OsDOF18, a DOF transcription factor from rice confers abiotic stress tolerance in Escherichia coli.
DNA-binding with one finger proteins (DOF) belongs to a group of plant-specific transcription factors that are characterised by the presence of conserved CX2CX21CX2C zinc finger of 50-52 amino acids containing DNA-binding DOF domain at the N-terminus. This DOF domain associates with a basic region and interacts with T/AAAAG core sequence in the promoters of target genes (Umemura et al., 2004; Yanagisawa, 2002). In comparison, the C-terminal DOF region is highly variable involved in protein-protein interaction and other regulatory activities. For example, AtDOF4.2, AtDOF4.4 and ZmDOF1 contain threonine, methionine and aspartate motifs, located in the C-terminal, are responsible for activation of target gene expression. Subsequently, DOF transcription factors display a complex modular structure by forming homo and heterodimeric complexes, involve in multiple controlling processes, by acting as transcriptional activator or repressor of target genes (Yamamoto et al., 2006). The regulatory action facilitated by DOF proteins is bifunctional which means it can interact with DNA as well as also with other regulatory proteins including basic leucine zippers (bZIPs) and myeloblastosis oncogenes (MYBs) (Diaz et al., 2002; Washio, 2001).
DOF transcription factors have been appeared to be broadly distributed in the plant domain, for instance DOF proteins have been identified in unicellular algae, moss, and vascular plants. Since the first DOF protein identified from maize, several DOF transcription factors have been found in other plants including Arabidopsis, rice, poplar, barley, wheat, maize, Chinese cabbage, tomato, pepper, banana and potato (Feng et al., 2016; Wu et al., 2016; Gupta et al., 2015; Ma et al., 2015; Venkatesh and Park, 2015 Cai et al., 2013; Noguero et al., 2013; Yanagisawa, 2002) after the availability of partial or complete information about genome sequences. Efforts have been made to envisage the DOF genes based on in silico studies. In Arabidopsis, 36 DOF genes have been identified, 31 in wheat, 54 in maize and 30 genes have been identified in the rice genome (Shaw et al., 2009; Lijavetzky et al., 2003). Furthermore, genetic and molecular studies have advocated that DOF proteins are implicated in the regulation of biological processes exclusive to plants, for instance, light-responsiveness, photoperiodic flowering, tissue differentiation, dormancy, seed germination and maturation, metabolic regulation and nitrogen assimilation and phytochrome and phytohormone signalling (Gupta et al., 2015; Noguero et al., 2013; Yanagisawa et al., 2004). Freshly, DOF proteins were described to be involved in biotic and abiotic stress responses, perhaps via the stimulation of various stress-responsive genes (Gupta et al., 2015; Sasaki et al., 2015; Corrales et al., 2014).
The cultivated crops particularly rice, wheat, maize and barley inhabit significant place are grown over a large area worldwide and more than half of the world population feeds on these crops to fulfill their energy demands. Although quite a few DOF family members have been functionally characterised in rice, for instance, OsDOF3 was found to be involved as a pyrimidine box binding factor in the germinated aleurone (Washio, 2003), OsDOF12 was stated to control heading date (Li et al., 2009) and OsDOF25 is reported in carbon and nitrogen metabolism in Arabidopsis (Santos et al., 2012) and acts as a transcriptional activator of C4 photosynthesis gene, OsC4PPDK in rice (Zhang et al., 2015). The functions of most of the rice DOF proteins remain unknown especially in abiotic and biotic stresses. The present study involves the cloning and in silico characterisation of OsDOF18 by carrying out phylogenetic and motif scan analysis followed by secondary and tertiary structure predictions. Expression pattern was investigated by RT-qPCR and effect of over-expression of OsDOF18 gene was studied on E. coli growth under different abiotic stresses.
Materials and Methods
In Silico analysis. The OsDOF18 gene sequence was used as a query in BLAST with NCBI database for homology search. InterProScan and MOTIFSCAN tools were used to locate and analyze conserve DNA binding domain. CLUSTALW online tool was used to align the protein sequences showing similarity with OsDOF18 followed by construction of phylogenetic tree using Neighbor-Joining method (Kumar et al., 2016) by employing MEGA7 software. Expasy's ProtParam Proteomic server was used for computing basic physiochemical properties of OsDOF18. Primary sequence of OsDOF18 was analysed by DISOPRED (http://bioinf.cs.ucl.ac.uk/psipred/?disopred=1) to measure the degree of intrinsic disorder.
Prediction of OsDOF18 three dimensional structure.
An attempt was made to predict the structure of OsDOF18 using I-TASSER (Iterative Threading ASSEmbly Refinement) web server (Yang et al., 2015). For energy minimization, the rough model was then subjected to GROMOS96 43B1 executed using Swiss-PDBViewer version 4.0.1. To validate the backbone conformation of the predicted structure, the Phi/Psi Ramachandran plot was obtained through PROCHECK server (Laskowski et al., 2001). Moreover, the quality of DOF 18 protein model was evaluated using Qualitative Model Energy Analysis (QMEAN) server (http:// swissmodel.expasy.org/qmean/cgi/index.cgi) which tells about the degree of nativeness of predicted model. The PDB sumserver (https://www.ebi.ac.uk/thorntonsrv/databases/ pdbsum/Generate.html) was used for structural motif analysis of modelled DOF protein. The final model of DOF protein was then subjected to raptorX tool (http://raptorx.uchicago.edu) for identifying the possible binding sites. Metal detector V1.0 (http:// metaldetector. dsi.unifi.it/) was used for metal detection studies.
Plant materials and stress treatments. Oryza sativa cv. KS282 obtained from Rice Program, Crop Science Institute, National Agricultural Research Centre (NARC) Islamabad Pakistan was selected for expression analysis of OsDOF18 gene. Seeds were germinated on half strength MS medium and kept at 25[degrees]C. Ten day old seedlings were subjected to different abiotic stresses. For drought, seedlings were placed on aluminium foil till visible leaf rolling and seedlings were then transferred to 4[degrees]C for 48 h for cold treatment. After 48 h of chilling treatment, seedlings were moved to the control environment and samples were collected after 24 h. Plant roots were immersed in 200 mM NaCl solution for 3 h for salt stress. Seedlings were subjected to 45[degrees]C for 6 h heat stress. For wounding stress, rice leaves were cut into pieces and then left in water for 6 h at room temperature. After respective treatments, samples were collected separately, frozen in liquid nitrogen and preserved at -80[degrees]C till analysis. Untreated plants were used as control. Experiments were performed in replicates to confirm precision and reproducibility.
RNA isolation and RT-qPCR. Total RNA was isolated from different stressed and control plants using the RNeasy Plant Mini Kit (Qiagen). RNA purity and integrity was ensured by running 1% agarose gel. Primers were designed using PrimerBLAST tool. Quantitative real time PCR was performed using Brilliant II SYBR Green RT-qPCR master mix Kit (Agilent Technologies). Primers used are mentioned in Supplementary Table 1. Samples were assayed in a 10 [micro]L reaction mixture containing 5 [micro]L of 2x reaction mix, 0.5 [micro]L (5 [micro]mol) of each forward and reverse primers, 2 [micro]L of RNA (100 ng), 0.1 [micro]L of reverse transcriptase and 1.8 [micro]L of nuclease free water. No template and no primer controls were also included. The thermal profile consists of 30 min of reverse transcription at 50[degrees]C one cycle and 10 min of polymerase activation at 95[degrees]C, followed by 40 cycles of PCR at 95[degrees]C for 30 sec, 53[degrees]C for 1 min and 72[degrees]C for 30 sec. Melting curve analysis (60 to 95[degrees]C after 40 cycles) and agarose gel electrophoresis were performed to examine the amplification specificity. The relative change in transcript level was calculated using 2-CT method (Schmittgen and Livak, 2008) with actin as internal standard to determine relative expression levels. RT-PCR assays were repeated at least twice and each repetition had three replicates.
Cloning and expression in E. coli. Oryza sativa cDNA clone J065152E11 with assigned accession number AK241364.1 was acquired from NIAS Databank of Japan and primers were designed specific for full length and DNA binding domain containing region of OsDOF18 containing BamHI and XhoI sites to aid in the cloning (Supplementary Table 1). PCR amplification was performed using 25 ng of cDNA clone with 66[degrees]C and 63[degrees]C annealing temperature for full length and DNA binding domain containing region, respectively. The amplified product was analysed on agarose gel, amplicon was gel eluted using Gene JET Gel Extraction Kit (Thermo Scientific), and cloned in GST expression vector (pGEX4T-1). Putative cloned DOF18 gene was commercially sequenced. The putative recombinant plasmids were confirmed by PCR amplification, restriction digestion and commercial sequencing. The pGEX4T/OsDOF18 expression was induced with 1.0 mM isopropyl b-D-thiogalactoside (IPTG) for 6 h at 37[degrees]C and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Assays for abiotic stress tolerance in E. coli. Spot assay was performed to establish the function of OsDOF18 gene in E. coli cells. BL21 (DE3) cells were subjected to transformation pGEX4T1-OsDOF18 and control plasmid (pGEX4T-1). Cells were allowed to grow in LB broth till OD600 reached 0.6. Afterward, expression of recombinant protein was induced by 1 mM IPTG and cells were incubated for further 4 h at 37[degrees]C. [OD.sub.600] was measured and cultures were diluted to [OD.sub.600] 1. Then cells were diluted to 50-fold, 100-fold and 200-fold. Then diluted samples were spotted on IPTG agar plates (100 [micro]g/mL ampicillin and 1.0 mM IPTG) supplemented with 400, 500, and 600 mM concentration gradient of NaCl for salt stress and 500 mM, 800 mM and 1 M mannitol for drought. For heat stress, 1 mL of each sample was kept at 50[degrees]C and 100 [micro]L of each sample was taken at different periods of 1, 2 and 3 h, successively. Samples were diluted by 50-fold, 100-fold and 200-fold and 10 [micro]L of each sample was spotted onto IPTG LB agar plates. For cold stress, samples were placed at -80[degrees]C for 24 h. Then samples were allowed to thaw at 35[degrees]C for 1 h and took 100 [micro]L at different periods of 2, 4, 6 and 8 h, successively. Samples were diluted by 50-fold, 100-fold and 200-fold and 10 [micro]L of each sample was spotted onto IPTG LB agar plates. All these plates were incubated overnight at 37[degrees]C and photographed.
Results and Discussion
Plant growth and survival are greatly affected by abiotic stresses. However, plants have the ability to endure the stress condition by cascade of events at cellular and molecular level. A number of genes are involved in providing adaptation to plants against abiotic stresses for instance, genes involved in ion homeostasis, osmoprotectants, and free redical-scavenging and genes encoding transcription factors. It is important to characterise the unknown proteins to decipher their role in providing tolerance to plants. In present study, OsDOF18 cDNA fragment of entire open reading frame (ORF) and DNA binding domain containing region were cloned and characterised.
Cloning and sequence analysis of OsDOF18 gene.
The OsDOF18 gene was amplified by PCR from cDNA clone using specific primer pair. The eluted PCR product was inserted into pGEX4T-1 vector in between BamHI and XhoI restriction sites. Then, cloned products were subjected to transformation in E. coli cells, and cloned gene was confirmed by digesting the plasmid with suitable restriction enzymes (Fig. 1) and commercial sequencing. Sequence analysis revealed that the complete open reading frame of OsDOF18 is 831 bp and predicted protein consists of 276 amino acids having molecular weight of 28.9 kDa and the isoelectric point (pI) of 6.75. It was observed that it is intronless gene which is a key feature of prokaryotic genes. Although there are many intronless genes in eukaryotes and to study those genes might help in understanding the evolutionary array of related genes and genomes. Research also revealed the conservation of many intronless genes in archaea, bacteria, fungi, plants and other eukaryotes during the process of evolution. The translated protein sequence of OsDOF18 gene was then subjected to BLAST search for finding homology with already available sequences in NCBI databases. Search showed similarity with DOF protein like sequences belonging to different species including maximum similarity with ZmDOF protein (57%), SiMNBIA-like (57%) and SbDOF22 (48%). The OsDOF18 ortholog sequences were retrieved and multiple sequence alignment was carried out using CLUSTALW tool followed by phylogenetic analysis which showed the clustering of OsDOF18 with Zea mays and Sorghum bicolor whereas other DOF proteins were clustered into different groups (Fig. 2).
Tertiary structure prediction. Although protein sequences of a number of DOF proteins is available in protein sequence database, but the information about the 3D structure of DOF proteins is missing in protein database. The absence of 3D structure has encouraged us to build the 3D structure of OsDOF18 protein. Information about molecular function and active site residues can be gathered from 3D structure. 3D structure was acquired by multiple threading from online accessible I-TASSER server utilizing diverse threading templates as given in Table 1. C-score of the I-TASSER models was used to deduce the final result from the consensus of top structural matches. C-score is calculated on the basis of threading template alignment and the conjunction parameters of the structure assembly imitations and describe about the quality of predicted models. Template modeling score (TM-score) was used to evaluate the structural similarity between templates and models and the sequence identity was determined in the structurally aligned region.
I-TASSER server predicted five structural models and on the basis of maximum C-score and maximum number of decoys for evaluation and verifications, most appropriate predicted structure was chosen. The selected model has predicted TM score of 0.37 [+ or -] 0.13 and RMSD (root mean square deviation) score of 13.3 [+ or -] 4.1 [Angstrom] and found in correct topology C-score, TM-score, and RMSD value. Swiss-pdbViewer was then used for stabilizing their stereochemical properties by energy minimization. Kushwaha et al. (2013) also predicted the structure of four DOF proteins from Sorghum in the same way.
Validation of the predicted 3D structure. PDB files were subjected to PDBsum and PROCHECK server for predicted model authentication. Ramachandran plot and % of the residues in the core region, allowed regions and in the disallowed region are shown in Fig. 3A. Maximum likelihood of finding residues of protein (>90%) in the core regions proposes better stereochemical quality. PROCHECK study revealed the low percentage of residues having phi/psi angles in the disallowed region suggesting the acceptability of Ramachandran plots. The percentage of residues in the allowed/core region were found to be 98.7% while residues in disallowed regions were found to be 1.3% as shown in Table 2. Additionally, the quality of 3D structure was estimated using QMEAN server. The QMEAN Z score were found to 0.262 (Z-score: -5.76). The presence of significant QMEAN Z score suggested the predicted model quality to be acceptable. The final modeled structure is shown in Fig. 3B.
Structural motif, active site and metal binding site analysis. The PDB file of putative OsDOF18 protein was subjected to PDBsum server for structural motif analysis. OsDOF18 protein contains more frequency of [alpha]-helix, [beta]-sheets, turns and coil. The DNA binding domain region primarily contains turns but sheets, helix and coil are also present. The predicted structure is shown in Fig. 4. Motif scan analysis showed the presence of amino acid-rich profiles along with zinc finger DOF type profile. Alanine-rich (28-57), serine-rich (114-125) and threonine-rich (145-198) profiles were found in motif scan output. The alanine-rich and serine rich profiles mainly consist of [beta]-turns and sheets while threonine-rich profile consists of helix, [beta]-turns and sheets.
The binding site of targeted protein was predicted by raptor X tool and shown in 3D structure of DOF domain 1 in Fig. 5. Use of metal detector tool showed that cysteine residues were actively involved in coordination with metal on the basis of greater metal score and lesser free score value with presence of disulphide bridges. The DOF domain was truly functioning as Cys2/Cys2-type zinc finger proteins.
Intrinsic disorder in OsDOF18. A large degree of intrinsic disorder (ID) is observed in eukaryotes as bioinformatic analysis revealed the presence of 30 or more disordered residues in 30-60% of proteins. Plant transcription factors have significant degrees of intrinsic disorder regions (IDRs) which play vital role in interaction with DNA and other regulatory proteins (Kragelund et al., 2012). The ID prediction profile showed that OsDOF18 has high degree of IDR in both N and C termini. A region at the N-terminus represents putative protein-protein interaction region. Degree of IDs is 65% in OsDOF18 including a region of high ID of 122 amino acids at C terminus adjacent to DNA binding domain. Only the region of DNA binding domain containing zinc finger is structured (Fig. 6A&B).
Expression analysis of OsDOF18 by RT-qPCR. RT-qPCR assay was used to investigate OsDOF18 expression in rice under drought, cold, heat, salinity and wounding/mechanical stress. Analyses of available microarray data demonstrated that expression of OsDOF18 gene is regulated by various abiotic stress conditions. RT-qPCR analysis showed up-regulation of OiDOF 18 transcript level in response to salt and drought stresses (Fig. 7A&B) whereas, GENEVESTIGATOR data showed the significant up-regulation under cold stress. In salinity and drought, transcript level was peaked up to 4 fold in comparison to control plants. There was no significant increase in heat, cold and wounding stresses. Corrales et al. (2014) reported that tomato cycling DOF factor transcript was up-regulated in response to salt, drought and threshold temperatures suggesting its role in multiple stresses. Ma et al. (2015) also observed the up-regulation of DOF genes in Chinese cabbage against cold, salt, heat and drought stresses.
Over-expression of OsDOF18 in E. coli improves growth during abiotic stresses. An attempt was made express the pGEX vector containing the full coding sequence of OsDOF18. It ended in failure, perhaps because the expression of the whole coding region of DOF protein is leaky and toxic for E. coli cells (Yanagisawa and Schmidt, 1999) or it may have produced degraded forms of DOF18 proteins. However, a plasmid that allowed the expression of DOF18 DNA binding domain only with some flanking region was constructed. The recombinant plasmid pGEX-OsDOF18 and empty vector pGEX4T-1, used as a control, were transformed into E. coli cells. The recombinant protein was induced by IPTG treatment as confirmed by SDS-PAGE (Fig. 8). Cultures of BL/pGEX4T-1 and BL-OsDOF18 were spread on different plates to investigate the consequences of OsDOF18 over-expression on E. coli cells against different stresses. Figure 8 showes that recombinant and control cells have similar growth on LB medium in overnight grown culture. When OsDOF18 was over-expressed in E. coli cells, BL/ OsDOF18 cells showed better tolerance in high salt and desiccation treatment as compared to vector alone. At low and high temperature, bacterial growth was similar in BL/OsDOF18 and BL/pGEX4T-1 cells. These results revealed that OsDOF18 gene significantly induced tolerance under salt and dehydration stresses. The improved tolerance to different abiotic stresses may be a consequence to the binding of OsDOF18 DNA in binding domain to the stress inducible promoters of different functional genes in E. coli. OsDOF18 proteins belong to [C.sub.2][C.sub.2] type zinc finger proteins initially considered restrained to the eukaryotes, the first prokaryotic [C.sub.2][H.sub.2] zinc finger protein was identified in 1998 in the transcriptional regulator protein (Ros) in Agrobacterium tumefaciens (Chou et al., 1998). This protein contains the sequence [CX.sub.2][CX.sub.3][FX.sub.2][LX.sub.2][HX.sub.2]HH located at the residues 79-97 and considerably bears a resemblance to the consensus sequence of a eukaryotic [C.sub.2][H.sub.2] zinc finger domain (Malgieri et al., 2007; Kado, 2002). Structural and evolutionary studies suggest that eukaryotic zinc finger domains were evolved from Ros homologues (Moreira and Rodriguez-Valera, 2000). This imitates that there might be some similarity in regulatory systems of both eukaryotes and prokaryotes at some point of communication, and might also share similar components between them. Therefore, it is credible to presume that OsDOF18 is making interaction with transcriptional network in the bacterial cells and aid in stress tolerance. Like our study, few other researchers also reported that survival of E. coli cells gets better by over-expressing plant stress-related genes. Over-expression of a LEA protein from soybean results in salt tolerance in E. coli (Liu and Zheng, 2005). Over-expression of phytochelatin synthase conferred tolerance to E. coli against various stresses including pesticide, UV exposure, heat and salt (Chaurasia et al., 2008). SbDREB2A transcription factor over-expression resulted in better E. coli growth under different stress conditions (Gupta et al., 2010). Similarly, SbSI-1 gene over-expression confers salt and drought tolerance in E. coli (Yadav et al., 2012). Recently, Jin-long et al. (2012) demonstrated that the expressed novel dirigent protein ScDir from sugarcane had enhanced the E. coli tolerance to PEG and NaCl.
There is no 3D structure available for Dof proteins. A Dof transcription factor was cloned and characterised from rice. this paper reports the in silico prediction of three dimensional structure of OsDOF18 validated by PROCHECK server and Ramachandran plot analysis, which suggested predicted model to be satisfactory. For functional characterisation, RT-qPCR analysis revealed the upregulation of OsDOF18 by salt and drought stresses. OsDOF18 DNA binding domain was transformed in E. coli and recombinant E. coli cells showed higher tolerance to desiccation and salinity compared to vector alone. The present study demonstrates that OsDOF18 gene might play an important positive modulation role in abiotic stress tolerance and suggest that it could be a potential bioresource for engineering abiotic stress tolerance in crop plants.
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Farah Deeba (a), Tasawar Sultana (a), Ghazala Kaukab Raja (a) and Syed Muhammad Saqlan Naqvi (b)*
Dapartment of Biochemistry, PMAS Arid Agriculture University, Rawalpindi--46300, Pakistan
(received June 23, 2016; revised November 29, 2016; accepted December 03, 2016)
* Author for correspondence; E-mail: email@example.com
Caption: Fig. 1(A-F). Cloning and over-expression of OsDOF18 full length and DNA binding domain.
Caption: Fig. 2. The phylogenetic analysis of DOF proteins from rice and other plant species by MEGA 7 from CLUSTALW alignments. The neighbor-joining method was used to construct the tree with p-distance.
Caption: Fig. 3(A-B). Prediction and validation of 3D structure of OsDOF18 protein.
Caption: Fig 4. Secondary structure of OsDOF18 protein predicted by PDBsum server. The DOF domain region is highlighted in green box.
Caption: Fig. 5. Identified binding sites of DOF domain 3D structure by raptorX tool.
Caption: Fig. 6. Intrinsic disorder (ID) prediction for OsDOF18 (A) Intrinsic disorder analysis by DISPORED. A threshold was applied with disorder assigned to values greater than or equal to 0.05 (black bar). (B) Diagrammatic representation of OsDOF18 structure. It comprises of zinc finger DNA binding domain.
Caption: Fig. 7(A-B). Expression analysis of OsDOF18.
Caption: Fig. 8. Growth performance of BL/pGEX4T-1 and BL/OsDOF18 recombinants
Table 1. Primers used in present study Primers name Primers sequences OsDOF18(1-277)-For 5'-CGCGGATCCATGCAGGAGCAGCAGCCG-3' (BamHI) OsDOF18(1-277)-Rev 5'-CCGCTCGAGTCATGGGAGGTTGAGGAACAC-3' (XhoI) OsDOF18(1-148)-For 5'-CGCGGATCCATGCAGGAGCAGCAGC-3' (BamHI) OsDOF18(1-148)-Rev 5'-CCGCTCGAGTCACTCGGGAGTCGTGACG-3' (Xho1) RTActin-F 5'-GAAGATCACTGCCTTGCTCC-3' RTActin-R 5'-CGATAACAGCTCCTCTTGGC-3' RTDOF18-F 5'-AAGACGACGACTTCCACAAC-3' RTDOF18-R 5'-AGACTCTTGGTGATGGACGG-3' Primers name Product Accession numbers size (bp) OsDOF18(1-277)-For 831 NM_001068645.2 OsDOF18(1-277)-Rev OsDOF18(1-148)-For 444 NM_001068645.2 OsDOF18(1-148)-Rev RTActin-F 226 X16280.1 RTActin-R RTDOF18-F 182 NM_001068645 RTDOF18-R Table 2. Ramachandran plot statistics No. of Percentage residues Most favoured regions 178 76.7% [A, B, L] Additional allowed regions 45 19.4% [a, b, l, p] Generously allowed regions 6 2.6% [~a, ~b, ~l, ~p] Disallowed regions [XX] 3 1.3% Non-glycine and non-proline 232 100 % residues Glycine residues 14 Proline residues 29 Total number of residues 276