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 C-terminus 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 (Chen et al., 2015). Our result also showed five candidate chloroplast-located SlWRKYs (Fig. 2). Nevertheless, four of them were confirmed 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 identified 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.
Table 2
The chilling related cis-acting element prediction in the promoter region of the 27 SlWRKYs
Chilling related cis-acting element | Gene names of WRKYs |
Core sequence | Cis-acting element |
RCCGAC | DRECRTCOREAT | WRKY2, WRKY21, WRKY24, WRKY35, WRKY43, WRKY50, WRKY59, WRKY69 |
GTCGAC | CRTDREHVCBF2 | WRKY2, WRKY23, WRKY43, WRKY50, WRKY59, WRKY62 |
CCGAC | LTRECOREATCOR15 | WRKY1, WRKY2, WRKY8, WRKY21, WRKY23, WRKY24, WRKY35, WRKY43, WRKY49, WRKY50, WRKY59, WRKY61, WRKY69, WRKY79 |
CCGAAA | LTRE-1 | WRKY1, WRKY2, WRKY8, WRKY21, WRKY23, WRKY24, WRKY35, WRKY43, WRKY49, WRKY50, WRKY59, WRKY61, WRKY69, WRKY79 |
The interaction proteins of WRKYs are essential to affect the DNA binding and transcription regulatory activities of WRKYs, thus controlling its downstream genes expression. WRKY-WRKY, WRKY-VQ and WRKY-MAPK interactions are the most common interaction relationship of WRKY (Chi et al., 2013). In Arabidopsis, MPK3, MPK4, and MPK6 could be activated by biotic and abiotic stresses and their functional analyses indicate their critical roles in plant disease resistance and stress tolerance (Pitzschke et al., 2009). AtWRKY33 acts as a downstream component of MPK3/MPK6 cascade in regulation of the pathogen-induced defense responses (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 homologous gene in Arabidopsis, AtWRKY70 can play a role in plant immunity and response to salicylic acid (SA), reactive oxygen species (ROS) and jasmonic acid (JA) which are also induced by chilling stress (Chen et al., 2017; Lortzing et al., 2018; Zhou et al., 2018; Chae et al., 2020). These suggest that SlWRKY21 may play an essential and complex role in chilling response pathway. Otherwise, SlWRKY23 was predicted to interact with a critical cold-responce factor ICE1 (Induced of CBF expression1, Lee et al., 2005) which showed its potential function under chilling stress.
Although chilling response WRKYs were studied in several species, the mechanism of how WRKYs respond to chilling stress and the specific regulatory mechanism to their downstream genes are remain poorly understood. In this study, we identified 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.