The Role of OsWRKY Genes in Rice When Faced with Single and Multiple Abiotic Stresses

: The WRKY genes are one of the largest families of transcription factors (TFs) and play a crucial role in certain processes in plants including stress signaling, regulation of transcriptional reprogramming associated with stress responses, and other regulatory networks. This study aims to investigate the WRKY gene family in the C 3 model plant, Oryza sativa L., using a genome-wide in silico expression analysis. Firstly, 104 WRKY TF family members were identiﬁed, and then their molecular properties and expression signatures were analyzed systematically. In silico spatio-temporal and hormonal expression proﬁling revealed the roles of OsWRKY genes and their dynamism in diverse developmental tissues and hormones, respectively. Comparative mapping between OsWRKY genes and their synteny with C 4 panicoid genomes showed the evolutionary insights of the WRKY TF family. Interactions of OsWRKY coding gene sequences represented the complexity of abiotic stress (AbS) and their molecular cross-talks. The expression signature of 26 novel candidate genes in response to stresses exhibited the putative involvement of individual and combined AbS (CAbS) responses. These novel ﬁndings unravel the in-depth insights into OsWRKY TF genes and delineate the plant developmental metabolisms and their functional regulations in individual and CAbS conditions. biosynthesis of amino acids, cellular and physiological processes, and


Introduction
As sessile organisms, plants are continuously exposed to adverse environmental conditions which may cause deleterious impacts on their growth, development, and productivity. Abiotic stresses (AbS) are predominant among various environmental stresses, which include drought, low temperature or cold stress, salinity, submergence, heavy metal, and other forms of oxidative stress such as radiation. At present, global agriculture is facing a serious threat from climatic changes, which is another aggravating challenge that affects the sustainability and productivity of crop plants [1]. Plants have well-developed defense responses to ensure survival under these environmental stresses and exhibit stress avoidance/stress tolerance through acclimation and adaptation mechanisms [2]. On deeper insight of stress, initiation of complex abscisic acid (ABA) -dependent and/or -independent signal transduction pathways and its manifestation at physiological, molecular, and metabolic responses that ultimately elevate the stress tolerance in plants [3].
with their IDs were used to fetch the corresponding genomic, transcriptomic, and coding sequences along with their chromosomal positions were collected from the RGAP (Rice Genome Annotation Project Database) (http://rice.plantbiology.msu.edu/, accessed on 24 December 2020) [56].

Spatio-Temporal and Phytohormone Expression Analysis
OsWRKY genes were exported to the spatio-temporal (RXP_0001) dataset and plant hormone (RXP_1001~RXP_1012) dataset of rice expression profile database (RiceXPro) (http://ricexpro.dna.affrc.go.jp/, accessed on 16 January 2020) [57] for analyzing the spatio-temporal gene expression profile covering different tissues/organs and cell types at various developmental stages, and plant hormone responses, respectively using publicly available microarray.

Gene Features and Phylogenetic Analysis
Gene properties including amino acid length, molecular weight (M.Wt), isoelectric point (pI), instability index, aliphatic index, and grand average hydropathicity (GRAVY) were predicted using the online ExPASy proteomics server (http://web.expasy.org/pro tparam/, accessed on 2 February 2020) [58]. The OsWRKY TF family members and their respective amino acid sequences in other C 4 panicoid sequenced grass species such as foxtail millet (Setaria italica), sorghum (Sorghum bicolor), and maize (Zea mays) were also identified by BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Genes, accessed on 8 February 2020) analysis. The accession numbers of newly predicted WRKY TFs family members from C 4 grass species were assigned as SiWRKY (S. italica), SbWRKY (S. bicolor), ZmWRKY (Z. mays) TF family members and their identity score values were tabulated (Supplementary Table S1). The predicted WRKY TF gene sequences confirmed the presence of WRKY DNA-binding domain and hAT family C-terminal dimerization domain by HMMSCAN (Supplementary Table S2). The amino acid sequences of OsWRKY along with SiWRKY, SbWRKY, ZmWRKY were imported into MEGA v7.0 (Philadelphia, PA, USA) [59] and multiple sequence alignment was performed using ClustalW. The parameters used in the alignment were as follows: gap opening: 10.00, and gap extension: 0.10. The alignment file was imported to construct a phylogenetic tree by the maximum-likelihood method and bootstrap analysis was performed with 1000 replicates.

Gene Structure Analysis and Gene Ontology Annotation
Understanding the gene organization will aid to reveal the function, regulation, and evolution of genes. Arrangements of exons and introns were predicted by comparing the coding sequences with their genomic sequences using Gene Structure Display Server (GSDS) v2.0, a web-based bioinformatics tool (http://gsds.cbi.pku.edu.cn/, accessed on 16 February 2021) [60]. Potential candidate genes and their corresponding RAP IDs were subjected to the ShinyGO v0.61 database (http://bioinformatics.sdstate.edu/go/, accessed on 7 March 2021) to obtain gene ontology (GO) annotation against O. sativa subsp. japonica. GO enrichment was calculated by the p-value cut-off (FDR) at 0.01 for the genes.

Molecular Interactome and Enrichment Analysis
Protein-Protein Interaction (PPI) analysis was performed using STRING v11.0 (https: //string-db.org/, accessed on 20 March 2021) [61] with a high confidence score of 0.7. The functional enrichment analysis of the interactome was done through the level of 0.01. Active interaction based on the various sources, including text mining, experiments, gene fusion, databases, and co-expression, and an interaction score > 0.4 were applied to construct the PPI network. This interactome map was used to identify the physical and functional role of the key candidate genes involved.

Gene Synteny Analysis
In order to unravel the genomic distribution of WRKY TFs, comparative mapping/gene synteny analysis was performed. OsWRKY and their orthologous genes in C4 grasses such as S. italica, S. bicolor, and Z. mays were identified by Gramene-BLASTP (reciprocal) analysis of the gene sequences against these panicoid genomes. All orthologous gene sequences and all hits with E-value 1e−0 (1 × 10 0 ) and minimum 60% similarity were treated as significant. The chromosomal synteny between O. sativa and these C4 grass species was then visualized by Circos v0.55 (Switzerland) [62].

Identification of the WRKY Family Transcription Factors in Rice Genome
The GRASSIUS Grass Regulatory Information Server identified a total of 104 OsWRKY TF family members (Supplementary Table S3). These 104 genes were subjected to RiceXPro for meta-expression analysis (Supplementary Figure S1). Based on the heatmap profiling, 26 OsWRKY TF novel candidate genes were identified and these players are significantly involved in individual and CAbS responses (Table 1). Notably, these genes were localized in all the rice chromosomes except the 10th chromosome, which revealed these players divergence of chromosomes in the rice genome.

OsWRKY TF Genes with Their Properties
The candidate genes and their properties such as amino acid length, M. Wt, pI, aliphatic index, instability index, GRAVY, and subcellular localization of the OsWRKY were analyzed and are given in Table 2. Among the 26 OsWRKY genes, OsWRKY28 was the smallest gene with 190 amino acids whereas OsWRKY74 was the largest one with 862 amino acids. The pI ranged from 9.48 (OsWRKY23) to 10.06 (OsWRKY1) and the molecular weight of the genes also varied according to gene size ranging from 20.49 kDa (OsWRKY28) to 97.37 kDa (OsWRKY74) ( Table 2). The variation in the physiochemical properties of genes deciphered the presence of putative novel variants. Notably, many of the players were localized in the nucleus and it revealed that these OsWRKY TFs were involved in gene transcriptional and several biosynthesis processes.

OsWRKY TF Genes with Their Properties
The candidate genes and their properties such as amino acid length, M. Wt, pI, aliphatic index, instability index, GRAVY, and subcellular localization of the OsWRKY were analyzed and are given in Table 2. Among the 26 OsWRKY genes, OsWRKY28 was the smallest gene with 190 amino acids whereas OsWRKY74 was the largest one with 862 amino acids. The pI ranged from 9.48 (OsWRKY23) to 10.06 (OsWRKY1) and the molecular weight of the genes also varied according to gene size ranging from 20.49 kDa (OsWRKY28) to 97.37 kDa (OsWRKY74) ( Table 2). The variation in the physiochemical properties of genes deciphered the presence of putative novel variants. Notably, many of the players were localized in the nucleus and it revealed that these OsWRKY TFs were involved in gene transcriptional and several biosynthesis processes.

Phylogenetic Analysis of WRKY TFs
Retrieved WRKY amino acid sequences were imported into MEGA v7.0 software (Philadelphia, PA, USA) and the unrooted phylogenetic tree was constructed by the maximum-likelihood method to study the evolutionary organization of the potential 26 WRKY TF family members (Figure 4). The unrooted tree confirmed the homology between the OsWRKY TF family members with SiWRKY, SbWRKY, and ZmWRKY using phylogenetic tree analysis.

Gene Organization Analysis
Gene structure analysis revealed the number and distribution of exons and introns in the OsWRKY TF genes ( Figure 5). The distribution of introns ranged from one to seven amid exonic sequences which may be due to evolutionary changes that have occurred in the OsWRKY TF family members. The majority of the genes contained two introns, whereas six genes (OsWRKY14, OsWRKY23, OsWRKY55, OsWRKY79, OsWRKY83, and OsWRKY94) had only one intron. A maximum of seven introns was found to be present in OsWRKY1 ( Figure 5).

Functional GO Analysis of OsWRKY TFs
OsWRKY TF genes and their functional ontology were predicted by the ShinyGO database which showed the involvement of these genes in various biological processes and molecular functions. OsWRKY novel candidate genes were imputed to be involved in stimulus, chemical, regulation of transcription, metabolic and biosynthetic processes ( Figure 6). The significant molecular functions of these candidate genes were encoded for different types of sequence-specific, DNA, heterocyclic and regulatory region binding activities (Figure 7).

Gene Organization Analysis
Gene structure analysis revealed the number and distribution of exons and introns in the OsWRKY TF genes ( Figure 5). The distribution of introns ranged from one to seven amid exonic sequences which may be due to evolutionary changes that have occurred in the OsWRKY TF family members. The majority of the genes contained two introns, whereas six genes (OsWRKY14, OsWRKY23, OsWRKY55, OsWRKY79, OsWRKY83, and OsWRKY94) had only one intron. A maximum of seven introns was found to be present in OsWRKY1 ( Figure 5).

Functional GO Analysis of OsWRKY TFs
OsWRKY TF genes and their functional ontology were predicted by the ShinyGO database which showed the involvement of these genes in various biological processes and molecular functions. OsWRKY novel candidate genes were imputed to be involved database which showed the involvement of these genes in various biological processes and molecular functions. OsWRKY novel candidate genes were imputed to be involved in stimulus, chemical, regulation of transcription, metabolic and biosynthetic processes ( Figure 6). The significant molecular functions of these candidate genes were encoded for different types of sequence-specific, DNA, heterocyclic and regulatory region binding activities (Figure 7).

OsWRKY Gene Interaction Network Analysis
Potential 26 WRKY TF family encoding genes were obtained from O. sativa ssp. Japonica AbSR OSWRKY TF genes and molecular interaction network was analyzed using the STRING v11.0 database. The gene network had 46 nodes, 74 weighted edges, and an enrichment p-value score <0.01 (Figure 8). The average nodal degree between the neighboring genes was 3.22. This interaction network revealed the complexity of AbSR OsWRKY, hence it proved the nature of multi-gene.

OsWRKY Gene Interaction Network Analysis
Potential 26 WRKY TF family encoding genes were obtained from O. sativa ssp. Japonica AbSR OSWRKY TF genes and molecular interaction network was analyzed using the STRING v11.0 database. The gene network had 46 nodes, 74 weighted edges, and an enrichment p-value score <0.01 (Figure 8). The average nodal degree between the neighboring genes was 3.22. This interaction network revealed the complexity of AbSR OsWRKY, hence it proved the nature of multi-gene.

Orthologous Relationships of OsWRKY Genes
Gramene-BLASTP analysis revealed chromosomal collinearity among 26 potential OsWRKY genes with those of C4 panicoid grass species such as S. italica, S. bicolour, and Z. mays. The chromosomal ideogram exhibited the maximum relationship that occurred between O. sativa and C4 grass plant species [26 OsWRKY

Orthologous Relationships of OsWRKY Genes
Gramene-BLASTP analysis revealed chromosomal collinearity among 26 potential OsWRKY genes with those of C4 panicoid grass species such as S. italica, S. bicolour, and Z. mays. The chromosomal ideogram exhibited the maximum relationship that occurred between O. sativa and C4 grass plant species [26 OsWRKY

Discussion
WRKY TFs are a class of DNA-binding proteins that play a major role in physiological processes, plant growth and development, signal transduction, senescence, seed dormancy, responses to a diverse biotic and AbS, biosynthesis and hormonal regulations and stress signaling through auto-and cross-regulation [63][64][65]. Compared to biotic stresses, so far, only limited information is available on the WRKY TF family members' role in AbS. Considering the importance of WRKY TF genes from various plant species and their crucial roles under various environmental conditions, it remains a big challenge to unveil their mechanisms in AbS. The functions of WRKY TF genes in defense signal transduction pathways came from the analysis of dicot plants, such as tomato, Arabidopsis, potato, and tobacco, and to date, less information was reported in rice and other monocot plants.
Few studies have demonstrated that many WRKY genes are predominantly expressed in response to AbS such as cold, salinity, drought, flooding and submergence [66,67], extreme levels of light (high and low), sugar starvation [63], phosphate deprivation [68], radiation (UV-B and UV-A) [69], and wounding [70]. However, the mode of action of WRKY TFs among the diverse signaling pathways and self-regulation is still not clearly understood.
The regulation and fine-tuning of WRKY TFs during plant stress responses contribute to the establishment of complex signaling networks and their crucial roles in plant AbS responses that make them potential candidates for imparting stress tolerance. More than 100 WRKY genes were predicted in rice [71]. They are upregulated in response to salinity, drought, and ABA, and downregulated in response to cold [71,72]. However, out of the extensive list of rice WRKY TFs studied, only a few genes have been functionally characterized with their response to AbS. The overexpression of OsWRKY genes, known to increase the sensitivity to cold and salt stresses [73], involved in the ABA signaling [12], enhanced drought and heat tolerance after heat pre-treatment as compared to wild-type plants [74]. When OsWRKY genes were over-expressed in Arabidopsis, besides an improvement of lateral root number and primary root length in the transgenic plants under osmotic stress, no clear phenotype regarding survival under AbS was shown [48,75] and increased sensitivity to ABA, salt, and osmotic stress [63]. The overexpression of OsWRKY TFs has induced higher sensitivity or enhanced stress tolerance, thus acting as both positive and negative regulators in stress signaling pathways [76].
This study is the foremost one to report an integrated genome-transcriptome-wide systematic analysis in C 3 model plant rice and also C 4 grass species. A deeper view of its importance in rice stress and systems biology particularly on AbS was investigated to identify and annotate the key players by computational omics approaches and examine their orthologs, differential expression signatures of spatio-temporal and plant hormones levels, interactome map, and molecular properties of the WRKY TFs in response to AbS. The systematic analysis provides insights for the molecular basis of WRKY TFs in O. sativa to stress responses, notably on plant AbS tolerance.
Based on the publicly available RNA-seq transcriptome data of OsWRKY genes, 26 out of 104 OsWRKY TFs showed a high and low level of hormonal expression. This expression signature data revealed that 26 potential OsWRKY genes for phytohormones such as ABA, JA, auxin, GA, CK, and BRs in the root and shoot of the rice plant at various time points. Thus, the obtained results revealed the lower expression of auxin and JA under field conditions. On the other hand, under a stressful environment, these two hormones are expressed in elevated levels and they play an important role in biotic and AbS conditions. From the heat map analysis, 26 potentially expressed AbSR OsWRKY TF genes were used for further functional analysis.
Spatio-temporal expression profiling of 26 OsWRKY genes showed the differential expression in 48 different tissue/organ-specific and developmental stages at individual abiotic stress conferring the higher expression level of OsWRKY genes from various tissuespecific and organs-specific expression dynamism under field conditions. This analysis revealed that OsWRKY TFs could be potential candidates for further functional characterization and distinct expression patterns for explaining their roles in AbS signaling. Further, this data provides the support for conducting overexpression studies and metabolic engineering in different plant tissues in order to increase the number of AbSR gene content and also to enhance the nutrition content in rice. Moreover, the properties of the genes have many differences in M.Wt, amino acid length, aliphatic index, and pI of these genes which contain novel variants, which need to be delineated further for validation.
A phylogenetic tree of OsWRKY and WRKY TFs from C4 plants such as SiWRKY, SbWRKY, and ZmWRKY was constructed in accordance with the multiple sequence alignment of their corresponding WRKY domains. The position of WRKY DNA-binding domain (WRKY) and hAT family C-terminal dimerization domain (Dimer_Tnp_hAT) in OsWRKY, SiWRKY, SbWRKY, and ZmWRKY have been analyzed by HMMSCAN. This unrooted tree showed the distribution and divergence of WRKY domains and conserved regions present in candidate genes. Phylogenetic analysis showed that the predicted gene sequences highly diverge to S. italica, S. bicolor, and Z. mays. OsWRKY and their respective molecular cross-talks and functional relationships unraveled the complexity of unique and combined abiotic stress upon evolutionary seed gene modules and their connecting nodes, edges, and genes that were expressed in AbS studies [4,77].
Comparative mapping of WRKY and their respective genes on rice, sorghum, maize, and foxtail millet were performed to unveil the collinearity between the rice and C 4 grass species. OsWRKY showed maximum orthology of genes with S. italica, Z. mays, and S. bicolor owing to their wide range of chromosomal synteny. This analysis clearly shows that OsWRKY genes are highly similar to SbWRKY, SiWRKY, and ZmWRKY TFs and this close evolutionary relationship revealed the putative novel variants about C 3 and C 4 model crop plants, particularly grass species. This gene synteny information could pave the way for understanding the molecular evolutionary analysis and also could be used to conduct the over-expression and molecular breeding studies of OsWRKY genes among Poaceae members.
Comparing with rice (104 WRKYs), Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) contains a proportionate number of WRKY genes (72 AtWRKYs and 171 TaWRKYs) [78,79], among which a certain number of genes play an important role in AbS. Drought is one of the most common AbS, that has a severe impact on crop growth and yield [80]. Rice OsWRKY11 and OsWRKY72 play an important role in drought tolerance [81,82] in analogous with Arabidopsis genes such as AtWRKY57, AtWRKY63 [83,84], and wheat genes TaWRKY14, TaWRKY90, TaWRKY8, TaWRKY122, and TaWRKY45 [85]. In addition, 12 TaWRKYs were recognized as the candidate drought-responsive genes, which are orthologous to genes in Arabidopsis and enhances during water deprivation [78]. Thus, the characterization of WRKYs in rice will help to unravels the AbS associated regulatory networks.

Conclusions
In this study, we have identified 26 OsWRKY genes that are responsible for various AbS via the computational systems biology approach. The gene properties, evolutionary analysis, gene structure, gene ontology annotation, and gene interaction networks of OsWRKY were evaluated. OsWRKY TFs and their spatio-temporal and phytohormonal expression of these candidate genes showed their differential expression signatures in various rice plant tissues and plant growth hormones, respectively. In addition to that, comparative mapping analysis exhibited that the maximum similarity with C 4 grass species such as S. italica, S. bicolor, and Z. mays. Thus, provides an important indication of their regulatory functions in AbS stress conditions. This study also provides depth information about OsWRKY TF genes and delineates the plant developmental metabolisms and their functional regulations under AbS conditions. This holistic study also hypothesizes that the identified candidate players may interact with various stress responsible TF family members and activates the transcriptional regulation, antioxidant enzymes, ROS scavenging mechanisms, biosynthesis of amino acids, cellular and physiological processes, and synthesis of polyamines in response to AbS tolerance. Further functional analyses are needed to unravel the novel avenues of the identified players.