MYB4 transcription factor, a member of R2R3-subfamily of MYB domain protein, regulates cadmium tolerance via enhanced protection against oxidative damage and increases expression of PCS1 and MT1C in Arabidopsis

Highlights

• MYB4 regulates Cd-tolerance via anti-oxidant defense and GSH-dependent pathway.

• MYB4 overexpression results in appreciable Cd tolerance than wild-type plants.

• MYB4 loss of function mutant lines (atmyb4) showed hypersensitivity to Cd-stress.

• MYB4 overexpression line showed enhanced Cd accumulation than wild-type plants.

• MYB4 overexpression specifically activates PC synthesis related genes expression.

Abstract

Here, we describe functional characterization of Arabidopsis thaliana MYB4 transcription factor, a member of R2R3-subfamily of MYB domain protein, in the regulation of Cd-stress tolerance in Arabidopsis. Transgenic Arabidopsis plants overexpressing MYB4 showed appreciable Cd tolerance than wild-type plants, while MYB4 loss of function mutant lines (atmyb4) showed increased sensitivity to Cd-stress. MYB4 overexpression lines showed strong activation of anti-oxidant defense components and increased Cd accumulation than wild-type and atmyb4 mutant lines under Cd-stress. MYB4 overexpression resulted in the coordinated activation of the expression of phytochelatin (PC) synthesis related genes and specifically enhanced the transcript abundance of phytochelatin synthase 1 (PCS1) and metallothionein 1C (MT1C) genes under Cd-stress. In contrast, atmyb4 mutant lines showed reduced Cd accumulation and compromised expression of PC-synthesis related genes. Electrophoretic gel mobility shift assays have demonstrated specific binding activity of recombinant AtMYB4 to the putative MYB4-binding motifs ACCAACCAA and GGTAGGT identified in the promoters of PCS1 and MT1C genes, respectively. Further analyses have revealed that MYB4 binds directly to PCS1 and MT1C promoters in vivo and positively regulates their transcriptional expression, suggesting that PCS1 and MT1C are the key targets of MYB4. Overall, our results have provided evidence that MYB4 regulates Cd-tolerance via the coordinated activity of improved anti-oxidant defense system and through the enhanced expression of PCS1 and MT1C under Cd-stress in Arabidopsis.

Introduction

Enhanced accumulation of heavy metal elements in the environment represents one of the major stresses for plants, which, being sessile in nature, cannot avoid the harmful toxic effects of heavy metals [[1], [2], [3]]. Previously, several reports have indicated the toxic effects of the non-essential mineral elements, such as cadmium (Cd), lead (Pb), mercury (Hg) and chromium (Cr) in plant cell [4,5]. After the entry into the plant cells through the membrane bound transporters, the heavy metal ions bind to the functional sites of various functional and structural proteins and membrane lipids, leading to the alteration in their structural conformation and frequently damage cellular components due to oxidative damage through generation of reactive oxygen species (ROS), thus disrupting normal functioning of plant cell [3,6,7].

Cadmium (Cd) and lead (Pb) are the two most commonly found elements in heavy metal contaminated soils and after uptake by plants frequently cause water imbalance, reduction in photosynthesis and nutrient assimilation and chlorosis, leading to plant growth inhibition and yield loss [8]. Cadmium has been shown to cause disruption of protein structure and function via binding to the sulfhydryl groups, causing cellular damage due to dissociation of cofactors from various proteins, including enzymes and transcription factors [1]. Low level of cadmium accumulation in plant cells also induces oxidative stress through the generation of reactive oxygen species (ROS). ROS eventually causes oxidative damage to DNA bases, protein side chains and destruction of phospholipids [2,9]. In heavy metal contaminated soil, Pb frequently accumulates in the roots stems and other parts of plants and adversely affects plant growth, development and productivity [10]. As like Cd, Pb also may enter into animal and human bodies through food chain, leading to increasing risk of dietary consumption of such harmful toxic elements [11].

Plants have developed extensive mechanisms for detoxification of heavy metal ions and development of tolerance to heavy metal stress [9,12,13]. The detoxification mechanisms include sequestration of heavy metal ions into vacuole, pumping out through plasma membrane and chelation or binding to different thiol compounds in the cytosol [14,15]. Induction of antioxidant mechanisms also represent one of the major signaling mechanisms activated under Cd and Pb stress in plants [2,4,16,17]. Several studies have demonstrated pivotal role of glutathione (GSH) in the detoxification of harmful effects of heavy metals, such as Cd [[18], [19], [20]].In addition, the phytochelatins (PCs), the thiol group containing low molecular pepetides, are synthesized from glutathione by the activity of phytochelatin synthase (PCS) and play crucial role in vacuolar sequestration of Cd and other heavy metal ions [18,21,22]. On the other hand, the metallothioneins (MTs), another family of small, low molecular weight cysteine rich compounds, also participates in detoxification of various heavy metal ions, predominantly Cd, Cu, Zn, and As via sequestration and transport [2]. MTs also regulate the redox homeostasis in plants by ROS scavenging [23,24].

Previous studies have identified several genes involved in Cd detoxification and tolerance, including AtPDR12, AtPDR8, AtATM3, AcBP1, ZNt1, OsHMA9, OSNrAMP5 etc. [22,[25], [26], [27], [28], [29]]. However, information on the transcriptional regulation of Cd and Pb detoxification mechanism remains still limited. In Arabidopsis, WRKY17, MYB39, MYB45, MYB63, MYB93 and MYB94 have been shown to play important role in the detoxification of heavy metal ions [30,31]. Expression studies in Pb-tolerant maize inbred line have identified 262 differentially expressed transcription factors in response to Pb-treatment with the basic leucine zipper domain transcription factors (bZIP) found as the predominant class related to Pb-tolerant trait among the inbred maize lines [10]. Recent studies in Arabidopsis have indicated important role of a Zinc-finger transcription factor, ZAT6, in the regulation of Cd-stress tolerance through glutathione-dependent pathway [13].

In Arabidopsis, the transcription factor MYB4 is a member of R2R3-subfaqmily of MYB domain proteins and plays important role in the regulation of accumulation of UV-B absorbing phenylpropanoids. MYB4 acts as transcriptional repressor of C4H gene, which encodes Cinnamate-4-Hydroxylase, the second enzyme in the phenylpropanoid pathway [29]. UV-B light has been shown to negatively regulate MYB4 transcript accumulation in wild-type Arabidopsis, thus causing increased C4H expression and phenylpropanoid production under high fluence of UV-B light [32]. MYB4 protein interacts with an importin β-like protein sensitive to ABA and Drought 2 (SAD2) via the terminal conserved GY/FDFLGL motif, which facilitates MYB4 transportation into the nucleus. Interestingly, recent studies have revealed strong induction of MYB4 transcript in Arabidopsis in response to heavy metal elements, including Cd and Zn [2,33]. However, role of MYB4 in metal homeostasis or tolerance has not been characterized. In this study, we report functional characterization of Arabidopsis MYB4 (AtMYB4) in the context of plant response to Cd stress. We demonstrate that transgenic Arabidopsis plants overexpressing AtMYB4 manifested enhanced Cd tolerance, whereas loss of function of AtMYB4 led to increased Cd sensitivity. Our results provide evidence that AtMYB4 mediates Cd-tolerance via enhanced anti-oxidant activity and also involves the glutathione pathway through transcriptional activation of PCS1 and MT1C under Cd-stress in Arabidopsis.