Transcription Factors PvERF15 and PvMTF-1 Form a Cadmium Stress Transcriptional Pathway

Abstract

In plants, cadmium (Cd)-responsive transcription factors are key downstream effectors of Cd stress transcriptional pathways, which are capable of converging Cd stress signals through triggering the expression of Cd detoxification genes. However, the upstream transcriptional regulatory pathways that modulate their responses to Cd are less clear. Previously, we identified the bean (Phaseolus vulgaris) METAL RESPONSE ELEMENT-BINDING TRANSCRIPTION FACTOR1 (PvMTF-1) that responds to Cd and confers Cd tolerance in planta. Here, we demonstrate an upstream transcriptional regulation of the PvMTF-1 response to Cd Using a yeast one-hybrid system, we cloned the bean ETHYLENE RESPONSE FACTOR15 (PvERF15) that binds to the PvMTF-1 promoter. PvERF15 was strongly induced by Cd stress, and its overexpression resulted in the up-regulation of PvMTF-1 DNA-protein interaction assays further revealed that PvERF15 binds directly to a 19-bp AC-rich element in the PvMTF-1 promoter. The AC-rich element serves as a positive element bound by PvERF15 to activate gene expression. More importantly, knockdown of PvERF15 by RNA interference resulted in reduced Cd-induced expression of PvMTF-1PvERF15 seems to be involved in Cd tolerance, since knockdown of PvERF15 by RNA interference in bean leaf discs decreased Cd tolerance in a transient assay. Since PvERF15 is a component of the Cd stress transcriptional pathway in beans and PvMTF-1 is one of its downstream targets, our findings provide a PvERF15/PvMTF-1 transcriptional pathway and thereby contribute to the understanding of Cd stress transcriptional regulatory pathways in plants.

Figure 1.

PvERF15 is a Cd-responsive transactivator that binds to the PvMTF-1 promoter. A, Y1H binding assay of PvERF15 to the PvMTF-1 promoter (ProPvMTF-1). A yeast strain with the HIS3 gene driven by ProPvMTF-1 was transformed with a plasmid encoding the GAL4 activation domain (AD) alone or with a PvERF15 fusion (AD-PvERF15). Interaction is indicated by the ability of cells to grow on His-deficient medium (−His) with or without 3-amino-1,-2,-4-triazole (3-AT). Three independent yeast clones are shown. B, Subcellular localization of PvERF15-GFP and GFP transiently expressed from the 35S promoter in N. benthamiana epidermal cells. DAPI, 4,6-Diamidino-2-phenylindole (nuclei staining). C, The transcriptional activation activity of PvERF15 was analyzed in yeast cells. LacZ reporter gene expression is indicated by blue color on synthetic defined (SD) medium lacking Trp (SD/-Trp) containing 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside (X-α-gal). The Gal4 DNA-binding domain (BD) alone was used as a negative control. Three independent yeast clones are shown. D, Time-course analysis of Cd-inducible gene expression in bean leaf discs. Bean leave discs were treated with 200 μm CdCl₂ for 0, 6, 12, and 24 h. RNAs were then extracted and subjected to qRT-PCR. PvERF15 and PvMTF-1 mRNA abundance was expressed as a ratio relative to the pretreatment level (0 h), which was set to a value of 1. Data shown are averages of three independent qRT-PCR experiments for each time point. Error bars represent sd. Significance between experimental values was assessed using Student’s t test (*, P < 0.05; and **, P < 0.01).

Figure 2.

PvERF15 binds an ACE within the PvMTF-1 promoter. A, Immunoblot (IB) confirming the expression of PvERF15-GFP (56.75 kD) in bean leaf discs. Untransformed bean leaf discs (Wild-type) were used as a negative control. Two independent 35S:PvERF15-GFP lines (1# and 2#) were included. A Coomassie Brilliant Blue (CBB)-stained gel served as a loading control. B, ChIP experiments were used with GFP antibody and mouse IgG (mock control). The diagram of the PvMTF-1 gene indicates the amplicons (P1, P2, and 3′ UTR) used for subsequent qPCR analysis. Relative enrichment was calculated by comparing GFP antibody-immunoprecipitated DNA with those immunoprecipitated with the IgG control (binding ratio of GFP antibody to IgG). 3′ UTR was used as a negative control. Error bars represent sd. Significance between experimental values was assessed using Student’s t test (*, P < 0.05). ORF, Open Reading Frame. C, Diagram of the PvMTF-1 promoter subfragments as probes in EMSA. F1 sequences (wt) and mutations introduced into F1 (m1, m2, and m3) are shown at bottom. ACE is boxed. D, EMSA was performed using biotin-labeled probes with the affinity-purified recombinant GST-PvERF15 and GST (mock proteins). The bound complex is indicated by the arrow. E, Competition experiments using a 1,000-fold excess of unlabeled competitors (wt, m1, m2, and m3). The bound complex is indicated by the arrow. F, Y1H binding assay of PvERF15 to wild-type ACE or a mutant version (ACEm; mutations shown in lowercase letters) bait. Other details are given in the legend to Figure 1.

Figure 3.

Transient assays in bean leaf discs confirm ACE as a positive element. A, Schematic diagrams of the test constructs in the transient assays. B, Immunoblot analysis of plants expressing GUS. The bean leaf discs transiently transformed with each of the constructs were treated without (−Cd) and with 200 μm CdCl₂ (+Cd) for 24 h. GUS expression was determined by immunoblot (IB) assays using anti-GUS and anti-tubulin (as a loading control) antibodies. The experiments were repeated independently two times with similar results.

Figure 4.

GUS transient assays of PvERF15 transcriptional activity in N. benthamiana leaf discs. A, Schematic diagram of constructs used in the experiments. B, N. benthamiana leaf discs were cotransformed with combinations of these constructs, as indicated. The expression of GUS, PvERF15-GFP, or GFP was determined by immunoblot (IB) using anti-GUS and anti-GFP antibodies. Transcriptional activity is measured as the relative immunoblotting signal intensity of GUS to GFP and represents an average from three independent experiments. Error bars represent sd. Significance between experimental values was assessed using Student’s t test, and P values are provided.

Figure 5.

PvERF15 is a transcriptional regulator of PvMTF-1. A, Schematic diagram of plasmids used in the transient assay. 35S:RNAi_PvERF15 contains a 327-bp coding sequence (black box) of PvERF15 in the sense and antisense orientation interspersed by intron 1 (intron) of the potato GA20 OXIDASE gene. Arrows indicate the primers used in the RT-PCR assay for PvERF15. B and C, Each of the plasmids was transformed into bean leaf discs to generate PvERF15 overexpression (OE), PvERF15 RNAi (RNAi), and 35S:GUS (Control) plants. PvERF15 overexpression and knockdown were confirmed by RT-PCR using a pair of primers, ORF-F and ORF-R. The ACTIN gene was used as a loading control. PvERF15 relative abundance is shown at bottom, and the control was set as 1. B, PvMTF-1 expression levels in overexpression and RNAi plants relative to the control (set as 1) determined by qRT-PCR analysis. C, Control and RNAi plants treated with Cd for 6 h and then subjected to qRT-PCR analysis for Cd-inducible PvMTF-1 expression relative to pretreatment (0 h; set as 1). sd values are from three technical replicates of one biological experiment. The experiments were repeated independently two times with similar results. Significance between experimental values was assessed using Student’s t test (**, P < 0.01).

Figure 6.

Transient expression assays in bean leaf discs for Cd tolerance. PvERF15 overexpression (OE), PvERF15 RNAi (RNAi), and 35S:GUS (Control) bean leaf discs were generated by A. tumefaciens-mediated plant transformation. A, Histochemical staining in the 35S:GUS leaf discs but not in untransformed control to monitor the validity of transformation. At least six leaf discs were examined, and a typical disc is presented. Bar = 2.5 mm. B, PvERF15 accumulation levels in overexpression and RNAi plants relative to the control (set as 1) determined by qRT-PCR analysis. C and D, Phenotype (C) and chlorophyll content (D) from overexpression, RNAi, and control bean leaf discs treated with 200 μm CdCl₂ (+Cd) or without (−Cd) for 48 h. Chlorophyll content (μg per leaf disc) is given as means ± sd of three independent experiments (at least 30 leaf discs were analyzed for each transgenic line in an experiment). Significance between experimental values was assessed using Student’s t test, and P values are provided.