Characterization and analysis of some chilling-response WRKY transcription factors in tomato


 WRKY transcription factors play various important roles in biotic and abiotic stress. In present study, a total of 81 WRKYs in tomato (Solanum lycopersicum) was identified and their gene structure, phylogeny and sub-location were analyzed. Here, we further analyzed their expression and potential roles under chilling stress. Nevertheless, the predicted chloroplast-located WRKYs are failed to be detected in the chloroplast. Then, 27 SlWRKYs with high chilling-induced mRNA levels (more than 3 fold to the control) are selected from these WRKYs. Promoter analysis showed that some cold stress-related cis-acting elements (CBFs binding site) existed in many promoter regions of these chilling response WRKYs (WRKY2, WRKY50, WRKY59 etc.), implying that these WRKY transcription factors may participate in CBFs mediated pathway under chilling stress. The interaction proteins of WRKYs are essential to affect the DNA binding and transcription regulatory activities of WRKYs, thus controlling its downstream genes expression. Therefore, we predicted and analyzed the protein-protein interactions of those chilling related WRKY transcription factors and then speculated the complex regulatory and functional network of WRKY transcription factors under chilling stress. A better understanding of SlWRKYs would be helpful for providing a theoretical basis for further illustrating the regulatory mechanism of SlWRKYs under chilling stress.

Abstract WRKY transcription factors play various important roles in biotic and abiotic stress. In present study, a total of 81 WRKYs in tomato (Solanum lycopersicum) was identi ed and their gene structure, phylogeny and sub-location were analyzed. Here, we further analyzed their expression and potential roles under chilling stress. Nevertheless, the predicted chloroplast-located WRKYs are failed to be detected in the chloroplast. Then, 27 SlWRKYs with high chilling-induced mRNA levels (more than 3 fold to the control) are selected from these WRKYs. Promoter analysis showed that some cold stress-related cis-acting elements (CBFs binding site) existed in many promoter regions of these chilling response WRKYs (WRKY2, WRKY50, WRKY59 etc.), implying that these WRKY transcription factors may participate in CBFs mediated pathway under chilling stress. The interaction proteins of WRKYs are essential to affect the DNA binding and transcription regulatory activities of WRKYs, thus controlling its downstream genes expression. Therefore, we predicted and analyzed the protein-protein interactions of those chilling related WRKY transcription factors and then speculated the complex regulatory and functional network of WRKY transcription factors under chilling stress. A better understanding of SlWRKYs would be helpful for providing a theoretical basis for further illustrating the regulatory mechanism of SlWRKYs under chilling stress.

Background
Chilling stress is a major environmental factor that limits the agricultural productivity and geographical distribution of many plant species (Sanghera et al., 2011). Tomato (Solanum lycopersicum), as a typical warm-season vegetable crops, is very sensitive to chilling stress (0-12 °C) which is common during their growing season. The chilling stress will rigidify the cell membrane, destabilize protein complexes, and impair photosynthesis of tomato (Martin et al., 1981;Ruelland et al., 2009). Under chilling stress, the complex mechanisms have been evolved to improve plant chilling tolerance including multiple signal transduction, transcription factor regulation and protein-protein interactions (Hannah et al., 2015;Chinnusamy et al., 2007;Shi et al., 2018).
As one of the largest transcriptional factors (TFs) families in plants, WRKY TFs with a conserved WRKYGQK motif and a novel zinc-nger-like motif contain more than 70 memebers in Arabidopsis thaliana and S. lycopersicum (Wu et al., 2005;Madhunita et al., 2014;Chen et al., 2015). Based on its number of WRKY domain and structure of zinc-nger motifs, WRKY proteins are initially classi ed into three groups (Group I-III) (Eulgem et al., 2000). Following analyses have shown that Group II WRKY proteins are further divided into ve subgroups (IIa, IIb, IIc, IId, and IIe) based on the primary amino acid sequence (Rushton et al., 2010;Madhunita et al., 2014). The green algae Chlamydomonas reinhardtii, non-photosynthetic slime mold Dictyostelium discoideum, and unicellular protest Giardi alamblia all contain a single Group I WRKY gene, suggesting that Group I WRKY proteins with two WRKY domains are the ancestors to the other groups of WRKY proteins and the WRKYs origin is before the emergence of photosynthetic eukaryotes (Zhang and Wang, 2005). In Arabidopsis, AtWRKY40, a Group IIa member, can be recruited from nucleus to cytosol to interact with a chloroplast envelope and the cytosolic C-terminus spanned magnesium-protoporphyrin IX chelatase H subunit (ABAR), thus playing a negative role in response to ABA signaling (Shang et al., 2010). This suggests that WRKY family may evolve to be involved in maintaining the connection between the chloroplast and nucleus during the long evolutionary process. However, the roles of WRKYs in chloroplasts are unclear.
WRKY transcription factors function as important components in the regulation of transcriptional reprogramming during plant stress responses (Madhunita et al., 2014). Extensive studies show that many WRKYs are responsive to pathogens or pathogen elicitors. Studies using overexpression lines or mutants of WRKYs have shown that WRKYs can positively or negatively regulate the expressions of hormonerelated or pathogen defence genes (Dong et al., 2003;Lai et al., 2008Lai et al., , 2011. In addition, WRKYs are involved not only in biotic stress responses but also in abiotic stress responses and adaptations. In Arabidopsis, AtWRKY25, AtWRKY26, and AtWRKY33 enhance the tolerance to heat stress through regulating the expression of heat-induced genes, such as AtHSPs . Overexpression of GsWRKY20 reduces the stomatal density and water loss e ciency, thus improving plant drought tolerance (Luo et al., 2013). While GhWRKY68 reduces the resistance to salt and drought stress (Jia et al., 2015). Tomato is one of the most sensitive crops to chilling stress. Although the function of many individual WRKY genes have been analyzed in tomato (Table 1, most of them function in responses to biotic stress), the chilling responsive pathway mediated by WRKYs remain unclear. Here, we identi ed 27 chillingresponse WRKY genes by qRT-PCR and RNA-seq assay. Then, the prediction analysis of their functional interaction networks, phosphorylation sites and cis-acting element suggested their potential roles under chilling stress. Gene structure analysis, phylogenetic tree and motif composition of SlWRKYs The gene structures of SlWRKYs were determined by comparing their genomic sequences with predicted coding sequences using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). The conserved motifs analysis of SlWRKY proteins were done by MEME (http://meme-suite.org/tools/meme). In  Table S3.

Results
The phylogenetic analysis, protein and gene structure analysis of WRKY family members of tomato The DNA and protein sequences of 81 SlWRKYs were obtained from PlantTFDB (http://planttfdb.cbi.pku.edu.cn/). The 81 transcripts were named from SlWRKY1 to SlWRKY81 based on their order on the tomato chromosomes. Based on these, phylogenetic tree, conserved protein domains and gene structure analysis were shown in Fig. 1. This analysis showed that SlWRKYs with two WRKY domains (motif1 and motif5) were clustered together except WRKY32 and WRKY8 whose motif5 is not very conservative (Fig. 1). WRKY proteins with two WRKY domains are the ancestors to the WRKY proteins with one WRKY domain (Eulgem et al., 2000;Zhang and Wang, 2005). SlWRKY32 and SlWRKY8 may have speci c roles in terms of evolution. In addtion, according to gene structure analysis of the SlWRKYs, the SlWRKY members with motif 4 and motif 10 have at least two introns besides SlWRKY46 and SlWRKY10, but most of the other members have only two introns (Fig. 1).

The subcellular localization analysis of SlWRKYs
The subcellular localization of each SlWRKY was predicted by LOCALIZER and TargetP 1.1 Server. Previous studies showed that most WRKY family members were targeted to nucleus and cytoplasm (Ruelland et al., 2009;Bakshi et al., 2014). Chloroplast is one of the most sensitive sites to chilling stress in plant cells. Here, we want to identify whether there is some chloroplast-located SlWRKYs. Five SlWRKYs (SlWRKY14, SlWRKY33, SlWRKY49, SlWRKY68, SlWRKY78) were predicted to target in the chloroplast (Fig. 2Table). However, the transient expression assay in Nicotiana benthamiana con rmed that SlWRKY14, SlWRKY33, SlWRKY68, SlWRKY78 were all located to the nucleus ( Fig. 2A) and we failed to detect the subcellular localization of SlWRKY49.

Analysis of putative relationship between these chilling response SlWRKYs and CBFs
In Arabidopsis, C-repeat/DREB binding factors (CBFs) as key transcription factors that function in cold stress (Shi et al., 2018). CBF proteins recognize the CRT/DRE cis-acting element (for example, CCGAC). In addition, WRKY gene have CRT/DRE cis-acting element. Thus, we speculate WRKY gene function as cold response by CBF combining with CRT/DRE cis-acting element of WRKY gene. Most of the promoters of 27 SlWRKYs contain these CBFs binding elements, while SlWRKY6, SlWRKY11, SlWRKY15, SlWRKY27, SlWRKY29, SlWRKY56, SlWRKY60, SlWRKY63, SlWRKY74 do not contain those elements (Table. 2). By contrast, we further analyzed whether the promoter regions of SlCBFs contained WRKY bind element (TTGACC/T, Ciolkowski et al., 2008). The analysis showed that all the three SlCBFs promoter regions process the WRKY binding region suggesting that some SlWRKYs, especially these WRKYs without the CBFs binding elements as described above, were the pontensial upstream regulator of SlCBFs (data was not shown).

Interaction network of these chilling response SlWRKY proteins
Exploring the interaction proteins of chilling response SlWRKYs is important to understand their regulatory function mechanism under chilling stress. Therefore, we constructed an SlWRKY proteins interaction network based on the data of Arabidopsis homologous proteins using STRING 11.0 to systematically analyze the interaction proteins of these chilling response SlWRKYs (Fig. 4). Chi et al., (2013) summarized that WRKY-WRKY, WRKY-VQ and WRKY-MAPK interactions were the most common interaction relationship of WRKY. Their corresponding genes in Arabidopsis were shown in Table. S1. As shown in Fig. 4, SlWRKY21 seems a central factor to interact with a large number of other chilling response WRKYs including SlWRKY6, SlWRKY24, SlWRKY43, SlWRKY49, SlWRKY60 and SlWRKY61, which may play the most important roles under chilling stress. Besides, MPK3, MPK2 and MPKA1 may function as a key complex to interact with a large number of chilling response SlWRKYs directly (SlWRKY21, SlWRKY24, SlWRKY49, SlWRKY50, SlWRKY64, SlWRKY74 and SlWRKY79) and then phosphorylated them to affect their transcription regulatory activities (Fig. 4). The post-translational modi cation analysis showed that all chilling response WRKYs had phosphorylation sites (Table. S3). As expected, these WRKYs, which interact with MPKs as description above, process at least 8 phosphorylation sites, suggesting their interaction relationship (Table. S3). VQ proteins were found to speci cally interact with the WRKY domain of WRKY proteins (Cheng et al., 2012). In this analysis, VQ proteins are also predicted to interact with chilling response SlWRKYs (SlWRKY11, SlWRKY24, SlWRKY49, SlWRKY56, SlWRKY68 and SlWRKY74) (Fig. 4). In addition, many other chilling related transcription factors (like ICE1, ERF, AP2, JAZ1 etc.) were found to interact with chilling response SlWRKYs, which was independent with the interaction as described above (Fig. 4). The interaction network of these chilling response SlWRKYs provides new research ideas for exploring the new chilling related mechanism of tomato in the future.

Page 8/18
Almost all studied WRKY proteins can bind to the core W-box promoter elements of their downstream genes in the nucleus to participate in plant growth, development, and responses to biotic and abiotic stress, respectively (Bakshi et al., 2014). As a nucleus and cytosol dual-located WRKY member, AtWRKY40 could be from nucleus to cytosol to interact with a chloroplast envelope located cytosolic Cterminus spanned magnesium-protoporphyrin IX chelatase H subunit (ABAR) (Shang et al., 2010). This suggests that WRKY family may be involved in maintaining the connection between the chloroplast, cytosol and nucleus. However, the affection of WRKYs to the chloroplast is not directly. In tomato, SlWRKYs were predicted to target to the nucleus, cytosol, chloroplasts and mitochondria . Our result also showed ve candidate chloroplast-located SlWRKYs (Fig. 2). Nevertheless, four of them were con rmed to target to the nucleus (Fig. 2). Only the sub-location of SlWRKY49 remains unclear.
CBF, also known as dehydration responsive element (DRE) binding factor (DREB) proteins, are key factors in plant cold response (Liu et al., 1998;Shi et al., 2018). In rice, WRKY genes expressed highly under cold stress and may be involved in ICE-CBF-COR pathway (Guo et al., 2019). In this study, we mainly identi ed 27 chilling response SlWRKY genes based on RNA-seq and qRT-PCR analysis (Fig. 3). Promoter analysis of SlWRKYs and SlCBFs showed most SlWRKYs may have close regulatory relationship with SlCBFs mediated chilling response pathway. The promoter regions of 18 chilling response SlWRKYs (SlWRKY1, SlWRKY2, SlWRKY8, SlWRKY21, SlWRKY23, SlWRKY24, SlWRKY35, SlWRKY43, SlWRKY45, SlWRKY49, SlWRKY50, SlWRKY59, SlWRKY61, SlWRKY62, SlWRKY64, SlWRKY69, SlWRKY79 and SlWRKY80) all contained the CBFs binding element, suggesting that they may be the directly downstream signaling factors under chilling stress ( Table 2). The SlWRKY6, SlWRKY11, SlWRKY15, SlWRKY27, SlWRKY29, SlWRKY56, SlWRKY60, SlWRKY63, SlWRKY74, which were left behind, may act as upstream regulatory factors of SlCBFs due to the W-box elements in SlCBFs promoters. Meanwhile, we can not fully rule out that these chilling response SlWRKYs function in a CBFs independent pathway under chilling stress.  (Mao et al., 2011). In our analysis, these chilling response SlWRKYs were also predicted to interact with some kinase (MPK3, MPK2 and MPKA1), which may function as a key complex in whole SlWRKYs interaction network under chilling stress in tomato (Fig. 4). In addition, SlWRKY21 seems a central factor due to its interaction with the most chilling response WRKYs. As its Although chilling response WRKYs were studied in several species, the mechanism of how WRKYs respond to chilling stress and the speci c regulatory mechanism to their downstream genes are remain poorly understood. In this study, we identi ed 27 obvious chilling-induced SlWRKYs and analyzed their potential roles and interaction network under chilling stress, which provides an important reference for future studies on their biological functions under chilling stress in tomato.

Declarations
Availability of data and material: The data that support the ndings of this study are available from the corresponding author upon request.
Competing interests: There is no competing interests about this research and all authors agree to publish.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.