box containing double helix DNA were docked. The docking analysis showed that PvDREB1A AP2 domain could bind to the major groove of the DNA by three-stranded antiparallel beta sheets, with residues Gly86 or Thr87 in β1-sheet and Arg63 or Arg64 in β3-sheet. The docked complex also indicated that AP2 domain has a preferential for the binding of GCC stretches in the double helix DNA. A total of 36 reliably estimated hot spots residues were identified with high mutability grade but none of these residues was essential for the protein function since they are located at outside the DNA-binding AP2 domain of PvDREB1A.

Comparative Analysis and Modeling of Superoxide Dismutases (SODs) in Brachypodium distachyon L.

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme catalyzing the dismutation of superoxide radical to hydrogen peroxide and dioxygen. To date, four types of SODs — Cu/ZnSOD, MnSOD, FeSOD, and NiSOD — have been identified. In this study, SOD proteins of Brachypodium distachyon (L.) Beauv. were screened by utilization of bioinformatics approaches. According to our results, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found to be in basic and acidic character, respectively. Domain analyzes of SOD proteins revealed that iron/manganese SOD and copper/zinc SOD were within studied SOD proteins. Based on the seconder structure analyzes, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found as having similar sheets, turns and coils. Although helical structures were noticed in the types of Mn/FeSODs, no the type of Cu/ZnSODs were identified having helical structures. The predicted binding sites of Fe/MnSODs and Cu/ZnSODs were confirmed for having His-His-Asp-His and His-His-His-Asp-Ser residues with different positions, respectively. The 3D structure analyzes of SODs revealed that some structural divergences were observed in patterns of SODs domains. Based on phylogenetic analysis, Mn/FeSODs were found to have similarities whereas Cu/ZnSODs were clustered independently in phylogenetic tree.

Genome-Wide Identification and Comparative Analysis of Copper Transporter Genes in Plants

Copper (Cu) transporters have primary importance in maintenance of physiological limits of Cu homeostasis in plants. However, structural characterization of Cu transporters in many plant species is still limited. In this study, a total of 78 potential Cu transporter genes were identified from 18 different plant species. Study revealed that Cu transporters could be characterized with a CTR protein family (PF04145) domain, three putative transmembrane domains (TMDs), a single exon number, and a basic character. Met-rich motifs at N-terminal region, MXXXM motif in TMD-2, and GXXXG motif in TMD-3 could be essential for Cu transport since they were highly conserved in all analyzed species. In phylogeny, a clear distinction was observed between Cu transporter sequences of lower and higher plants. General topological features of Cu transporters in higher plants—monocots and dicots—were highly conserved compared to lower plants. Identification of Cu transporter homologous in various plant species and their comparative analysis at gene and protein levels will become valuable theoretical basis for future studies aiming to further characterization and molecular manipulation of Cu transporters.

Genome-wide exploration of silicon (Si) transporter genes, Lsi1 and Lsi2 in plants; insights into Si-accumulation status/capacity of plants

Silicon (Si) is a nonessential, beneficial micronutrient for plants. It increases the plant stress tolerance in relation to its accumulation capacity. In this work, root Si transporter genes were characterized in 17 different plants and inferred for their Si-accumulation status. A total of 62 Si transporter genes (31 Lsi1 and 31 Lsi2) were identified in studied plants. Lsi1s were 261–324 residues protein with a MIP family domain whereas Lsi2s were 472–547 residues with a citrate transporter family domain. Lsi1s possessed characteristic sequence features that can be employed as benchmark in prediction of Si-accumulation status/capacity of the plants. Silicic acid selectivity in Lsi1s was associated with two highly conserved NPA (Asn-Pro-Ala) motifs and a Gly-Ser-Gly-Arg (GSGR) ar/R filter. Two NPA regions were present in all Lsi1 members but some Ala substituted with Ser or Val. GSGR filter was only available in the proposed high and moderate Si accumulators. In phylogeny, Lsi1s formed three clusters as low, moderate and high Si accumulators based on tree topology and availability of GSGR filter. Low-accumulators contained filters WIGR, AIGR, FAAR, WVAR and AVAR, high-accumulators only with GSGR filter, and moderate-accumulators mostly with GSGR but some with A/CSGR filters. A positive correlation was also available between sequence homology and Si-accumulation status of the tested plants. Thus, availability of GSGR selectivity filter and sequence homology degree could be used as signatures in prediction of Si-accumulation status in experimentally uncharacterized plants. Moreover, interaction partner and expression profile analyses implicated the involvement of Si transporters in plant stress tolerance.

Genome-wide analysis of iron-regulated transporter 1 (IRT1) genes in plants

Iron (Fe) is an essential micronutrient required in a number of biological processes in plant species. Fe transporters are a type of broad-range metal transporter and have different families functioning in different compartments. This study focused on iron-regulated transporter 1 (IRT1), which are mainly responsible for Fe uptake from root, in 17 selected plant species with an emphasis on Brachypodium distachyon, Chlamydomonas reinhardtii, Solanum lycopersicum and Populus trichocarpa species. All IRT1 proteins were observed to belong to the ZIP (PF02535) protein family with eight transmembrane (TM) domains, and have a similar molecular weight (33.86–42.72 kDa, except for C. reinhardtii with 65.83 kDa) and amino acid length (324-408 aa, except for C. reinhardtii with 639 aa), with pI values of 5.31–7.16. The sub-cellular localization of these proteins was predicted to be the plasma membrane. Similar exon numbers were also detected with most genes having 2–3, except for C. reinhardtii (5), Physcomitrella patens (5) and Vitis vinifera (4). In a phylogenetic tree, monocot-dicot separation was not observed in main groups but some subgroups included only monocot or dicot proteins. Predicted interaction partner analysis of AtIRT1 (AT2G30080.1) pointed to main interaction partners either directly related with iron transport or that of other metal ion. The results of this study provide a theoretical reference for elucidating the structural and biological role of IRT1 genes/proteins in plant species.

Genome-wide identification of galactinol synthase (GolS) genes in Solanum lycopersicum and Brachypodium distachyon

GolS genes stand as potential candidate genes for molecular breeding and/or engineering programs in order for improving abiotic stress tolerance in plant species. In this study, a total of six galactinol synthase (GolS) genes/proteins were retrieved for Solanum lycopersicum and Brachypodium distachyon. GolS protein sequences were identified to include glyco_transf_8 (PF01501) domain structure, and to have a close molecular weight (36.40-39.59kDa) and amino acid length (318-347 aa) with a slightly acidic pI (5.35-6.40). The sub-cellular location was mainly predicted as cytoplasmic. S. lycopersicum genes located on chr 1 and 2, and included one segmental duplication while genes of B. distachyon were only on chr 1 with one tandem duplication. GolS sequences were found to have well conserved motif structures. Cis-acting analysis was performed for three abiotic stress responsive elements, including ABA responsive element (ABRE), dehydration and cold responsive elements (DRE/CRT) and low-temperature responsive element (LTRE). ABRE elements were found in all GolS genes, except for SlGolS4; DRE/CRT was not detected in any GolS genes and LTRE element found in SlGolS1 and BdGolS1 genes. AU analysis in UTR and ORF regions indicated that SlGolS and BdGolS mRNAs may have a short half-life. SlGolS3 and SlGolS4 genes may generate more stable transcripts since they included AATTAAA motif for polyadenylation signal POLASIG2. Seconder structures of SlGolS proteins were well conserved than that of BdGolS. Some structural divergences were detected in 3D structures and predicted binding sites exhibited various patterns in GolS proteins.

Characterization of wound-induced serine protease inhibitor (wip1) genes and proteins in Turkish maize varieties

Protease inhibitors (PIs) are generally small proteins that have been identified in plants. The wip1 gene codes for wound-induced protein, which is similar to serine PIs of the Bowman-Birk family (BBIs). In this study, we analyzed 10 wip1 genes of Turkish maize varieties to understand the structure and characteristics of the wip1 genes and proteins in maize. We found that genetic variability of wip1 genes was higher (π: 0.0173) than reported in previous studies. Tajima’s D value was found to be positive (1.73), suggesting over-dominant selection in these loci. According to phylogenetic analysis of wip1 proteins, monocot and dicot BBIs were separated independently, and Turkish varieties were clustered with each other generally. The 3D structures of wip1 proteins indicated that several wip1 proteins had structural divergence in active loops, containing various numbers of cysteine residues ranging between 7 and 9. Particularly, Cys74 was identified in Kocbey and Gozdem varieties, whereas Cys98 was only in the Gozdem variety. Also, a critical serine residue (Ser98) was observed in two varieties — Antbey and Batem Efe. These results can contribute to understanding the role of wip1 genes and corresponding proteins in maize.