TaWRKY70 positively regulates TaCAT5 enhanced Cd tolerance in transgenic Arabidopsis
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.
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These authors contributed equally to this work.