TaWRKY70 positively regulates TaCAT5 enhanced Cd tolerance in transgenic Arabidopsis

https://doi.org/10.1016/j.envexpbot.2021.104591Get rights and content

Highlights

  • TaWRKY70 increased Cd tolerance of transgenic Arabidopsis.

  • Expression of TaWRKY70 in Arabidopsis suppress Cd transfer coefficient.

  • TaWRKY70 binds to the promoter of TaCAT5 as well as regulating the transcription of TaCAT5.

Abstract

The WRKY transcription factors (TFs) are involved in plant responses to multiple biotic and abiotic stressors; however, the role of WRKY TFs in the regulation of Cd stress in wheat remains unknown. In this study, we investigated the mechanism of the wheat TaWRKY70 TF in Cd stress. TaWRKY70 expression in Arabidopsis was used for the functional analysis. TaWRKY70 regulated Cd tolerance with Cd accumulating in roots but not in the leaf tissues of transgenic Arabidopsis. The net Cd2+ influx into Arabidopsis roots decreased when TaWRKY70 was expressed. The quantitative real-time-polymerase chain reaction (qRT-PCR) results showed that the expression levels of heavy metal ATPase (AtHMA3), natural resistance-associated macrophage protein (AtNRAMP5), yellow stripe1-like (AtYSL3), and iron transport protein (AtIRT1) decreased under Cd-stress condition. Electrolyte leakage, as well as malondialdehyde and hydrogen peroxide contents, were lower in transgenic Arabidopsis than those in the wild-type (WT), whereas antioxidant enzyme activities were higher. Electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay, and transient transactivation assay confrmed that TaWRKY70 could directly bind to the TaCAT5 promoter. Our findings provide a novel understanding of the WRKY TFs involved in the response to heavy metal stress.

Introduction

Abiotic stressors are adverse environmental factors that inhibit plant growth and development. Common abiotic stressors include drought stress, salt stress, waterlogging, abnormal temperature, and heavy metal pollution (Pandey et al., 2009; Prabu et al., 2011). Soil heavy metal pollution has been recently attracting increasing attention. Heavy metals in the soil not only reduce food production but are transferred to humans through the food chain and threaten human health (Zhang et al., 2020). Cadmium (Cd) is one of the most widespread heavy metal pollutants. In the past few decades, the intensification of human industrial activities has led to serious Cd contamination of agricultural systems (Wang et al., 2020). Hence, it is necessary to study the biological functions and the mechanisms of Cd-regulated genes to enhance crop adaptation to the Cd environment and reduce Cd accumulation in edible parts.

Plants have evolved various strategies to cope with abiotic stimuli. Several key genes encoding metal transporters and transcription factors have been reported to participate in Cd detoxification and tolerance in plants (Sasaki et al., 2014; Cai et al., 2017; Luo et al., 2019; Mekawy et al., 2020). Yellow stripe-like (YSL) transporters, iron-regulated transporter (IRT), natural resistance-associated macrophage proteins (NRAMP), and heavy metal ATPase (HMA) have been reported as key transporters involved in ion absorption and transport (Connorton et al., 2017). Transcriptional regulation mechanisms play a critical role in plant response to environmental stress (Miao et al., 2004; Zhang et al., 2011). Abiotic stressors initiate the synthesis of transcription factors (TFs), which bind to the promoter region of a downstream gene and are involved in different signaling pathways (Eulgem and Somssich, 2007). The WRKY proteins are a superfamily of plant TFs that act as key regulators of multiple biotic and abiotic stressors (Eulgem and Somssich, 2007; Pandey and Somssich, 2009; Miao et al., 2004; Luo et al., 2005). The WRKY proteins are characterized by a specific WRKY domain consisting of approximately 60 amino acid residues at the end of the N-terminus (Prabu et al., 2011; Zhang et al., 2011). WRKY proteins generally bind with high affinity to the specific DNA cis-acting element W-box (C/TTGACT/C) in the promoter region and regulate the expression of downstream genes (Ding et al., 2016). Several studies have shown that WRKY proteins participate in the response to Cd stress by regulating the expression of target genes, such as ThWRKY7, AtWRKY12, AtWRKY13, CaWRKY41, and GmWRKY142 (Yang et al., 2016; Han et al., 2019; Sheng et al., 2019; Dang et al., 2019; Cai et al., 2020). However, little is known about the role of the WRKY proteins of wheat in Cd tolerance. An increase of reactive oxygen species (ROS) occurs in plant tissues under stress conditions, leading to cellular structure damage and decreased photosynthetic capacity, which consequently affects the nutritional status and growth of plants (Mengutay et al., 2013; Boaretto et al., 2020). Scavenging ROS represents a promising strategy to improve the stress tolerance of plants under unfavorable environmental conditions (Jalmi et al., 2018; Wang et al., 2020). Amino acid transporters (AATs) are membrane-localized proteins that mediate the movement of amino acids from biosynthetic organs to utilization organs (Svennerstam et al., 2011). The cationic amino acid transporter (CAT) belongs to the AAT family in plants and plays an important role in amino acid transport, nitrogen homeostasis, and abiotic stress responses (Wu et al., 2015; Akbudak and Filiz, 2020; Tian et al., 2020). However, the regulatory mechanism and CAT pathway involved in Cd stress are unclear.

Wheat is consumed in large quantities worldwide. Cultivating Cd tolerant wheat cultivars and reducing the Cd concentrations in wheat grains are solutions that could potentially alleviate the risks to human health. Therefore, it is necessary to investigate genes involved in Cd tolerance and understand the mechanisms of Cd tolerance in wheat. In this study, we identified TaWRKY70 as highly responsive to Cd stress in wheat. The downstream gene TaCAT5, which is regulated by TaWRKY70, was further identified and the functions of TaWRKY70 in Cd stress were characterized in transgenic Arabidopsis. The data suggest that TaWRKY70 acts as a critical regulator of Cd stress by regulating TaCAT5.

Section snippets

Plant materials

Guinong 19, a widely cultivated wheat cultivar in Guizhou Province, China, was used to analyze the response of TaWRKY70 to Cd stress. Guinong 19 shows moderate sensitivity to Cd and the expression of TaWRKY70 was upregulated in the Guinong 19 transcriptome data under Cd stress (unpublished data). Arabidopsis thaliana (Columbia) was used to study gene function.

Plant growth conditions

Plant cultivation was performed according to Qiao et al., (2019). The Cd concentration was set according to the Environmental Quality

TaWRKY70 localizes to the nucleus

To analyze the subcellular localization of TaWRKY70, the 35S:TaWRKY70-GFP fusion construct was transferred into onion epidermal cells, and the 35S:GFP vector was used as a control. TaWRKY70-GFP fluorescent signals were transiently expressed only in nuclei, but they were distributed in the cytoplasm and nuclei of the negative controls (Fig. 1a), suggesting that TaWRKY70 is a nuclear protein.

TaWRKY70 is induced by Cd in roots and shoots of wheat

The expression of TaWRKY70 in wheat root and shoot tissues under Cd stress was analyzed by qRT-PCR. The

Discussion

Heavy metals are an abiotic stressor that largely restricts wheat productivity and quality. Thus, improving wheat tolerance to heavy metal stress and reducing heavy metal accumulation in wheat grains is an effective strategy for promoting grain output and food safety. Cd is a prevalent heavy metal that harms wheat growth and human health (Clemens et al., 2013; Zare et al., 2018). The accumulation of Cd inhibits seed germination, destroys the photosynthetic system, and increases cell damage,

Author statement

Zhenzhen Jia: Investigation, Writing-Original draft preparation, Muzi Li: Investigation, Hongcheng Wang: Conceptualization, Investigation, Bin Zhu: Conceptualization, Lei Gu: Formal analysis, Xuye Du: Supervision, Writing- Reviewing and Editing, Mingjian Ren: Supervision

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

This work was financially supported by Guizhou Normal University (2018-5769-16) and Guizhou Provincial Science and Technology Foundation (2019-1236). The authors would like to thank TopEdit (www.topeditsci.com) for linguistic assistance during preparation of this manuscript.

References (49)

  • A.A. Zare et al.

    Root uptake and shoot accumulation of cadmium by lettuce at various Cd:Zn ratios in nutrient solution

    Ecotoxicol. Environ. Saf.

    (2018)
  • W.W. Zhang et al.

    MhMAPK4 from Malus hupehensis Rehd. decreases cell death in tobacco roots by controlling Cd2+ uptake

    Ecotox. Environ. Safety

    (2019)
  • J. Zhang et al.

    A novel plasma membrane-based NRAMP transporter contributes to Cd an Zn hyperaccumulation in Sedum alfredii Hance

    Environ. Exp. Bot.

    (2020)
  • P. Agarwal et al.

    WRKY: its structure, evolutionary relationship, DNA-binding selectivity, role in stress tolerance and development of plants

    Mol. Biol. Rep.

    (2011)
  • R.P. Birkenbihl et al.

    Induced genome-wide binding of three Arabidopsis WRKY transcription factors during early MAMP-triggered immunity

    Plant Cell

    (2017)
  • R.M. Boaretto et al.

    The possible role of extra magnesium and nitrogen supply to alleviate stress caused by high irradiation and temperature in lemon trees

    Plant Soil

    (2020)
  • Z.D. Cai et al.

    Transcription factor GmWRKY142 confers cadmium resistance by up-regulating the cadmium tolerance 1-like genes

    Front. Plant Sci.

    (2017)
  • J.M. Connorton et al.

    Wheat vacuolar iron transporter TaVIT2 transports Fe and Mn and is effective for biofortification

    Plant Physiol.

    (2017)
  • F.F. Dang et al.

    A feedback loop between CaWRKY41 and H2O2 coordinates the response to Ralstonia solanacearum and excess cadmium in pepper

    J. Exp. Bot.

    (2019)
  • W.W. Ding et al.

    Wheat WRKY type transcription factor gene TaWRKY1 is essential in mediating drought tolerance associated with an ABA-dependent pathway

    Plant Mol. Biol. Rep.

    (2016)
  • L. Feng et al.

    Identification and characterization of cationic amino acid transporters (CATs) in tea plant (Camellia sinensis)

    Plant Growth Regul.

    (2018)
  • Y.Y. Han et al.

    WRKY12 represses GSH1 expression to negatively regulate cadmium tolerance in Arabidopsis

    Plant Mol. Biol.

    (2019)
  • S.K. Jalmi et al.

    Traversing the links between heavy metal stress and plant signaling

    Front. Plant Sci.

    (2018)
  • M. Luo et al.

    MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis

    Proc. Natl. Acad. Sci. U.S.A.

    (2005)
  • Cited by (19)

    • TaMYC8 regulates TaERF6 and inhibits ethylene synthesis to confer Cd tolerance in wheat

      2022, Environmental and Experimental Botany
      Citation Excerpt :

      Images of these cells were captured to record transcriptional activation. The promoter sequence (1600 bp) of TaERF6 was retrieved from the WheatOmics database (http://202.194.139.32/), and the cDNA library used in this work for screening the TF target TaERF6 promoter was described in our previous work (Jia et al., 2021). The pHIS2 vector (Clontech, Shiga, Japan) harboring the TaERF6 promoter was used as a bait vector, while the pGADT7 vector harboring the TaMYC8 ORF was used as a prey vector.

    • AetSRG1 contributes to the inhibition of wheat Cd accumulation by stabilizing phenylalanine ammonia lyase

      2022, Journal of Hazardous Materials
      Citation Excerpt :

      The H2O2-specific fluorescent probe H2DCF-DA (Invitrogen, USA) was used to detect H2O2 accumulation in wheat. The antioxidants enzyme activity, including SOD, POD, and CAT were measured according to our previous work (Jia et al., 2021). MDA contents were determined using thiobarbituric acid as described earlier (Wang et al., 2017), chlorophyll was measured by 80% aqueous acetone (v/v) as described by Arnon (1949).

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text