Phylogenetic analysis and drought-responsive expression profiles of the WRKY transcription factor family in maize

Accepted Manuscript Phylogenetic analysis and drought-responsive expression profiles of the WRKY transcription factor family in maize

Ting Zhang, Dengfeng Tan, Li Zhang, Xiaoyan Zhang, Zhaoxue Han PII: DOI: Reference:

S2352-2151(17)30001-6 doi: 10.1016/j.aggene.2017.01.001 AGGENE 39

To appear in: Received date: Revised date: Accepted date:

11 August 2016 11 January 2017 23 January 2017

Please cite this article as: Ting Zhang, Dengfeng Tan, Li Zhang, Xiaoyan Zhang, Zhaoxue Han , Phylogenetic analysis and drought-responsive expression profiles of the WRKY transcription factor family in maize. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aggene(2017), doi: 10.1016/j.aggene.2017.01.001

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ACCEPTED MANUSCRIPT Phylogenetic analysis and drought-responsive expression profiles of the WRKY transcription factor family in maize Ting Zhang1,3#, Dengfeng Tan1,3#, Li Zhang2, Xiaoyan Zhang3*, Zhaoxue Han1,2*

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State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F

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University, Yangling, Shaanxi, China 712100 Department of Biochemistry & Molecular Biology, College of Life Sciences,

Department of Agronomy, College of Agronomy, Northwest A&F University,

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Northwest A&F University, Yangling, Shaanxi, China 712100

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Yangling, Shaanxi, China 712100

These authors contributed equally to the paper as first authors.

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*To whom correspondence should be addressed:

Xiaoyan Zhang, E-mail: [email protected]

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Zhaoxue Han, E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract: WRKY transcription factors play diverse roles in biotic and abiotic stresses. However, little comprehensive study has been presented about maize WRKY genes in drought stress response. In the present study, the phylogenetic relationships between ZmWRKYs and known WRKYs were analyzed, and it was shown that the gene structure and motif compositions were conserved within a group or a subgroup

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identified. And then, the expression profiling of ZmWRKY genes based on the global microarray data revealed eight genes responded to drought stress. Additionally,

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RNA-Seq profiling showed that 58 ZmWRKY genes were induced in drought stress. Real-time quantitative RT-PCR was used to verify the expression patterns of several

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candidate drought-responsive ZmWRKY genes. The cis-elements analysis of ten candidate ZmWRKY genes showed that the putative promoter of each gene includes at

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least a drought-responsive MBS element. Furthermore, the protein-protein interaction analyses revealed the intricate co-regulatory and co-expression network, which was

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consistent with the drought-responsive expression profiles of ZmWRKYs. Thus, these

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results provide a fundamental clue for cloning functional maize WRKY genes.

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Keywords: Maize; WRKY Transcription factor; Drought stress; Expression profiles;

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Protein interaction analyses

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ACCEPTED MANUSCRIPT

1 Introduction Plants have evolved intricate mechanisms to cope with various adverse conditions, such as drought, high salinity, and extreme temperature (Chen et al., 2012). At the molecular level, the induction of stress-related genes contributes to the plant’s ability to adapt to unfavorable environmental factors, and numerous transcriptional

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regulatory networks are activated in this process. Among these networks, stress-responsive transcription factors (TFs), such as ERF/AP2, bZIP, MYB, MYC,

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HSF, NAC and WRKY, are key transcriptional regulators that act as activators or

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repressors in altering plant stress responses (Golldack et al., 2011).

The WRKY family is one of the ten largest TF families and exists exclusively in

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plants (Rushton et al., 2010). The first WRKY cDNA was cloned from sweet potato (designated SWEET POTATO FACTORS, SPF1) (Ishiguro and Nakamura, 1994).

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Similar proteins were subsequently found in several other plant species (Eulgem et al., 2000). The common feature of these proteins is that they all contain a conservative

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WRKY domain at their N-terminal end, and it has approximately 60 amino acid

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residues with highly conserved amino acid sequences (WRKYGQK), together with a zinc-finger motif. The zinc-finger structure is either Cx4–5Cx22–23HxH or Cx7Cx23HxC, which could integrate with Zn2+ to facilitate the process of binding DNA (Rushton et

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al., 2010). In addition, the WRKY proteins still contain the following structures: putative basic nuclear localization signals, leucine zippers, serine-threonine-rich glutamine-rich

region,

proline-rich

region,

kinase

domains

and

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region,

TIR-NBS-LRRs (Chen et al., 2012). These complete structures make WRKY proteins participate in various regulatory pathways in plants. WRKY proteins play pivotal roles in various stress responses and developmental processes in plants (Ryu et al., 2006; Golldack et al., 2011; Yu et al., 2012). Previous studies have mainly concentrated on the roles of WRKY TFs in defense responses or developmental processes in maize. For example, Wei et al. (Wei et al., 2012) mainly focused on the spatial and temporal expression patterns of 116 maize WRKY genes in developmental regulation and defense response through microarray-based expression

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ACCEPTED MANUSCRIPT analysis in maize. Fountain et al. (Fountain et al., 2013) reported that six WRKY genes were involved in resistance to A. flavus infection in maize. Recently, substantial progress about WRKY proteins in plant processes such as germination, senescence, and responses to various abiotic stresses such as drought and cold has been achieved (Rushton et al., 2010). For example, it was reported that the

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overexpression of three GmWRKYs, TaWRKY2, TaWRKY19, and ZmWRKY33 conferred tolerance to abiotic stresses including drought or salt in transgenic

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Arabidopsis plants (Zhou et al., 2008; Niu et al., 2012; Li et al., 2013). Chen et al. (Chen et al., 2010) reported that three Arabidopsis WRKY TFs form a highly

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interacting regulatory network that modulated gene expression in both plant defense and abiotic stress responses. In addition, the activated expression of WRKY57

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improves drought tolerance by elevation of ABA levels in the Arabidopsis gain-of-function mutant (Jiang et al., 2012). Likewise, transgenic tobacco plants

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overexpressing TaWRKY10 or GhWRKY68 exhibits enhanced and reduced tolerance, respectively, to drought and salt stresses compared with controls (Wang et al., 2013;

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Liu et al., 2015). Overexpression of ZmWRKY58 (named ZmWRKY61 in our study)

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enhances drought tolerance in transgenic rice (Cai et al., 2014). However, our understanding of the functions of the majority of the WRKY family members and their roles in signaling crosstalk is still limited, especially in

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maize. There is little functional analysis about maize WRKY genes related to abiotic stresses. Additionally, although more than 100 members of the maize WRKY family

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have been proposed (Wei et al., 2012), the complete list of maize drought-responsive WRKY genes has not been explored. So the basic information about maize WRKY family remains to be discovered. With the utilization of high-throughput sequencing techniques, RNA-Seq has been an effective method to investigate gene expression patterns and identify candidate genes (Bhargava et al., 2013). In the present study, we analyzed the phylogenetic relationships between ZmWRKYs and known functional WRKY proteins, and characterized the expression profiles of the maize drought-responsive WRKY gene family based on RNA-Seq data and publicly available microarray data.

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ACCEPTED MANUSCRIPT Additionally, we investigated the stress-responsive cis-acting elements and protein interactions of these ZmWRKYs. Finally, real-time quantitative RT-PCR was used to verify the expression patterns of candidate drought-responsive ZmWRKY genes. These results can be used for further functional validation studies of the maize WRKY genes and increase our understanding of the roles of plant WRKYs.

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2 Materials and Methods

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2.1 Plant growth, drought treatment, and tissue collection

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Maize (Zea mays cv B73) seeds were soaked in deionized water for 12 hours and then placed on a sheet of moist filter paper in a Petri dish and germinated at 28℃ for

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three days. Germinated seeds were transferred to pots (inside diameter=25cm, depth=20cm) containing clay under a controlled growth chamber (28℃ day/26℃

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night, 16 h light /8 h dark photoperiod, 30–50% relative humidity). The soil field moisture capacity of clay was 26.2% in field condition. The seedlings were daily ensured a normal water supply with 80% of soil field moisture capacity for about two

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weeks. Then three-leaf seedlings were subjected to light, moderate and severe drought

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stress by withholding water, and the soil relative water contents were 60%, 45% and 30% of soil field moisture capacity, respectively. 80% of field moisture capacity was

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a control test. After drought stress, the seedlings were re-watered. The third leaves of the control, stressed, and re-watered seedlings were harvested in three biological

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replicates, respectively, immediately frozen in liquid nitrogen, and stored at -70℃ for RNA extraction.

2.2 Identification of WRKY genes in maize The latest publicly available maize genome sequences were downloaded from phytozome10.2 (http://www.phytozome.net) (Schnable et al., 2009). Putative maize WRKY genes were identified using BLASTP (Altschul et al., 1997) and HMMER (http://hmmer.org/) programs based on the conserved DNA-binding WRKY domain (Pfam: PF03106) obtained from Pfam database (http://pfam.xfam.org) (Finn et al., 2015). First, the local BLASTP search with a threshold of 1E-5 was used for initial

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ACCEPTED MANUSCRIPT identification of the homologous WRKY genes in the maize genome. Then the raw HMM profile was downloaded by searching the WRKY family in the Pfam database, and the WRKY proteins were extracted from the protein database by hmmsearch with an E-value threshold of 1E-5. Finally, The Pfam was used to verify the presence of complete WRKY domain in their protein structure.

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2.3 Phylogenetic analysis, motifs discovery, and gene structure Multiple sequence alignment of putative ZmWRKY domains and AtWRKY

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domains was performed by using the MAFFT program with default parameters

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(Katoh and Standley, 2013). The unrooted tree was constructed by using MEGA v6.0 (Tamura et al., 2013) with the neighbor-joining (NJ) method. The p-distance model

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and complete deletion mode were used to produce the result. The stability of internal nodes was assessed by bootstrap analysis with 1,000 replicates.

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All non-redundant candidate maize WRKY proteins were surveyed to further verify whether they contained conserved motifs using the online Multiple Expectation

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maximization for Motif Elicitation (MEME) v4.9 program (http://meme.sdsc.edu;

motifs

were

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maximum number of motifs=10; expected value<1E-4) (Bailey et al., 2015). The annotated

according

to

InterProScan

(http://www.ebi.ac.uk/Tools/pfa/iprscan).

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Gene structures of the WRKYs were analyzed on the Gene Structure Display Server 2.0 (GSDS, http://gsds.cbi.pku.edu.cn) by aligning the full-length cDNA with

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their corresponding genomic DNA sequences (Hu et al., 2015). 2.4 Chromosome mapping and gene duplications Both tandem duplication and chromosomal segmental duplication contribute to gene family expansion in plants (Shiu and Bleecker, 2003). The chromosomal location information of ZmWRKY genes was obtained from the B73 gene 5b+ annotation file (v3) and converted by the Ensemble Assembly Converter (http://ensembl.gramene.org) online tool. The segment duplication data of maize (v2) was downloaded from SYMAP Synteny Browser (Soderlund et al., 2011). The

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ACCEPTED MANUSCRIPT distribution of the ZmWRKY genes on the ten maize chromosomes was visualized by using Circos (Krzywinski et al., 2009). 2.5 RNA extraction and qRT-PCR analysis Total RNA was extracted from plants using TRIZOL reagent (Invitrogen, California, USA). The DNase treatment for total RNA and the synthesis of the

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corresponding cDNA were performed following the manufacturer’s protocols (TaKaRa, Dalian, China). The gene-specific primers of ZmWRKY genes for qRT-PCR

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were designed with the Primer Premier 5 software (Table S1). The qRT-PCR

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reactions were performed in a final volume of 20 μL following the manufacturer’s protocol (Roche, Shanghai, China) on the CFX96 cycler (Bio-Rad, California, USA).

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The amplifying conditions consisted of two minutes at 95℃ followed by 40 cycles of 95℃ for 10 seconds, 58℃ for 20 seconds and 72℃ for 30 seconds. The maize

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GADPH gene (Accession number X07156.1) was used as internal control. Amplification specificity was verified with a heat dissociation protocol (melting

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curves in 65–95℃ range) in the final step of PCR. All primer pairs showed a single

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peak on the melting curve, and a single band of the expected size was visualized after separation by agarose gel electrophoresis. The expression data were calculated using the formula (Pfaffl, 2001):

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Ratio = (Etarget)ΔCptarget(control-treatment)/ (Eref)ΔCpref(control-treatment)

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2.6 Stress-responsive expression profiles of ZmWRKY genes in maize Maize microarray data for the Affymetrix Maize Genome Array about drought stress were extracted from Gene Expression Omnibus (GEO accession: GSE16567) (Zheng et al., 2010). The hierarchical clustering with Euclidean Distance were conducted with GENEVESTIGATOR V3 (https://www.genevestigator.com/gv) (Hruz et al., 2008). List of ZmWRKY genes and their probe IDs derived from the databases integrated in GENEVESTIGATOR V3 were shown in Table S3. In addition, the expression of ZmWRKYs was also profiled using RNA-Seq data. The data about dehydration stress memory genes in maize (GEO accession:GSE48507)

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ACCEPTED MANUSCRIPT (Ding et al., 2014) and the effect in maize reproductive and leaf meristem tissue under drought stress (GEO accession:GSE40070) (Kakumanu et al., 2012) were analyzed for differential expression patterns of ZmWRKY genes using Genevestigator. The drought-responsive expression pattern analysis of ZmWRKY genes also used the high-throughput RNA-sequencing data in our lab. Total RNA from two maize

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samples, a control sample and a severe drought stress sample by withholding water, were extracted for sequencing using Illumina® HiSeq 2000 (Beijing Genomics

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Institute, Shenzhen, Guangdong, China). The Reads/Kb/Million (RPKM) normalized data for drought stress-responsive ZmWRKYs transcripts are available. The RPKM

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putative functional genes could be selected.

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normalized read count data of expressed genes were log2-transformed, and the

2.7 Identification of stress-responsive cis-regulatory elements in the putative

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promoter region of ZmWRKYs

The 1.5 kb region upstream of the annotated transcription start site for candidate

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genes was evaluated for putative cis-regulatory elements. Based on comparison of the

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genomic DNA with CDS sequences of ZmWRKYs from maize 5b+ database, the putative promoter sequences were retrieved from B73 genome using Perl scripts. Promoter sequences that included many N residues were excluded from the analysis.

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The cis-regulatory elements were identified using the Plantcare program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002).

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2.8 Protein-Protein Interaction analyses To

better

understand

the

differentially expressed

WRKY

genes,

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Protein-Protein Interaction networks data from the STRING database (Search Tool for the Retrieval of Interacting Genes/Proteins, http://string-db.org/) were used to detect the relationships among different WRKY genes. This database lists protein associations based on multiple sources: known experimental evidence from primary databases; pathway knowledge parsed from databases; text-mining; and using genomic information (Szklarczyk et al., 2014). There are confidence scores from

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ACCEPTED MANUSCRIPT medium (score above 0.4) to highest (above 0.9) to assess the interactions based on the evidence. 3 Results 3.1 Identification of putative WRKY-encoding genes in maize

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We first used the known AtWRKY protein sequence (AT2G30250) (http://www.arabidopsis.org/) to search the consensus of the HMM of WRKY domain

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at the Pfam database. The local BLASTP between the consensus of WRKY domain

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sequence and maize protein sequences was run, resulting in 155 ZmWRKY protein sequences. Then, the hidden Markov model-based profile (HMM-profile) of WRKY

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domain from the Pfam database was used to search the possible ZmWRKY proteins with HMMsearch program, and 166 sequences were selected. Based on the above

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results, we removed redundant protein sequences, and the WRKY domain of each predicated ZmWRKY protein was verified by searching against the PFAM database. Finally, we identified 120 putative WRKY gene loci that may encode WRKY proteins. All

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the identified members in this report were named according to their sequential

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locations on the chromosomes (Fig. 1, Table S2). In our study, most WRKY domains contained the highly conserved WRKYGQK

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sequences. Interestingly, 14 members showed several mismatched amino acids in their conserved signature and elongate domains. For example, in ZmWRKY27, 41, 69,

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74, 86, 91, and 97, the WRKY domain had the sequence WRKYGKK; in ZmWRKY7, 60 WKKYGQK; and in ZmWRKY1, 19, 53, 55, and 94, WRKYGEK. In addition, in ZmWRKY18, 54, 57, and 119 proteins, the complete WRKY domains had the length of 75, 85, 92, 100 amino acid residues, respectively, which were longer than the common WRKY domain that has 60 residues. 3.2 Chromosomal distribution and gene duplication of ZmWRKY genes The 120 ZmWRKY genes are dispersedly distributed across all chromosomes in maize (Fig. 1). Every chromosome contains at least ten ZmWRKYs except chromosomes 5, 7, 9. Chromosomes 3 and 8 show the most intensive physical

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ACCEPTED MANUSCRIPT distribution of ZmWRKY genes. Gene duplication events are important in the rapid expansion and evolution of gene families (Cannon et al., 2004). We investigated the genome duplications of ZmWRKYs across the genome. Fine mapping analysis in this study revealed eight pairs of ZmWRKY genes that located within 100 kb of each other, probably arising

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through tandem duplications. Three gene pairs were found on chromosome 3 and 8 (Fig. 1, shown in red). Then, we investigated segmentally duplicated blocks across the

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whole maize B73 genome. The result showed that there were 45 segmental duplicated blocks that contain WRKY homologs in the maize genome, and these duplication

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events were distributed across all chromosomes, but the chromosome 8 had the largest

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segmental duplicated blocks (Fig. 1).

3.3 Phylogenetic tree, encoded amino acid motif, and gene structure of ZmWRKY

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genes

The phylogenetic relationship of the WRKY proteins was examined by NJ

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method with aligned sequences of ZmWRKY domains and several AtWRKY

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domains which represent different groups and subgroups (Fig.S1, Fig.2A). Based on the AtWRKY classification (Wu et al., 2005), ZmWRKYs were also classified into three major groups, and several subgroups were clearly formed on the basis of the

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phylogenetic analysis (Fig. 2A). Among the three groups, there were 15 ZmWRKYs members in group I, 75 in group II, and 30 in group III (Table S2). The sequence

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alignment figure (Fig. S1) was generated utilizing ESPrit 3.0 (Robert and Gouet, 2014). Furthermore, amino acid motifs of ZmWRKY proteins and gene structure of ZmWRKY genes were analyzed and their schematic diagrams were shown together (Fig. 2B, 2C). A total of ten conserved motifs in ZmWRKYs were found using MEME software and annotated by online InterProScan. As shown in Fig. 2B, the amino acid motifs were evidently different among the groups. There were five WRKY DNA-binding motifs which constitute the WRKY domain. All ZmWRKY proteins contained at least one of them. Moreover, groupⅢ, group Ⅱd/Ⅱe, group Ⅱb/Ⅱa, and groupⅠ/Ⅱc had their unique motifs. There were common motif compositions

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ACCEPTED MANUSCRIPT between the subgroups Ⅱa and Ⅱb, containing the coiled-coil domain. The subgroups Ⅱd and Ⅱe had the Plant zinc cluster domain, indicating these subgroups have a close phylogenetic relationship. It was noteworthy that group I and IIc both contained three similar motifs, and group I also had other different motifs. The exon/intron analysis showed clear differences in both exon positions and

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exon numbers across the different groups or subgroups (Fig. 2C). Most of the ZmWRKY genes in the same groups or subgroups had relatively conserved exon

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numbers. For example, members of group II d had three exons except for two genes, and 24 of the 28 genes in group III also had three exons. In addition, exon positions

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were also relatively conserved within a group or a subgroup. Notably, in addition to their differences in exons, the introns in different groups or subgroups also varied

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significantly. Most WRKY genes had two introns. The intron in conserved WRKY domain could be classified as the R-type intron and V-type intron. The R-type intron,

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a phase-2 intron, is spliced exactly at the R residue, while the V-type intron, a phase-0 intron, is localized before the V residue, which is at the sixth amino acid after the

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second Cys residue in the Cys2His2 motif in the zinc finger region (Wu et al., 2005).

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In this study, the phase-0 intron was only observed in subgroup IIa, IIb, and IC, which was consistent with those in grape (Wang et al., 2014). On the contrary, the phase-2 intron was widely distributed in other subgroups. In addition, there were several

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phase-1 introns in groupⅠand groupⅢ, and four genes had no intron. These results indicate that the ZmWRKYs clustered together in the phylogenetic tree tend to have

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similar intron-exon structure and protein motifs. 3.4 Drought-responsive expression profiling of ZmWRKYs Using maize microarray data (ZM_15k array) based on maize genotypes Han21 (drought-tolerant) and Ye478 (drought-susceptible) in the Genevestigator V3 database, we examined the expression patterns of 26 ZmWRKY genes (Fig. 3A). We found that there were eight ZmWRKY genes with significant responses to drought stress in Han21and Ye478. ZmWRKY53 and ZmWRKY78 were up-regulated only in Ye478 compared with Han21 under moderate drought (MD) and severe drought (SD)

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ACCEPTED MANUSCRIPT conditions. In addition, under SD stress, ZmWRKY75 was up-regulated only in Han21, while ZmWRKY25 and ZmWRKY47 were up-regulated only in Ye478. In contrast, ZmWRKY73 and ZmWRKY113 were obviously down-regulated under MD and SD stresses in the two maize lines, and ZmWRKY40 was down-regulated under MD stress in Han21 and SD stress in Ye478. Moreover, there was a higher amount of expression

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level under SD stresses than that under MD. After re-watering (RW) treatment, all these genes were down-regulated or not responsive, except for ZmWRKY40,

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ZmWRKY59 and ZmWRKY73 in two maize lines (Fig. 3A, Table S3).

According to the available RNA-Seq gene expression data in Genevestigator

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database, thirty-five ZmWRKYs showed the response to drought stress in 14-day-old maize seedlings after air-drying for 2h and three dehydration-rehydration cycles (3×

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2h). Thirteen of 35 ZmWRKYs had similar expression patterns under 1×2h and 3×2h conditions (Fig. 3B). Six of 35 ZmWRKYs, WRKY25, WRKY53, WRKY58, WRKY83,

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WRKY86, and WRKY106, exhibited higher expression levels under 3×2h condition. Additionally, thirty-six differentially expressed ZmWRKY genes were identified in the

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ovary tissue and the basal leaf meristem of three youngest leaves undergone 3 or 4 d

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of withholding irrigation stress (Fig. 3C, Table S4). Compared with leaf base, the caryopsis at maize silking stage under drought stress showed a larger number of differentially expressed genes, which indicates some functional divergence of

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ZmWRKY genes at different development stages. Based on the maize RNA-Seq data (SRA data No. SRP070859) generated in our

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lab, we found that 13 ZmWRKY genes were differentially expressed under SD condition compared with control. All these ZmWRKY genes were up-regulated under severe soil dehydration stress (Table S5). 3.5 The qRT-PCR confirms the expression of candidate ZmWRKYs during dehydration stress Based on phylogenetic analysis among ZmWRKYs and previously reported known drought-responsive WRKY genes (Fig. S3, Table S6), we screened 16 candidate ZmWRKY genes which were distributed in seven subfamilies including known

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ACCEPTED MANUSCRIPT functional WRKY genes at whole-genome level. Then we performed qRT-PCR analysis of candidates with gene-specific primers (Table S1) in shoot tissues of maize plants subjected to soil withholding water treatment. The expression of eight genes was detectable. As shown in Fig. 4, the expression of ZmWRKY20 and ZmWRKY106 are up-regulated more than 2-fold under SD or MD stress, respectively, and

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ZmWRKY97 is up-regulated under MD and SD stresses, which is consistent with those of RNA-Seq data in our lab. By contrast, ZmWRKY9 and ZmWRKY39 are

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down-regulated under light drought (LD) stress and have no response to SD stress, which is different from the RNA-Seq results in our lab. Additionally, ZmWRKY80 and

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ZmWRKY113 are down-regulated under LD, MD and SD stresses. Furthermore,

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ZmWRKY47 don't show any detectable expression.

3.6 Identification of abiotic stress-responsive cis-elements in the promoter

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regions of ZmWRKYs

To understand the molecular mechanism of ZmWRKY genes response to abiotic

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stresses, we selected the ten ZmWRKY genes which were drought-responsive at least

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in three studies to investigate possible drought-responsive cis-elements using the PlantCARE program (Fig. S3). Seven types of cis-elements, including the C-repeat/DRE (cold, salt, and dehydration-responsive element), MBS (MYB binding

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site, involved in drought-inducibility), LTR (low-temperature response), HSE (heat stress response), ARE (anaerobic response), GC-motif (anoxic inducibility), and

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W-box (defense and stress responsiveness and WRKY binding site) were detected (Fig. 5). These results show that each of the ZmWRKY genes contains one or more MBS cis-elements in their promoter region. The ZmWRKY97 and ZmWRKY106 contain one C-repeat/DRE. The results suggest a potential relationship between drought stress-responsive cis-elements and gene expression. 3.7. Protein-Protein Interaction analyses To further explore the interactions among drought-responsive ZmWRKY genes, we generated protein-protein interaction networks of ZmWRKY proteins by using

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ACCEPTED MANUSCRIPT STRING10 program (Fig. 6). A total of 21 ZmWRKY proteins were predicted in the network when the confidence value was set as 0.4. The ZmWRKY proteins also showed comprehensive associations with other proteins. We found that there were two interaction groups of up-regulated and down-regulated proteins, respectively. ZmWRKY62 and ZmWRKY109 were located in the center of the network and

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interacted with 14 up-regulated ZmWRKY proteins, which indicated the two proteins might be the crucial factors and their involvement in common biological

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functions/processes. Additionally, The WRKY39 and ZmWRKY106 proteins were not only phylogenetically closely related to each other but also had up-regulated

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co-expression interaction. By contrast, the ZmWRKY43, ZmWRKY101, and ZmWRKY113 showed strong down-regulated co-expression interactions under

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drought stress. Furthermore, ZmWRKY16 and ZmWRKY118 showed the interaction with MYB transcription factors. We proposed that the two WRKY proteins may form

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functional homo- or heterodimers in drought stress response, which will require validation in vivo test.

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4 Discussion

4.1 Identification of maize drought-responsive ZmWRKY gene family In this study, in order to identify maize drought-responsive ZmWRKY gene

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family, 120 ZmWRKY genes were extracted from the latest B73 5b+ genome. Compared with the 119 ZmWRKY genes previously reported by Wei et al. (2012), we

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identified additional 3 new ZmWRKY genes (GRMZM2G103742, GRMZM5G849918, GRMZM2G452444) and re-annotated one gene (GRMZM2G060918), while GRMZM2G045560 identified by Wei et al. (2012) was not found in the latest B73 5b+ genome. Huang et al. (Huang et al., 2012) indicate that the characterized SlWRKYs with similar functions in stress resistance tend to be phylogenetically close to each other. Therefore, a phylogenetic tree, based on the 120 ZmWRKY proteins and 17 functional WRKY proteins from other species, was constructed (Fig. S3). The result showed the functional WRKY genes responding to drought stress mostly were

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ACCEPTED MANUSCRIPT clustered to the group I, Ⅱc, and Ⅲ. This suggests that the members of the three groups may contain a lot of drought-responsive ZmWRKY genes, and they may also be part of important stress-responsive signaling networks. A previous analysis has shown that group Ⅲ members are most adaptable in the face of external environmental stress among the whole WRKY family (Kalde et al., 2003).

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Then, we integrated the gene expression profiles analysis of ZmWRKYs collected from the microarray, three RNA-Seq studies, and the RT-PCR result. We found that 27

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genes showed a response to drought stress at least in two studies, and ten genes were drought-responsive at least in three studies (Fig. S3B). Additionally, the segmentally genes

ZmWRKY16/118,

ZmWRKY42/102,

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duplicated

ZmWRKY43/101,

ZmWRKY44/100 exhibit the similar response to drought stress (Fig. 3B, 3C).

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It was reported that ZmWRKY33 (ZmWRKY93 named in our study) could be induced by dehydration treatments (Li et al., 2013). In our study, we found that

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ZmWRKY93 was up-regulated under drought stress in two RNA-Seq profiles (Fig. 3B, 3C). Cai et al. (Cai et al., 2014) report that overexpression of ZmWRKY58 (named

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ZmWRKY61 in our study) enhances drought tolerance in transgenic rice. Our results,

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nonetheless, imply that this gene does not respond to drought stress. 4.2 The abiotic stress responsive regulation of ZmWRKY genes

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We selected ten ZmWRKY genes responding to drought stress to scan the cis-elements in their putative promoter regions. Each of the ten ZmWRKY genes

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contained at least one MBS element. Two of ten included C-repeat/DRE element, supporting their roles in involving in drought stress. Many WRKY proteins regulate gene expression by binding the conserved nucleotide consensus sequence (C/T)TGAC(T/C) (named W-box) of target genes (Eulgem et al., 2000). The W-box occurs in the promoter regions of many WRKY genes, which might reflect the functional mechanism. WRKY proteins can form functional homo- or heterodimers among some WRKY proteins to perform functions (Chen et al., 2012). There is a growing body of evidence for extensive WRKY– WRKY protein interactions. In this study, the promoters of ZmWRKY25, ZmWRKY47,

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group Ⅱa is the important component. The known protein-protein interaction network provides important clues to better

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understand the gene expression regulation. ZmWRKY39 and ZmWRKY106 as well as ZmWRKY62 and ZmWRKY109 show up-regulation, where each pair is from the same

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co-expression system (Fig. 6). Cluster analysis revealed that WRKY members belonging to the same group often exhibit similar expression patterns under different

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stress conditions (Zhao et al., 2015). This phenomenon was detected between (II e),

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between

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ZmWRKY109 (II a) in this study (Fig. S3A). In addition, various TFs would work together to control downstream gene expression. ZmWRKY16 and ZmWRKY118

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were predicted to interact with MYB transcription factors, suggesting their potential

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co-expression relationship (Fig. 6). A similar phenomenon is also reported that GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants (Wang et al., 2015).

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In conclusion, the current study characterized the expression patterns and regulatory network of maize WRKY genes under drought conditions. These results

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provide valuable clues about the biological functions of WRKYs in abiotic stress responses. However, more research is needed to determine the functions of the ZmWRKY genes.

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ACCEPTED MANUSCRIPT Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgments

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This work is supported by National Natural Science Foundation of China (No. 31201268), Natural Science Foundation of Shaanxi Province (No. 2011JQ3005), the Research Fund for Doctoral Programs of Higher Education of China (No. 20100204120037), and the Special Fund for Basic Scientific Research of Central College (No. QN2011114).

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Fig. 1 Chromosome location and duplications of maize ZmWRKY genes. Each circular segment represents maize chromosome. A pair of genes located within a 100 kb distance to each other are shown in red. The link lines connecting ZmWRKY genes stand for the segmental duplications of WRKY homologs in maize. The data of segmental duplications of ZmWRKY genes are downloaded from SYMAP Synteny Browser. The circle plot is produced using CIRCOS software.

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Fig. 2 Phylogenetic tree, encoded amino acid motif, and intron-exon structure of ZmWRKY genes. (A) The phylogenetic tree of 120 ZmWRKY genes (N-terminal WRKY domains of Group I are excluded) is constructed by the MEGA 6.0 program with the NJ method. Percentages of bootstrap scores higher than 50% are indicated above the branches. Roman numerals indicate different subfamilies. (B) The schematic diagram of amino acid motifs. The different-colored boxes named at the bottom represent conserved motifs. Gray lines represent the non-conserved sequences, and the position of each WRKY sequence is exhibited proportionally (The domain logo are shown in Fig. S2). (C) The corresponding exon-intron organization. Blue boxes indicate exons and single lines represent introns, and the untranslated regions (UTRs) are indicated by brown boxes and the numbers indicate the phases of the introns. The sizes of exons and introns can be estimated using the scale at the bottom.

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Fig. 3 Expression profiling of ZmWRKY genes in maize. In the figure, (A) Expression profiling of ZmWRKY genes based on microarray data under drought stress. (B) and (C) are shown above the figures for the ZmWRKY gene expression levels (log2-scale) in 14-day-old maize leaf tissues and for the ZmWRKY gene expression levels (log2-scale) in the ovary tissues and the basal leaf meristem of three youngest leaves, respectively.

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Fig. 4 Identification of dehydration-responsive ZmWRKY genes by qRT-PCR. The GADPH gene is used as an internal control. The x-axes are different drought stresses and re-watering. CT: control test; LD: light drought; MD: moderate drought; SD: severe drought; RW1: re-watering/untreated sample; RW2: re-watering/severe drought. The y-axes represent the relative expression. Mean relative expression levels in unstressed maize leaves (CT) are normalized to a value of 1. The standard errors of three technical replicates are shown.

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Fig. 5 Identification of stress-responsive cis-elements in the upstream regions of ZmWRKY genes. The 1500 bp upstream sequences are used to identify their cis-elements by running the PlantCARE program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). C-repeat/DRE: cold, salt, and dehydration responsiveness; MBS: drought inducibility; LTR: low-temperature responsiveness; HSE: heat stress responsiveness; ARE: anaerobic induction; GC-motif: anoxic specific inducibility; W-box: defense and stress responsiveness and WRKY binding site.

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Fig. 6 Interaction analyses of ZmWRKY proteins. The line indicates protein interaction with a confidence score >0.4 revealed by STRING Database v10. Different colors mean different types of evidence for the association: recurring Neighborhood in different genomes (green line), events of Gene Fusion (red), co-occurrence of those genes in the same organisms (dark blue), co-expression (black), experimental protein–protein interaction data (pink), pathway described by other databases (light blue), literature text-mining (yellow), and homology (purple lines) (Szklarczyk et al., 2015). The red triangle represents genes up-regulated, while the green triangle represents genes down-regulated.

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ACCEPTED MANUSCRIPT Abbreviations list

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BLASTP, Protein-Protein Basic Local Alignment Search Tool; GEO, Gene Expression Omnibus; GSDS, Gene Structure Display Server; HMM, Hidden Markov Model; MEME, Multiple Expectation maximization for Motif Elicitation; RT–PCR, real-time fluorescent quantitative polymerase chain reaction; TFs, transcription factors

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ACCEPTED MANUSCRIPT Highlights

1. Uncovering tens of candidate ZmWRKY genes related to drought stress. 2. Some candidate ZmWRKY genes include drought-responsive MBS element.

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3. The co-expression network was consistent with stress-responsive expression profiles.

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