Research article
CaWRKY27 negatively regulates salt and osmotic stress responses in pepper

https://doi.org/10.1016/j.plaphy.2019.08.013Get rights and content

Highlights

  • CaWRKY27 was induced by salinity, osmotic and ABA treatment.

  • Silence of CaWRKY27 by virus induced gene silencing (VIGS) conferred tolerance to salinity and osmotic stress in pepper.

  • Transgenic plants conferred CaWRKY27 sensitivity to salinity and osmotic stress dependent on ABA pathway.

Abstract

WRKY transcription factors are key regulatory components of plant responses to both biotic and abiotic stresses. In pepper (Capsicum annuum), CaWRKY27 positively regulates resistance to the pathogenic bacterium Ralstonia solanacearum and negatively regulates thermotolerance. Here, we report that CaWRKY27 functions in the response to salinity and osmotic stress. CaWRKY27 transcription was induced by salinity, osmotic, and abscisic acid (ABA) treatments, as determined using qPCR and GUS assays. Transgenic Arabidopsis thaliana and tobacco (Nicotiana tabacum) plants heterologously expressing CaWRKY27 had an increased sensitivity to salinity and osmotic stress, with a higher inhibition of both root elongation and whole plant growth, more severe chlorosis and wilting, lower germination rates, and an enhanced germination sensitivity to ABA than the corresponding wild-type plants. Furthermore, most marker genes associated with reactive oxygen species (ROS) detoxification and polyamine and ABA biosynthesis, as well as stress-responsive genes NtDREB3, were downregulated in plants transgenically expressing CaWRKY27 upon exposure to salinity or osmotic stress. Consistently, silencing of CaWRKY27 using virus-induced gene silencing conferred tolerance to salinity and osmotic stress in pepper plants. These findings suggest that CaWRKY27 acts as a molecular link in the antagonistic crosstalk regulating the expression of defense-related genes in the responses to both abiotic and biotic stresses by acting either as a transcriptional activator or repressor in pepper.

Introduction

In their natural habitats, plants inevitably encounter biotic stresses, such as pathogens, and abiotic stresses, such as salt, drought, heat, and cold. To withstand these stresses, which are often experienced concurrently, plants have evolved sophisticated defense mechanisms that perceive the stress, initiate defense signaling, and translate the signaling into an appropriate defense reaction. Increasing evidence suggests the existence of extensive crosstalk between the plant responses to biotic and abiotic stresses, which is believed to enable plants to fine-tune their responses to stress and ensure they react appropriately (Fujita et al., 2006). However, the detailed mechanisms underlying this crosstalk are yet to be elucidated.

Drought and salt stresses are two important abiotic stresses with similar adverse effects on the growth and development of plants and hence on crop yields. Plants exposed to these stresses generally undergo cellular dehydration and osmotic stress, and exhibit oxidative damage to membranes, proteins, RNA, and DNA caused by reactive oxygen species (ROS) bursts and accumulation (Miller et al., 2008; Møller and Sweetlove, 2010). To adapt to these stresses, plants initiate signaling pathways, including the abscisic acid (ABA)-dependent, ABA-independent, and salt overly sensitive (SOS) signaling pathways, which play crucial roles during the plant adaptive response to salt stress and drought (Huang et al., 2012; Yang and Guo, 2018; Zhu, 2002). These signals accumulate in the nucleus, where they induce transcriptional reprogramming via the action of various transcription factors (TFs) such as CBF/DREB1, DREB2, AREB/ABF, bZIP, MYB/MYC, and WRKY (Ding et al., 2015; Huang et al., 2012; Mondini et al., 2012; Zhu et al., 2019), leading to the reestablishment of cellular ionic, osmotic, and ROS homeostasis and ultimately enhancing plant tolerance to salt stress or drought (Mittler et al., 2004). Despite these insights, the mechanisms underlying plant responses to salt or drought stress have not been fully elucidated.

Members of one of the largest TF families, the WRKYs, are characterized based on the presence of one or two conserved WRKY domains, which bind to the highly conserved cognate W-box (TTGACC/T) (Eulgem et al., 2000; Rushton et al., 2010). Members of this family have been implicated in plant responses to stresses (Jiang et al., 2017), including pathogens (Amorim et al., 2017; Sarris et al., 2015), heat (Cai et al., 2015; Dang et al., 2013; Li et al., 2009, 2010, 2011), cold (Marè et al., 2004; Yokotani et al., 2013), salinity (Ding et al., 2015; Hichri et al., 2017), drought (Luo et al., 2013; Marè et al., 2004), and phosphate starvation (Baek et al., 2017). The roles of the WRKY TFs have been intensively investigated in plant immune responses to various pathogens with different lifestyles, and several WRKY genes have been found to be transcriptionally modulated by pathogen attack within a given plant species (Bencke-Malato et al., 2014; Jiang et al., 2014). Some of these pathogen responsive WRKYs function in networks as positive or negative regulators of plant immunity (Birkenbihl et al., 2017; Eulgem and Somssich, 2007), and some have been found to be induced by salinity and drought (Jiang et al., 2015; Wei et al., 2018). For example, FvWRKY42, a WRKY TF in the diploid woodland strawberry (Fragaria vesca), enhances resistance to powdery mildew and improves osmotic stress resistance (Wei et al., 2018), indicating its role in the crosstalk between the response to pathogens and other stresses and a possible function in coordinating the responses to different stresses. However, the roles of different WRKY TFs in terms of coordinating plant immunity and other biological processes have not been fully understood.

Pepper (Capsicum annuum) is a commercially important vegetable in the Solanaceae family. The growth and development of pepper plants are frequently challenged by attack from soil-borne pathogens, such as Ralstonia solanacearum (RS), and these plants are often exposed to abiotic stresses such as heat, drought, and salt. A subset of WRKY TFs, including CaWRKY6 (Cai et al., 2015), CaWRKY22 (Hussain et al., 2018), CaWRKY27 (Dang et al., 2014), CaWRKY40 (Dang et al., 2013), and CaWRKY40b, have been found to be modulated by RS and act as positive or negative regulators of the pepper response to this pathogen. Among these genes, CaWRKY6 (Cai et al., 2015), CaWRKY22 (Hussain et al., 2018), and CaWRKY40 (Dang et al., 2013) were also found to act as positive regulators of the pepper response to heat stress, while CaWRKY27 (Dang et al., 2018) acts as a negative regulator of this process, indicating a role of CaWRKY27 as a crucial node in the crosstalk between pepper immunity and thermotolerance. Here, we provide evidence that CaWRKY27 is also transcriptionally modulated by salt and drought stress and acts as a negative regulator of the response to these two abiotic stresses.

Section snippets

Plant materials and growth conditions

Seeds from Capsicum annuum 8# and tobacco (Nicotiana tabacum) cultivar K326 were soaked in water overnight at 26 ± 1 °C, and then sown in steam-sterilized soil mix (peat moss, vermiculite, and perlite; 2/1/1, v/v/v) in plastic pots. Plants were grown in a growth room maintained at 26 ± 1 °C with a light intensity of ~100 μmol photons m−2 s−1 and a relative humidity of 70% under a 16-h-light/8-h-dark cycle.

Wild-type (Col-0) and transgenic Arabidopsis thaliana seeds were treated at 4 °C for 3 d

CaWRKY27 expression is induced by both salt and drought stress

The CaWRKY27 promoter is enriched with various cis-elements, including TCA elements, ABREs, and CGTCA motifs, that are responsive potentially to phytohormones associated with plant immune and stress responses, including salicylic acid, ABA, and jasmonic acid (Narusaka et al., 2003). The presence of these cis-elements indicates that CaWRKY27 might play a role in coordinating the responses of pepper to multiple stresses. We previously demonstrated that CaWRKY27 is a positive regulator of the

Discussion

We previously revealed that CaWRKY27 is a positive regulator of the pepper response to RSI and a negatively regulator of the response to heat stress (Dang et al., 2014, 2018). In this study, we demonstrated that CaWRKY27 also acts as a negative regulator of the pepper response to salt stress and drought. CaWRKY27 expression is induced by both salt stress and drought, as well as by mannitol, which mimics osmotic stress. By contrast, silencing of CaWRKY27 significantly enhanced tolerance of

Author contributions

S. L. H. designed the experiments. J. H. L. and F. F. D. performed most of experiments and analyzed the data. The other authors assisted in experiments and discussed the results. J. H. L. and S. L. H. wrote the manuscript.

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31572136, 31372061).

References (58)

  • R. Mittler et al.

    Reactive oxygen gene network of plants

    Trends Plant Sci.

    (2004)
  • I.M. Møller et al.

    ROS signalling--specificity is required

    Trends Plant Sci.

    (2010)
  • Y. Peng et al.

    OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice

    Mol. Plant

    (2008)
  • P.J. Rushton et al.

    WRKY transcription factors

    Trends Plant Sci.

    (2010)
  • P.F. Sarris et al.

    A plant immune receptor detects pathogen effectors that target WRKY transcription factors

    Cell

    (2015)
  • W. Wei et al.

    Ectopic expression of FvWRKY42, a WRKY transcription factor from the diploid woodland strawberry (Fragaria vesca), enhances resistance to powdery mildew, improves osmotic stress resistance, and increases abscisic acid sensitivity in Arabidopsis

    Plant Sci.

    (2018)
  • D. Zhu et al.

    VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role under salinity stress

    Plant Sci.

    (2019)
  • M.S. Alves et al.

    Transcription factor functional protein-protein interactions in plant defense responses

    Proteomes

    (2014)
  • L.L.B. Amorim et al.

    Transcription factors involved in plant resistance to pathogens

    Curr. Protein Pept. Sci.

    (2017)
  • M. Bencke-Malato et al.

    Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection

    BMC Plant Biol.

    (2014)
  • H. Cai et al.

    CaWRKY6 transcriptionally activates CaWRKY40, regulates Ralstonia solanacearum resistance, and confers high-temperature and high-humidity tolerance in pepper

    J. Exp. Bot.

    (2015)
  • S.M. Clarke et al.

    Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

    Plant J.

    (2004)
  • F.F. Dang et al.

    CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection

    Plant Cell Environ.

    (2013)
  • F. Dang et al.

    CaWRKY27 negatively regulates H2O2-mediated thermotolerance in pepper (Capsicum annuum)

    Front. Plant Sci.

    (2018)
  • F. Dang et al.

    Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection

    Physiol. Plant.

    (2014)
  • Z.J. Ding et al.

    Transcription factor WRKY46 modulates the development of Arabidopsis lateral roots in osmotic/salt stress conditions via regulation of ABA signaling and auxin homeostasis

    Plant J.

    (2015)
  • L. Du et al.

    Ca2+/calmodulin regulates salicylic-acid-mediated plant immunity

    Nature

    (2009)
  • Y.T. Du et al.

    Identification and characterization of GmMYB118 responses to drought and salt stress

    BMC Plant Biol.

    (2018)
  • Y. Fujita et al.

    ABA-mediated transcriptional regulation in response to osmotic stress in plants

    J. Plant Res.

    (2011)
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