{'fraction_non_alphanumeric': 0.03994202651434418, 'fraction_numerical': 0.03405942282279722, 'mean_word_length': 4.967947087255151, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 4, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Genome-wide analysis of PHD finger gene family and identification of potential miRNA and their PHD finger gene specific targets in Oryza sativa indica

Rice (Oryza sativa L.) is one of the most important cereal crops for one third of the world population. However, the grain quality as well as yield of rice is severely affected by various abiotic stresses. Environmental stresses affect the expression of various microRNAs (miRNAs) which in turn negatively regulate gene expression at the post-transcriptional level either by degrading the target mRNA genes or suppressing translation in plants. Plant homeo-domain (PHD) finger proteins are known to be involved in the plant response to salinity stress. In the present study, we identified 44 putative OsPHD finger genes in Oryza sativa Indica, using Ensembl Plants Database. Using computational approach, potential miRNAs that target OsPHD finger genes were identified. Out of the 44 OsPHD finger genes only three OsPHD finger genes i.e., OsPHD2, OsPHD35 and OsPHD11, were found to be targeted by five newly identified putative miRNAs i.e., ath-miRf10010-akr, ath-miRf10110-akr, osa-miR1857–3p, osa-miRf10863-akr, and osa-miRf11806-akr. This is the first report of these five identified miRNAs on targeting PHD finger in Oryza sativa Indica. Further, expression analysis of 44 PHD finger genes under salinity was also performed using quantitative Real-Time PCR. The expression profile of 8 genes were found to be differentially regulated, among them two genes were significantly up regulated i.e., OsPHD6 and OsPHD12. In silico protein-protein interaction analysis using STRING database showed interaction of the OsPHD finger proteins with other protein partners that are directly or indirectly involved in development and abiotic stress tolerance.


Introduction
Rice (Oryza sativa L.) is one of the most important cereal crops and serves as the staple food for over one-third of the world's population [1]. However, the productivity of rice is greatly affected due to soil salinity which is the second most widespread soil problem next to drought in rice growing areas of the world [2,3].
Plant homeo-domain (PHD) finger proteins have been known to be involved in the plant response to various abiotic stress. The PHD finger is a chromatin interacting domain localized in the nucleus of eukaryotic organisms and involved in transcriptional regulation [4,5]. A typical PHD usually consist of 50-80 amino acid residues, stabilized by two zinc ions as metal binding RING domain in a "cross-brace" topology and possess a unique characteristic Cys4-His-Cys3 pattern [6][7][8]. It is conserved and has been found in more than 400 proteins in eukaryotes including the yeast [9][10][11]. The PHD finger protein plays diverse role in plants including development and plant growth [12][13][14][15][16][17]. In recent studies, it has been reported that PHD finger genes were also responsive under different abiotic stress treatment in P. trichocarpa [18], maize [5], carrot [19], tomato [20], Oryza sativa Japonica [21] and moso bamboo [22].
In plants, many studies have revealed that microRNAs (miRNAs) play a vital post-transcriptional regulatory role in gene expression by targeting mRNA cleavage or translational inhibition [23]. In plants, mature miRNAs are generated from the long stem-loop precursor (pre-miRNAs) by a DICER-like RNA endonuclease and then the RNA-Induced Silencing Complex (RISC) guided by ARGONAUTE 1 (AGO1) protein directs the miRNA to the complementary target mRNA sequence [23][24][25][26]. Plant miRNAs are reported to possess important functions in several metabolic and biological pathways such as tissue development and differentiation, biotic and abiotic stress responses, phytohormones signaling, and secondary metabolite production [27,28]. Nonetheless, the evolutionary highly conserved nature of an extensive number of miRNAs simplified the process of characterization of novel miRNA orthologs in new plant species through homologs identification [29]. Several abiotic stress-sensitive miRNAs have been reported over a period of time in various studies, e.g. In Arabidopsis thaliana, miRNA398 is involved in oxidative stress tolerance [30], and gene expression of 21 miRNAs are up-regulated in response to UV-B exposure [31].
Knowing the importance of miRNA and their roles in gene regulation, in the present investigation, experiments have been designed for in silico identification of miRNAs and their potential PHD targets in rice through computational approach. Further, to study the response of PHD genes under salinity stress, we performed gene expression analysis using qRT-PCR. Our results provide better framework to understand the role of PHD gene family in Indica rice and their potential role in salinity stress response.

Identification and domain analysis of PHD family genes in rice
We identified candidate PHD family genes by using Pfam ID "PF00628" as a keyword against the rice genome database in the Ensembl Plant (http://plants.ensembl.org/Oryza_indica/Info/Index). The amino acids, as well as the cDNA sequence of all the selected PHD proteins, were then retrieved from the Ensembl Plant database for further analysis. The amino acid sequences obtained were used for domain analysis using Pfam [32] and HMMER database [33]. Multiple sequence alignment was performed in MEGA X program using MUSCLE algorithm using default parameters and visualized by Jalview software [34][35][36]. The aligned PHD finger protein sequences were used for construction of phylogenetic trees using default parameters in MEGA X program using Neighbor Joining (NJ) algorithm. The default parameters of MEGA X used during alignment are as follows -gap penalty was set at gap opening penalty at -2.90, extension at 0 and hydro-phobicity multiplier at 1.2. The maximum memory usage was set at 2048 MB and the Maximum iterations performed were 16. The cluster method used for iteration was UPGMA (Unweighted Pair Group Method with Arithmetic mean) as a simple agglomerative (bottom-up) hierarchical clustering method. UPGMA with a lambda value of 24 (Minimum diagonal Length) was used. While phylogeny reconstruction, neighbor joining (NJ) was used as a bottom-up (agglomerative) clustering method for the creation of phylogenetic trees. The Poisson model was used as a substitution model in this case. Substitution rates among sites were considered to be uniform and the pattern along lineages were also taken as homogeneous. The gaps and missing data were treated as pair wise deletion in each case.

Identification of potential miRNAs and their target PHD gene
Workflow of the identification and characterization of potential miRNAs and their target genes is depicted in Fig. 1. A total of 10,898 mature miRNA sequences were retrieved from PMRD: Plant micro RNA Database (http://bioinformatics.cau.edu.cn/PMRD/) [37]. With identity value 90, CD-HIT-v4.5.4 was used to remove the redundancy in miRNA sequences [38]. In order to identify miRNA-targeted PHD genes of Indica rice, local BLAST was performed using Blast2GO version 5.2 [39]. BLASTx analysis (E-value ≤ 1e-10) was performed in order to remove protein-coding sequences from precursor sequences.

Prediction of the secondary structure of pre-miRNAs
Prediction of the secondary structure was done by using the software MFOLD 3.1 available at (http://www.bioinfo.rpi.edu/applications/ mfold/rna/form1.cgi) [40]. The following criteria were used for screening the candidates of potential miRNAs: minimum length of the pre-miRNA to be 60 nt; pre-miRNA should be folded into appropriate stem-loop hairpin secondary structure; mature miRNA sequence should be placed in one arm of the hairpin structure; not >6 nt mismatches in miRNA/miRNA duplex; No loops or breaks between the miRNA/miRNA duplex; A + U content within 30-70%; Predicted secondary structure should have higher minimal folding free energy index (MFEI) and negative minimal folding free energy (MFE) values [41]. The MFE or ΔG (-kcal/mol) values generated from the MFOLD web server of the stem-loop structures were used for calculating the MFE index values using the following formula:

Plant material, growth condition and stress treatment
Seeds of Oryza sativa Indica variety Pusa Basmati 1 were surface sterilized with 70% ethanol for 1 min followed with 0.1% Mercuric chloride (HgCl 2 ) for 5 min and then 0.2% Bavastin for 10 min. All seeds were placed in the dark for 2 days then allowed to germinate for 15 days under control condition maintained at 26 ± 2 • C with 16/8 h light/dark photoperiods cycle. For salinity stress treatment, 15 day old seedlings were treated with 200 mM NaCl for 24 h using hydroponic system, whereas seedlings maintained in distilled H 2 O were used as control. After 24 h of treatment, seedlings were harvested and immediately frozen into liquid nitrogen and stored at − 80 • C.

RNA extraction and qRT-PCR analysis
Total RNA was isolated from 200 mM NaCl treated as well as untreated seedlings and was used as a template for the cDNA synthesis following the manufacturer's protocol (Thermo Scientific, EU). Gene specific primers for all 44 OsPHD genes were designed manually using exon-exon junction region (Supplementary Table 1) to amplify 110-243bp (PCR amplicons) and their specificity were examined through Primer Blast of NCBI. The rice actin 1 gene was used as an internal control to normalize the gene expression level. The qRT-PCR was performed on an ABI 7900HT real time system (Applied Biosystems). The total reaction volume was 10 μl containing 5 μl of 2X KAPA SYBR FAST qPCR Master Mix Universal, 200 nM gene specific primers and 0.5 μl of cDNA in a total volume of 10 μl. The thermal cycle reaction conditions were 95 • C for 3min, followed by 40 cycles of 95 • C at 10 s and then 57 • C for 30 s. A melting curve was generated at the end of 40 cycles for analyzing the specificity of each gene. The experiment was conducted with 3 independent biological replicates and technical triplicates for each sample. The relative gene expression of individual gene was calculated via 2^-ΔΔCT method [42].

Statistical analysis
All the experimental data are means of triplicates and represented as mean ± standard deviation (SD). The significance was tested using SPSS (Statistical Package for the Social Sciences) software (version 21 for Windows; IBM Ltd., Japan) for calculating Student's t-test at significance level p ≤ 0.05.

Identification and phylogenetic analysis of PHD family genes in rice
In the present investigation, we have identified 44 putative PHD family genes in the Indica rice genome. Domain analysis using Pfam database showed the presence of PHD-finger domain (PF00628) in all the identified 44 putative PHD proteins and were named OsPHD1 to OsPHD44 on the basis of their chromosomal location ( Table 1). The length of OsPHD proteins was between 175 and 2275 amino acid. The Table 1 List of the PHD family proteins from Indica rice and their chromosome localization.
Phylogenetic tree analysis revealed that OsPHD proteins might be divided into ten major groups (Group I to X). Group I forms the largest clade with 9 members followed by group X which has 8 members. These two groups jointly represent 38.63% of total rice PHD proteins. The other groups like group IV-VI, II-III and VII had 5, 4 and 3 members respectively. Rest three groups V, VIII and IX remained the smallest ones with only 2 member each (Fig. 4).

Prediction of the secondary structure of potential miRNAs
The six non-coding miRNA sequences i.e., ath-miRf10010-akr, ath-miRf10110-akr, gma-miR4993, osa-miR1857-3p, osa-miRf10863-akr and osa-miRf11806-akr, were further used for secondary structure analysis including hairpin stem-loop structure using MFOLD version 3.1 (Fig. 5A-F). The putative miRNAs obtained varied in their lengths ranging from 21 to 26 nucleotides. The negative MFE (-ΔG) of the  miRNA precursors were also calculated to study the stability of the hairpin stem-loop structure (Table 3). In comparison to the length of miRNAs, the length of putative precursor miRNAs of rice also varied ranging from 70 to 130 nucleotides. The stability of the secondary hairpin structure of pre-miRNA was determined by MFE (-ΔG). The distribution of G, C, A, and U nucleotides in the pre-miRNA were found to be different, where it ranged from 14.29 to 33.85% for A, 12.75-34.13% for U, 17.69-35.71% for G and 17.46-36.27% for C, respectively (Table 4). In the present study, out of the six miRNAs, gma-miR4993 did not fulfil two criteria as it has >6 mismatches in miRNA/ miRNA* duplex and there are loops or breaks between miRNA/miRNA* duplex, hence was not considered for further study (Fig. 5C) (see Fig. 6.

Quantitative real-time PCR analysis of OsPHDs expression in response to salt stress
In order to study the potential response of all OsPHD genes under salinity stress we further conducted quantitative real-time PCR. Out of 44 genes 8 genes were differentially expressed (Log2 fold change > 1) under salt stress (Fig. 6). Two genes OsPHD6 and OsPHD12 were significantly up-regulated (>2 fold) from group I and VI, respectively. Other six genes i.e., OsPHD 5,7,8,15,19 and 43 corresponding to group II, III, VI and X showed the expression level between 1 and 2 fold change. The expression of remaining 36 OsPHD genes were slightly low (<1 fold change) under salinity stress (see Fig. 7).

Protein-protein interactions network analysis
Using the STRING database, protein-protein interaction of OsPHD6 protein was generated (Fig. 7A). The protein sequence of OsPHD6 showed similarity to uncharacterized PHD finger protein of Oryza sativa (OS01T0883400-00) with 373.2 bit score and 9e-104 E-value. OsPHD6 protein showed interaction with other proteins at confidence scores varied from 0.495 to 0.407 (Table 5A). Predicted functional partners of OsPHD6 includes Acetyltransferase (Os03g0747600), G-box-binding factor 1(OsJ_08237), EBS like protein (OsJ_27,353), small ubiquitin-like modifier (SUMO) E3 ligase (SIZ1 and SIZ2), PERSISTENT TAPETAL CELL 1 (Os09t0449000), Putative collagen protein (OS03T0716200) and uncharacterized protein (OS01T0367400). Among the parameters that describe the topology of the network, 11 nodes with a total of 20 edges, a 3.64 grade per node and a clustering coefficient of 0.851 were found. The totality of the network proteins is predicted computationally from its nucleic acid sequence and has not been demonstrated experimentally. Functional enhancement of the network was performed considering the information from the KEGG, PFAM, and INTERPRO databases, from which data on the biological routes and the predominant domains in the interacting proteins were collected. Two proteins i. e., SIZ1 and SIZ2, of the PPI network showed molecular function of SUMO ligase activity and involved in Ubi conjugation pathway.
Functional enhancement of the network proteins showed the involvement of two proteins (CKX2 and CKX4) in the Zeatin biosynthesis pathway. PPI network proteins were shown to have different biological process such as cytokinin metabolic process, organonitrogen compound metabolic process, cell communication and oxidation-reduction process.

Discussion
PHD finger transcription factor family have involved in numerous biochemical and physiochemical processes like transcription, chromatin structure regulation and few reports on involvement under abiotic stress response like drought, cold, heavy metals, ABA etc in different plant species [12,21,[44][45][46]. It is also involved in fruit development and lignin synthesis in Chinese pear [47]. However, in rice no report has been published yet on the role of PHD finger proteins under salt stress. Hence, in the present study comprehensive genome wide analysis of PHD finger genes has been performed in Indica rice and studied their expression under salinity stress.
A total of 44 members of PHD finger family have been identified and were divided into 10 groups on the basis of phylogenetic tree analysis. From previous studies, it has been evident that the PHD finger proteins are evolutionary divergent [5,18,19]. It has been reported that PHD finger is a common structural motif present in all eukaryotic organisms and it shows characteristic typical conserved motif i.e., Cys4-His-Cys3 [48]. The PHD finger domain has been explained in Daucus carota L. (carrot) as C-X (1-2)-C-X (8-14)-C-X (2-8)-C-X (4-6)-H-X2-C-X  Table 4 The distribution of G, C, A, and U in the identified pre-miRNAs of Indica rice.  . This suggests that during evolution of different plant species the PHD finger proteins got significantly changed. The further analysis of PHD finger protein reveals that the length of PHD finger genes and their encoded proteins varied greatly from each other. In this study, we found that the length of PHD protein sequence varied from 175 (OsPHD6) to 2275 aa (OsPHD32); similar finding has also been observed with Japonica rice variety where it varied from 175 to 2192 aa and in maize it was between 72 and 2379 aa [5,21]. During the evolution of PHD finger genes few characteristic features were relatively conserved. For example, the presence of some common domains within particular groups, like the Alfin domain was exclusively present on group I and BAH on group IV. Furthermore, their corresponding size (amino acid) within the groups was also similar except for OsPHD39 from group IV. In another study, BAH was found only in group X in maize, and in Japonica rice group A and F were dominated by Alfin  and BAH respectively [5,21]. Also, DDT and BAH were the most common additional domain found within plant PHD-finger proteins. The DDT domain is a DNA binding domain in different transcription and chromosome remodeling factors [49,50], however, the BAH domain acts as a protein-protein interaction module specialized in gene silencing and plays an important role by linking DNA methylation, replication and transcriptional regulation. Several studies have shown the involvement of rice miRNAs under various abiotic stress like drought [51][52][53], salinity [54][55][56], high temperature [57,58], low temperature [59], heavy metal [60][61][62], etc. In the present study, we identified miRNAs and their PHD-finger genes specific targets in Oryza sativa Indica using bioinformatics approaches. Using the Plant micro RNA Database, we obtained five newly identified putative miRNAs targeting three OsPHD genes i.e., OsPHD2, OsPHD35 and OsPHD11. Gene expression is tightly regulated by miRNAs at the post-transcriptional level by translational repression or target degradation and gene silencing [63][64][65]. Therefore, to study the expression pattern of all the 44 OsPHD genes under salinity stress, we performed qRT-PCR. We found that out of 44 genes, 8 genes were differentially expressed under salinity stress after 24 hours of treatment i.e., OsPHD 5,6,7,8,12,15,19 and 43. Among these 8 genes, two i.e., OsPHD 6 and 12, were significantly up-regulated in response to salinity stress treatment.
In the present study, the expression of OsPHD 6 and 12 were significantly up-regulated under salinity stress treatment. Proteinprotein interaction studies using STRING database showed several functional partners of OsPHD6 and OsPHD12. Interacting partners of OsPHD6 such as Acetyltransferase, G-box-binding factor 1, SUMO E3 ligase (SIZ1 and SIZ2), and Persistent Tapetal cell 1 are reported to provide abiotic stress tolerance in plants. Acetyltransferase is involved in transcriptional regulation of nitrogen-associated metabolism and growth [66]. In Arabidopsis, it has been reported to be involved in the synthesis of secondary cell wall [67]. Another partner, G-box-binding factor 1 is reported to be involved in transcriptional regulation of environmentally inducible genes in plants [68]. In maize, GBF 1 is known to regulate maize Adh1 gene under hypoxia condition [69]. Many self-fertilising crops are particularly sensitive to abiotic stress at the reproductive stage. In rice (Oryza sativa L.) and wheat (Triticum aestivum L.), for example, abiotic stress during meiosis and the young microspore stage indicates that the tapetum is highly vulnerable and that the developmental program appears to be compromised. One of the partners of OsPHD6 such as Persistent tapetal cell 1 reported to control the development and degeneration of tapetum as well as pollen development in rice [70]. In plants, the post-translational addition of SUMO is an essential protein modification that provides protection against numerous environmental challenges.
In the present study, we have identified SUMO E3 ligase (SIZ1 and SIZ2) as a partner of both the proteins OsPHD6 and OsPHD12. Role of SUMO E3 Ligase SlSIZ1 in reported to facilitate heat tolerance in several plants [71,72]. Overexpression of the rice gene OsSIZ1 improved drought-, heat-, and salt-tolerance in Arabidopsis simultaneously [73]. Another functional partners of OsPHD12 such as chloroplastic   monogalactosyldiacylglycerol and cytokinin dehydrogenase (CKX2 and CKX4) are shown to provide tolerance to heat and drought in plant [74,75]. Overexpression of cytokinin dehydrogenase gene in barley has shown to provide greater tolerance to drought stress [75]. Its overexpression has been reported to modify antioxidant defense against heat, drought and their combination in Nicotiana tabacum [76]. Expression of BGIOSGA001450 (OsPHD2), BGIOSGA009559 (OsPHD11) and BGIOSGA026108 (OsPHD35) genes showed low expression (<0.5 fold change) under salinity stress treatment indicating the higher expression level of miRNA ath-miRf10010-akr, osa-miRf10863-akr, osa-miR1857-3p, osa-miRf11806-akr and ath-miRf10110-akr, respectively. The expression pattern of miRNAs and their target genes was reported to be inverse in response to different abiotic stress treatment [77]. Interaction studies showed BGIOSGA001450 (OsPHD2) to interact with PERSISTENT TAPETAL CELL2 (PTC2) which is required for normal tapetal programmed cell death and pollen wall patterning [78]. BGIOSGA009559 (OsPHD11) interacts with WD-repeat (WDR) proteins which is involved in signal transduction, cytoskeletal dynamics, protein trafficking, nuclear export, and RNA processing, and are especially prevalent in chromatin modification and transcriptional mechanisms [79]. BGIOSGA026108 (OsPHD35) shown interaction with auxin response factors (ARFs) which bind to the auxin response elements and regulates the expression of genes in auxin responses and plant development [80].

Conclusion
In the present investigation, we identified 44 putative OsPHD finger genes in Oryza sativa Indica and five potential miRNA targeting specific OsPHD genes. This is the first report of miRNAs namely ath-miRf10010akr, ath-miRf10110-akr, osa-miR1857-3p, osa-miRf10863-akr and osa-miRf11806-akr, on targeting PHD finger gene in Oryza sativa Indica. Expression analysis of 44 OsPHD genes demonstrated that two genes OsPHD 6 and 12 showed up-regulation under salinity stress. Proteinprotein interaction analysis showed several interacting protein partners that are directly or indirectly involved in development and abiotic stress tolerance. Thus, using genetic engineering tools the identified PHD finger genes or miRNAs can be manipulated for increasing the tolerance against abiotic stress and crop yield.

Declaration of competing interest
The authors declare that there are no conflicts of interest.