transcription factors pverf15 and pvmtf-1 form a cadmium ... · et al., 2016). in fact, these...

Research Report

Transcription Factors PvERF15 and PvMTF-1 Form aCadmium Stress Transcriptional Pathway1[OPEN]

Tingting Lin2, Wanning Yang2, Wen Lu2, Ying Wang, and Xiaoting Qi*

College of Life Science, Capital Normal University, Beijing 100048, China

ORCID ID: 0000-0001-7172-2391 (X.Q.).

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, theupstream transcriptional regulatory pathways that modulate their responses to Cd are less clear. Previously, we identified thebean (Phaseolus vulgaris) METAL RESPONSE ELEMENT-BINDING TRANSCRIPTION FACTOR1 (PvMTF-1) that responds toCd and confers Cd tolerance in planta. Here, we demonstrate an upstream transcriptional regulation of the PvMTF-1 response toCd. Using a yeast one-hybrid system, we cloned the bean ETHYLENE RESPONSE FACTOR15 (PvERF15) that binds to thePvMTF-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 ofPvERF15 by RNA interference resulted in reduced Cd-induced expression of PvMTF-1. PvERF15 seems to be involved in Cdtolerance, since knockdown of PvERF15 by RNA interference in bean leaf discs decreased Cd tolerance in a transient assay. SincePvERF15 is a component of the Cd stress transcriptional pathway in beans and PvMTF-1 is one of its downstream targets, ourfindings provide a PvERF15/PvMTF-1 transcriptional pathway and thereby contribute to the understanding of Cd stresstranscriptional regulatory pathways in plants.

Cadmium (Cd) is highly toxic to plants. Genome-wide gene expression analyses in different plantspecies have revealed that plants alter transcriptomicexpression profiles in response to Cd stress (Weberet al., 2006; Gao et al., 2013; He et al., 2013; Liu et al.,2015; Xu et al., 2015; Oono et al., 2016). These findingsindicate that transcriptional regulation of Cd tolerance-related genes is a conserved strategy for plant responseto this heavy metal. Cd-responsive transcription isoften modulated by multiple transcription factors,some of which confer Cd tolerance through controllingthe expression of Cd detoxification genes. Previous studieshave characterized a few Cd-responsive transcriptionfactors, including wheat (Triticum aestivum) heat shocktranscription factor A4a (Shim et al., 2009), Indianmustard (Brassica juncea) BjCdR15 (Farinati et al., 2010),

Arabidopsis (Arabidopsis thaliana) basic helix-loop-helixtranscription factors bHLH29, bHLH38, and bHLH39(Wu et al., 2012), maize (Zea mays) zinc finger proteinOXIDATIVE STRESS2 (He et al., 2016), and Arabi-dopsis zinc finger transcription factor ZAT6 (Chenet al., 2016). In fact, these Cd-responsive transcriptionfactors are the key downstream effectors of Cd stresstranscriptional pathways. They trigger the expressionof Cd detoxification genes and, thus, converge Cd stresssignals (DalCorso et al., 2010; Chmielowska-Bąk et al.,2014). Although the downstream targets of these tran-scription factors have been identified extensively (Shimet al., 2009; Farinati et al., 2010; Wu et al., 2012; Sunet al., 2015; Chen et al., 2016; He et al., 2016), the up-stream transcriptional regulatory pathways that mod-ulate their responses to Cd remain unclear.

Wepreviously identified bean (Phaseolus vulgaris)METALRESPONSE ELEMENT-BINDING TRANSCRIPTIONFACTOR1 (PvMTF-1) as a Cd-responsive transcrip-tion factor (Sun et al., 2015). PvMTF-1 contains a zincfinger-like domain and confers Cd tolerance of tobacco(Nicotiana tabacum) plants through activating Trp bio-synthesis (Sun et al., 2015). The expression of PvMTF-1 isdriven by an intronic promoter located in the beanSTRESS-RELATEDGENE2 (PvSR2) locus (Qi et al., 2007;Sun et al., 2015; Yang et al., 2015). This intronic promoter(hereafter named PvMTF-1 promoter) is composed of a397-bp PvSR2 genomic sequence between the upstreamtranscription start site and the intronic transcription startsite (Supplemental Fig. S1). In bean, PvSR2 confersCd tolerance of tobacco plants (Chai et al., 2003) and is

1 This work was supported by the National Natural Science Foun-dation of China (grant nos. 31271293 and 30770181 to X.Q.) and theBeijing Natural Science Foundation (grant no. 5112005 to X.Q.).

2 These authors contributed equally to the article.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Xiaoting Qi ([email protected]).

T.L., W.Y., and X.Q. designed the research and wrote the article;T.L., W.Y., W.L., Y.W., and X.Q. conducted the experiments and an-alyzed the data; all authors reviewed and interpreted the data andedited the article; all authors reviewed the results and approved thefinal version of the article.

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one of the PvMTF-1 downstream target genes (Yang et al.,2015).

Interestingly, the expression of PvMTF-1 is inducedby Cd stress (Sun et al., 2015), suggesting that it shouldbe activated by putative upstream transcription factorsin the Cd response pathways. In this study,we identifiedand functionally characterized a bean ethylene responsefactor, PvERF15, involved in Cd-induced PvMTF-1 acti-vation and Cd tolerance. It binds to an AC-rich element(ACE) in the promoter of the PvMTF-1 gene and thenactivates its expression. These findings provide new in-sights into understanding the Cd stress transcriptionalpathways in plants.

RESULTS

PvERF15 Is a Cd-Responsive Transcription Factor ThatBinds to the PvMTF-1 Promoter

To identify putative transcription factors regulatingPvMTF-1, we constructed a cDNA library from Cd-treated bean seedlings and performed the yeast one-hybrid (Y1H) assay with the PvMTF-1 promoter asbait. A total of 33 104 clones were screened for growthon His-free plates in the presence of 2 mM 3-amino-1,2,4-triazole, and 51 colonies tested positive. Bysequencing cDNA from these clones, we isolatedseven independent clones of a transcription factor with

255 amino acids that belongs to the bean ERF tran-scription factor family. The AP2/ERF domain of thisbean ERF displays high similarity (62%) with that ofArabidopsis ERF15 (AtERF15; At2g31230); therefore,we designated this transcription factor as PvERF15(PHAVU_007G193300g; Supplemental Fig. S2). We se-lected it as a potential transcriptional regulator ofPvMTF-1 for further study. The Y1H assay was con-ducted to further confirm the screening results (Fig. 1A).

Next, we checked whether PvERF15 acts as a tran-scription factor. PvERF15 was localized in the nucleus ofNicotiana benthamiana cells (Fig. 1B). The GAL4-bindingdomain (BD)-PvERF15 fusion protein (BD-PvERF15)but not the BD alone (negative control) was able toactivate the expression of the GAL4 upstream acti-vating sequence-driven LacZ reporter gene in yeast(Saccharomyces cerevisiae) cells (Fig. 1C). These resultssuggested that PvERF15 is a transcriptional activatorin the nuclei. Since we sought to determine the tran-scriptional regulation of PvMTF-1 by Cd in this study,we checked whether PvERF15 also is regulated by Cd.A time-course analysis of Cd-inducible gene expres-sion in bean leaf discs was performed using real-timequantitative reverse transcription (qRT)-PCR over24 h. The mRNAs of these two genes were increasedduring a 6-h period (31.5- and 2.85-fold induction forPvERF15 and PvMTF-1, respectively) and graduallydecreased thereafter (Fig. 1D). These results suggest

Figure 1. PvERF15 is a Cd-responsive transactivator that binds to the PvMTF-1 promoter. A, Y1H binding assay of PvERF15 to thePvMTF-1 promoter (ProPvMTF-1). A yeast strain with the HIS3 gene driven by ProPvMTF-1 was transformed with a plasmidencoding the GAL4 activation domain (AD) alone or with a PvERF15 fusion (AD-PvERF15). Interaction is indicated by the abilityof cells to grow on His-deficient medium (2His) with or without 3-amino-1,-2,-4-triazole (3-AT). Three independent yeast clonesare shown. B, Subcellular localization of PvERF15-GFPand GFP transiently expressed from the 35S promoter inN. benthamianaepidermal cells. DAPI, 4,6-Diamidino-2-phenylindole (nuclei staining). C, The transcriptional activation activity of PvERF15 wasanalyzed 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-a-D-galactopyranoside (X-a-gal). The Gal4 DNA-binding domain (BD) alonewas used as a negative control. Three independent yeast clones are shown. D, Time-course analysis of Cd-inducible gene ex-pression in bean leaf discs. Bean leave discs were treatedwith 200mM CdCl2 for 0, 6, 12, and 24 h. RNAswere then extracted andsubjected to qRT-PCR. PvERF15 and PvMTF-1mRNA 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 barsrepresent SD. Significance between experimental values was assessed using Student’s t test (*, P , 0.05; and **, P , 0.01).

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that both PvMTF-1 and PvERF1 respond to Cd stress,but PvMTF-1 is more weakly induced by Cd thanPvERF15. Taken together, we concluded that PvERF15is a Cd-responsive transcription factor that binds tothe PvMTF-1 promoter.

PvERF15 Binds to an ACE in the PvMTF-1 Promoter

To demonstrate the in vivo binding of PvERF15 onthe PvMTF-1 promoter, we performed chromatin im-munoprecipitation (ChIP) using transgenic bean leafdiscs transiently expressing GFP-fused PvERF15 underthe control of the cauliflower mosaic virus (CaMV) 35S

promoter (35S:PERF15-GFP; Fig. 2A). The quantitativePCR (qPCR) analysis revealed significant enrichment(3.4-fold enrichment compared with that of the 39 un-translated region [UTR]; P , 0.05) of PvERF15 at thePvMTF-1 promoter region 1 (2397/2175; P1) but not atthe overlapping region 2 (2201/24; P2). However, nosignificant enrichment of PvERF15 at P1 and P2 wasobserved in the wild-type control samples (Fig. 2B).These results suggest that PvERF15 binds directly to the197-bp PvMTF-1 promoter region (2397/2201) in vivo.

However, we were more interested in the PvMTF-1promoter region, which contains unknown ERF-bindingcis-elements such as the GCC box and drought-responsive element (DRE). Therefore, we further

Figure 2. PvERF15 binds an ACE withinthe PvMTF-1 promoter. A, Immunoblot(IB) confirming the expression ofPvERF15-GFP (56.75 kD) in bean leafdiscs. Untransformed bean leaf discs(Wild-type) were used as a negative con-trol. Two independent 35S:PvERF15-GFPlines (1# and 2#) were included. ACoomassie Brilliant Blue (CBB)-stainedgel served as a loading control. B, ChIPexperiments were used with GFP anti-body and mouse IgG (mock control).The diagram of the PvMTF-1 gene indi-cates the amplicons (P1, P2, and 39 UTR)used for subsequent qPCR analysis. Rela-tive enrichment was calculated by com-paring GFP antibody-immunoprecipitatedDNAwith those immunoprecipitatedwiththe IgG control (binding ratio of GFP an-tibody to IgG). 39 UTR was used as anegative control. Error bars represent SD.Significance between experimentalvalueswas assessed using Student’s t test(*, P , 0.05). ORF, Open ReadingFrame. C, Diagram of the PvMTF-1 pro-moter subfragments as probes in EMSA.F1 sequences (wt) and mutations intro-duced into F1 (m1, m2, and m3) areshown at bottom. ACE is boxed. D, EMSAwas performed using biotin-labeledprobes with the affinity-purified recom-binant GST-PvERF15 and GST (mockproteins). The bound complex is indi-cated by the arrow. E, Competition ex-periments using a 1,000-fold excess ofunlabeled competitors (wt, m1, m2, andm3). The bound complex is indicated bythe arrow. F, Y1H binding assay ofPvERF15 to wild-type ACE or a mutantversion (ACEm; mutations shown inlowercase letters) bait. Other details aregiven in the legend to Figure 1.

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delineated the PvERF15-binding element into frag-ments by conducting an electrophoretic mobility shiftassay (EMSA). The promoter fragment was partitionedinto four ;50-bp regions (F1–F4; Fig. 2C). The gluta-thione S-transferase (GST)-PvERF15 fusion protein(GST-PvERF15), but not GST alone, specifically recog-nized the biotin-labeled F1 probes but not the F2 to F4probes (Fig. 2D).

Because F1 contained AC-rich sequences (78% ACcontent), we mutated C to T in the F1 and named theseas m1, m2, and m3 (Fig. 2C). The binding ability ofPvERF15 with these mutated probes was examined bythe competition assay (Fig. 2E). Like wild-type F1, m1andm3were able to compete the interaction of PvERF15with F1 probe. In contrast, m2 fails in inhibiting itsbinding to PvERF15. These results indicate that the 19-bpF1 sequence (2382/2364; 59-CCTAAACCCCAAAA-CAATC-39) is essential for the binding of PvERF15. Wethereby named this sequence as the ACE. To confirm thebinding of PvERF15 to this element, we performed aY1H assay using two copies of ACE as bait. The resultsshowed that PvERF15 was capable of binding to ACEbut not to the mutated ACE (Fig. 2F).

ACE Is a Non-Cd Response Element But Acts as aPositive Element

To investigate whether ACE acts as a Cd responseelement, we performed transient GUS assays in beanleaf discs as described by Peng et al. (2015). A GUS re-porter gene controlled by two copies of ACE and theminimal (246/+8) cauliflower mosaic virus 35S pro-moter [ACE-35S(246/+8):GUS; Fig. 3A] was used toquantify the activity of ACE. In transient GUS assays,we used GUS under the control of a minimal 35S pro-moter [35S(246/+8):GUS; Fig. 3A] as a vector control. The

bean leaf discs transiently expressing each of these con-structs were treated without or with Cd and then assayedfor GUS protein accumulation by western blot (Fig. 3B).Low GUS accumulation was found in 35S(246/+8):GUStransgenic control plants (Fig. 3B, lanes 3 and 4),reflecting a low activity of 35S(246/+8). However, plantsexpressing ACE-35S(246/+8):GUS exhibited higher GUSaccumulation than control plants (Fig. 3B, lane 1). Theseresults suggest that ACE acts as a positive element,since it confers gene expression when fused to aminimal promoter. ACE failed to confer Cd-responsiveexpression of the GUS gene, because the GUS accumu-lation level was not altered significantly by Cd treatmentin the plants expressing ACE-35S(246/+8):GUS (Fig. 3B,lanes 1 and 2). These results suggest that ACE is a non-Cd response element but acts as a positive element.

Figure 3. Transient assays in bean leaf discs confirm ACE as a positiveelement. A, Schematic diagrams of the test constructs in the transientassays. B, Immunoblot analysis of plants expressing GUS. The bean leafdiscs transiently transformed with each of the constructs were treatedwithout (2Cd) and with 200 mM CdCl2 (+Cd) for 24 h. GUS expressionwas determined by immunoblot (IB) assays using anti-GUS and anti-tubulin (as a loading control) antibodies. The experiments were re-peated independently two times with similar results.

Figure 4. GUS transient assays of PvERF15 transcriptional activity inN. benthamiana leaf discs. A, Schematic diagram of constructs used inthe experiments. B,N. benthamiana leaf discs were cotransformedwithcombinations 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 asthe relative immunoblotting signal intensity of GUS to GFP and repre-sents an average from three independent experiments. Error bars rep-resent SD. Significance between experimental values was assessed usingStudent’s t test, and P values are provided.


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PvERF15 Activates Gene Expression via ACE

To test whether PvERF15 depends onACE to activategene expression in planta, we performed transient GUSassays in N. benthamiana as described by Shim et al.(2013). ACE-35S(246/+8):GUS or its ACE mutant (C-to-T)version [ACEm-35S(246/+8):GUS; Fig. 4A] was used as areporter. 35S:PvERF15-GFP and 35S:GFP were used aseffector and control, respectively (Fig. 4A). The reporterand effector were coinfiltrated into N. benthamianaleaf discs and then subjected to GUS activity analysis(Fig. 4B). In theACE-35S(246/+8):GUS transgenic reporterplants, the expression of PvERF15-GFP resulted in amore than 3.5-fold increase of GUS expression com-paredwith the GFP-alone control, providing evidencefor a positive transactivation activity of PvERF15. Inthe ACEm-35S(246/+8):GUS transgenic reporter plants,however, PvERF15-GFP showed a GUS expressionlevel similar to that of the GFP control, suggestingthat mutations in the ACE completely abolished thetransactivation of PvERF15. These data suggestthat PvERF15 activates gene expression in an ACE-dependent manner.

PvERF15 Is Essential for Both Basal and Cd-InducibleExpression of PvMTF-1

PvERF15 is able to bind directly to the ACE-containingPvMTF-1 promoter in vivo (Fig. 2), and PvERF15activates gene expression via ACE (Fig. 4). These re-sults imply that PvERF15 possibly acts as a tran-scriptional regulator of PvMTF-1. To demonstrate

this, we examined the effects of overexpression andunderexpression of PvERF15 on the PvMTF-1 ex-pression in bean plants. 35S:PvERF15 (Fig. 5A) wasintroduced into bean leaf discs to generate PvERF15-overexpressing plants (OE). PvERF15 RNA interference(RNAi)-mediated knockdown plants were gener-ated by transfecting leaf discs with the RNAi con-struct (35S:RNAiPvERF15; Fig. 5A). This RNAi constructcontains a 327-bp PvERF15-specific DNA sequence(+1/+327, relative to the translational start codon).In transient assays, the 35S:GUS transgenic leafdiscs were used as a control. RNAi plants and over-expression plants had less than 25% and more than200% of the control PvERF15 mRNA level, respec-tively (Fig. 5B, top). This overexpression and under-expression of PvERF15 increased and suppressed thePvMTF-1 expression, respectively (Fig. 5B, bottom).These findings suggest that PvERF15 is required for thebasal expression of PvMTF-1.

Furthermore, we determined whether knockdownof PvERF15 affects Cd-induced PvMTF-1 expression.We generated RNAi plants that had less than 40% ofcontrol PvERF15 transcript level (Fig. 5C, top). BecausePvMTF-1 was strongly induced at 6 h of Cd stress(Fig. 1D), we selected this time point in this experi-ment. Approximately 4.1- and 2.35-fold induction ofPvMTF-1were detected in the control and RNAi plants,respectively (Fig. 5C, bottom). The result showedthat knockdown of PvERF15 decreased Cd-induciblePvMTF-1 expression level by approximately 43% com-pared with the control, indicating an important role ofPvERF15 in Cd-induced expression of PvMTF-1.

Figure 5. PvERF15 is a transcriptional regulator of PvMTF-1. A, Schematic diagram of plasmids used in the transient assay.35S:RNAiPvERF15 contains a 327-bp coding sequence (black box) of PvERF15 in the sense and antisense orientation inter-spersed by intron 1 (intron) of the potato GA20 OXIDASE gene. Arrows indicate the primers used in the RT-PCR assay forPvERF15. 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-PCRusing a pair of primers, ORF-F and ORF-R. The ACTIN gene was used as a loading control. PvERF15 relative abundance isshown at bottom, and the control was set as 1. B, PvMTF-1 expression levels in overexpression and RNAi plants relative to thecontrol (set as 1) determined by qRT-PCR analysis. C, Control and RNAi plants treated with Cd for 6 h and then subjected toqRT-PCR analysis for Cd-inducible PvMTF-1 expression relative to pretreatment (0 h; set as 1). SD values are from threetechnical replicates of one biological experiment. The experiments were repeated independently two times with similarresults. Significance between experimental values was assessed using Student’s t test (**, P , 0.01).





Knockdown of PvERF15 Decreases Cd Tolerance in aTransient Assay

To assess the functional relevance of PvERF15 in beanagainst Cd stress, we used a transient assay to check thecapability of PvERF15-OE and PvERF15-RNAi beanleaf discs to tolerate Cd stress. In the transient assays,the bean leaf discs transiently expressing 35S:GUSwereused as a control. The effectiveness of transformationand the level of PvERF15 transcript accumulation weredetected 24 h after transformation. Detectable GUS ac-tivity in the 35S:GUS control leaf discs confirmed the

validity of transformation (Fig. 6A). Further qRT-PCRanalysis showed that RNAi plants and overexpressionplants had less than 45% and more than 180% of thecontrol PvERF15 mRNA accumulation level, respec-tively (Fig. 6B). Compared with untreated leaf discs,the leaf discs showed chlorosis 48 h after Cd treatment(Fig. 6C), indicative of a symptom of Cd toxicity(Laspina et al., 2005). No distinguishable difference wasobserved among these Cd-treated leaf discs. To gainfurther insight into the toxicity of Cd, we determinedthe changes in the chlorophyll level of leaf discs in re-sponse to Cd (Fig. 6D). Under both Cd stress andnon-Cd stress conditions, overexpression plants showedno substantial difference (P . 0.05, Student’s t test)compared with the control plants. Under the conditionwithout Cd stress, there was no substantial difference(P. 0.05, Student’s t test) between the RNAi plants andthe control plants. After Cd treatment, however, chlo-rophyll content in the RNAi plants was much lower(P, 0.05, Student’s t test) than that of the control plants.This finding suggested that knockdown of PvERF15resulted in a decreased Cd tolerance, supporting a pos-sible role of PvERF15 in bean leaf tissues against Cd stress.

DISCUSSION

PvERF15 and PvMTF-1 Form a Cd Stress TranscriptionalRegulatory Pathway

PvMTF-1 is a key component of the Cd stress tran-scriptional pathway in plants. In this study, we dem-onstrate that PvERF15 is located upstream of PvMTF-1and acts as a transcriptional regulator of PvMTF-1 ex-pression via the ACE of its promoter. A working modelof PvERF15 on the PvMTF-1 response to Cd is pre-sented. Upon Cd stress, PvERF15 expression is inducedto bind a positive element ACE and enhance PvMTF-1expression. Several lines of evidence support thismodel: (1) PvERF15 is strongly induced by Cd stress,and its overexpression results in the up-regulation ofPvMTF-1; (2) knockdown of PvERF15 by RNAi resultedin reduced Cd-induced expression of PvMTF-1; (3) ACEacts as a positive element through which PvERF15 ac-tivates gene expression; and (4) PvERF15 seems to beinvolved in Cd tolerance, since knockdown of PvERF15in bean leaf discs decreased Cd tolerance in a transientassay. Although it is unknown how PvERF15 sensesstress signals and regulates the bean response to Cdstress, the results presented here strongly support thatPvERF15 is a component of the Cd stress transcriptionalpathway in beans and PvMTF-1 is one of its downstreamtargets. Our findings provide a PvERF15/PvMTF-1transcriptional pathway and thereby contribute tothe understanding of Cd stress transcriptional path-ways in plants. In this study, genetic evidence for thebiological relevance of PvERF15 is based mainly onRNAi-mediated knockdown of PvERF15 in bean leafdiscs. To have a more complete understanding ofPvERF15 function in the bean Cd response, stable

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-mediatedplant transformation. A, Histochemical staining in the 35S:GUS leafdiscs but not in untransformed control to monitor the validity of trans-formation. At least six leaf discs were examined, and a typical disc ispresented. Bar = 2.5 mm. B, PvERF15 accumulation levels in over-expression and RNAi plants relative to the control (set as 1) determinedby qRT-PCR analysis. C and D, Phenotype (C) and chlorophyll content(D) from overexpression, RNAi, and control bean leaf discs treatedwith 200mMCdCl2 (+Cd) orwithout (2Cd) for 48 h. Chlorophyll content(mg per leaf disc) is given as means 6 SD of three independent experi-ments (at least 30 leaf discs were analyzed for each transgenic line in anexperiment). Significance between experimental values was assessedusing Student’s t test, and P values are provided.


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PvERF15 knockout bean lines will be needed for futurestudy.

Cd-Induced Transcription Can Be Modulated by a Non-CdResponse Element

The working models of Cd-induced transcriptionremain largely unclear. We previously revealed aCd-responsive transcription factor (such as PvMTF-1)/Cdresponse element (such as the metal response element)working model (Sun et al., 2015). However, unlike themetal response element, ACE is a positive elementand interacts with a Cd-responsive PvERF15 tran-scription factor to regulate Cd-induced transcription.Our findings, therefore, suggest a non-Cd responseelement/Cd-responsive transcription factor workingmodel. Our results thereby extend our knowledge ofCd-induced transcriptional regulation.

ACE Is a New ERF-Binding Element

Interestingly, ACE is different from the known ERFtranscription factor-binding sites such as the GCC boxand DRE, indicating that it is a new ERF-binding ele-ment. It is well established that ERFs are involved indifferent biological processes through binding a diverseset of cis-elements present in downstream targets(Sasaki et al., 2007). In the future, experiments assessingthe core sequences of ACE and its role in the ethyleneresponse will be necessary.

MATERIALS AND METHODS

Plant Materials and Treatments

The leaves of 4- to 5-week-old Nicotiana benthamiana plants were used fortransient expression assays and subcellular localization of PvERF15-GFP pro-tein. The seeds of bean (Phaseolus vulgaris ‘Saxa’) were sown on a soil mix ofcommercial potting soil:perlite (3:1) at 22°C with a 16-h/8-h light/dark cycle.The detached leaves (8 –10 cm long and 4–5 cmwide) from bean seedlings wereused for transient expression assays and Cd stress. For Cd treatment, bean leafdiscs (5 mm diameter) were transferred to a filter paper soaked with one-quarter-strength Murashige and Skoog (MS) liquid medium supplementedwith 200 mM CdCl2 in the greenhouse. The samples were removed from thecultures and frozen in liquid nitrogen at 0, 6, 12, and 24 h.

Y1H Library Screening

Two-week-old bean seedlings were grown on MS liquid medium (control)with 200 mM CdCl2 for 24 h and then used to construct a GAL4 AD-cDNAlibrary using the yeast SfiI-digested pGADT7 vector and the Creator SMARTcDNA Library Construction Kit (Clontech). The resulting PvMTF-1 pro-moter bait plasmid and AD-cDNA library were introduced into the yeast(Saccharomyces cerevisiae) strain Y187 (Clontech). Because the bait plasmiddid not undergo self-activation in Y187 yeast cells, positive clones were isolatedby growth on His-free SD medium containing 1 mM 3-amino-1,2,4-triazole.Plasmid DNA was isolated from yeast cells and sequenced. The Y1H assayswere performed as described previously (Sun et al., 2015).

Primers

Oligonucleotide primers used for PCR and the construction of expressionvectors are listed in Supplemental Table S1.

Plasmid Construction

35S:PvERF15 and 35S:PvERF15-GFP were generated using the ClonExpressII One Step Cloning Kit (Vazyme Biotech). The coding region of PvERF15was PCR amplified using the primers 121-PvERF15-F and 121-PvERF15-Rand then cloned into the BamHI/SacI sites of pBI121 (Clontech). The codingregion of PvERF15 was PCR amplified using the primers 1302-PvERF15-F and1302-PvERF15-R and then cloned into the BglII/SpeI sites of pCAMIBA1302(Clontech). The 35S:RNAiPvERF15 plasmid was constructed by fusion PCR ofthree amplified overlapping PCR fragments. Using AD-PvERF15 as a template,the sense RNAiPvERF15 fragment was amplified using the primers ERF15-sF andERF15-sR and the antisense RNAiPvERF15 fragment was amplified using theprimers ERF15-asF and ERF15-asR. The intron of the potato (Solanum tuber-osum) GA20 OXIDASE gene was obtained from a pUCCRNAi plasmid by PCRusing the primers GA20in-F and GA20in-R. The final PCR was performedusing mixed PCR products (33 ng of each PCR product) using ERF15-sF andERF15-asR. The PCR products were cloned into the BamHI/SacI sites of pBI121(Clontech). For bait construction, the PvMTF-1 promoter was amplified frombean genomic DNA using the primers ProPvMTF-1-F and ProPvMTF-1-R.23ACE and 23ACEm DNA were synthesized by Shanghai Sangon Biotech-nology. These baits were then cloned into the EcoRI/SpeI sites of pHIS2.1(Clontech) to generate bait constructs. All of the constructs were verified bysequencing using the corresponding sequencing primers as described by Sunet al. (2015).

We used an asymmetric overlap extension PCR method to constructACE-35S(246/+8):GUS and ACEm-35S(246/+8):GUS plasmids using AC-35S-Fand ACEm-35S-F and a common reverse primer, AC-35S-R. The PCR pro-ducts were then cloned into the HindIII/XbaI sites of pBI121 instead of the 35Spromoter and sequenced using GUS-seqR (59-CCCACACTTTGCCGTAATGA-39).

Subcellular Localization

The 35S:PvERF15-GFP andpCAMBIA1302 empty vector (35S:GFP) constructswere transformed into N. benthamiana leaves by Agrobacterium tumefaciens infil-tration. The fluorescence signal was observed with a Zeiss 5 Live laser scanningconfocal microscope at 42 h after transformation.

Transcriptional Activation Activity Assay in Yeast Cells

The coding sequence of PvERF15 was PCR amplified and cloned intopGBKT7 (Clontech) to generate BD-PvERF15. Its transcriptional activation ac-tivity was determined in SD medium lacking Trp and containing 40 mg mL21

5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside (Sigma-Aldrich).

RNA Isolation and cDNA Synthesis

Total RNAwas extracted from plant material using the RNAprep pure plantkit with on-columnDNase digestion (Tiangen Biotech). RNA (2mg) was used tosynthesize the first-strand cDNA with oligo(dT) primers with the PrimeScriptfirst-strand cDNA synthesis kit (Takara).

A. tumefaciens-Mediated Gene Transfer by Infiltration ofPlant Leaf Discs

Various constructs tested were introduced into A. tumefaciens GV3101. Theleaf discs of bean orN. benthamianawere vacuum infiltrated with A. tumefaciensas described previously byMarion et al. (2008). Briefly,A. tumefaciens cells werecollected and resuspended at an optical density at 600 nm of 2 in one-quarter-strength MS liquid medium containing 5% (w/v) Suc, 200 mM acetosyringone,and 0.01% (v/v) Silwet. After vacuum infiltration, transformed bean leaf discswere transferred to a filter paper soaked with one-quarter-strength MS solutionmedium.

ChIP Assay

Bean leaf discs expressing PvERF15-GFP fusion proteins and non-transformed leaf discs (wild-type control) were subjected to ChIP using an anti-GFP antibody (ab290; Abcam) following the instructions of the EpiQuik PlantChIP Kit (Epigentek). Western blot analysis of the PvERF15-GFP protein wasperformed using the anti-GFP antibody (ab290; Abcam). For ChIP-qPCRanalysis, 1 mL of 20-fold diluted immunoprecipitated DNA was analyzed by






qPCR on a CFX96 Real-Time System (Bio-Rad) with the SYBR Premix Ex-TaqKit (Takara). Enrichment of the ChIP target was expressed as a binding ratiobetween GFP antibody-immunoprecipitated samples and those immunopre-cipitatedwith the IgG control. The binding ratio was calculated using the 22DDCt

method as described previously (Mukhopadhyay et al., 2008). The followingprimers were used: P1-F and P1-R for P1, P2-F and P2-R for P2, and 39-UTR-Fand 39-UTR-R for 39 UTR.

Fusion Protein Preparation and EMSA

AGST-PvERF15 fusionproteinwaspreparedby cloning thePvERF15 codingregion into the BamHI/SalI sites of the pGEX 4T-1 plasmid (GE Healthcare Bio-Sciences). GST-PvERF15 fusion proteins were isolated according to the manu-facturer’s protocol (GE Healthcare Bio-Sciences) and subsequently subjectedto an EMSA using the LightShift Chemiluminescent EMSA Kit (Pierce). The59-biotin-labeled DNA probes or completed DNA were synthesized byShanghai Sangon Biotechnology.

Transient Gene Expression Analysis in Bean andN. benthamiana Leaf Discs

For ACE functional analysis, transformedN. benthamiana leaf discs weretreated without (2Cd) and with 200 mM CdCl2 (+Cd) for 24 h. GUS ex-pression was determined by western blot using anti-GUS and anti-tubulin(as a loading control) antibodies. In GUS transient assays of PvERF15transcriptional activity, transformed N. benthamiana leaf discs were grownon one-quarter-strength MS for 2 d. GUS and GFP expression was deter-mined by western blot using anti-GUS or anti-GFP antibodies. To deter-mine the expression of PvMTF-1 regulated by PvERF15, transformed beanleaf discs were grown on one-quarter-strength MS solution medium for1 d. One-half of the samples were removed from the cultures and frozen inliquid nitrogen at 0 h, and the other half were transferred to a filter papersoaked with fresh one-quarter-strength MS solution with 200 mM CdCl2for 6 h.

Total RNA was isolated and then subjected to cDNA synthesis. The ex-pression of PvERF15 was confirmed by semiquantitative reverse transcription-PCR using the primers ORF-F andORF-R. qRT-PCR analysis was performed onthe CFX96 Real-Time System (Bio-Rad) with the SYBR Premix Ex-Taq Kit(Takara). Real-time qRT-PCR analysis of PvERF15 or PvMTF-1 transcripts wasperformed using PvERF15-rF and PvERF15-rR for PvERF15 and PvMTF-1-rFand PvMTF-1-rR for PvMTF-1. The bean ACTIN gene was used as an internalcontrol using the primers Actin-F and Actin-R.

Leaf Disc Assay for Cd Tolerance

Transformed bean leaf discs were grown on one-quarter-strength MSsolution medium for 1 d. GUS staining of bean leaf discs transientlyexpressing 35S:GUS was monitored for transformation efficiency. Real-time qRT-PCR analysis of PvERF15 transcripts was performed usingPvERF15-rF and PvERF15-rR. The bean ACTIN gene was used as an in-ternal control using the primers Actin-F and Actin-R. These bean leafdiscs were then transferred to a filter paper soaked with fresh one-quarter-strength MS solution without (control) or with 200 mM CdCl2for another 2 d. Chlorophyll was extracted in 80% acetone and quantifiedaccording to Arnon (1949). The concentration was calculated using the followingequation: total chlorophyll (mg mL21) = 20.23 OD663 + 8.023 OD645, where ODrepresents optical density at the given values.

Total Protein Extraction and Western Blot

Total protein from the plant samples was extracted using extraction buffer(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA,10% [v/v] glycerol,5 mM dithiothreitol, 0.5% [v/v] Triton X-100, 1 mM phenylmethylsulfonylfluoride, and 1% [v/v] Nonidet P-40). Proteins separated on a gel wereelectrophoretically transferred to a pure nitrocellulose blotting membrane(Pall Life Sciences). The membrane was cut across the molecular mass regionof corresponding proteins and separately probed with anti-GUS antibody(Clontech), anti-GFP antibody (Abcam), or anti-tubulin antibody (Sigma-Aldrich). Protein blots were developed with an ECL kit (Amersham Phar-macia Biotech), and images were obtained using the LAS3000 image-capturesystem (Fujifilm).

Statistical Analysis

Statistical analysis (SD and P values) was performed using Microsoft Excel2007. Significance (P , 0.05 and P , 0.01) was assessed using Student’s t test.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers XM_007144842 (PvERF15), AB067722(ACTIN ), and DQ109993 and U54704 (PvMTF-1).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Schematic representation of the PvMTF-1 genelocated within the PvSR2 locus.

Supplemental Figure S2. Nucleotide and deduced amino acid sequencesof PvERF15 cDNA.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Ligeng Ma (College of Life Science, Capital Normal University)for providing pUCCRNAi plasmid, anti-GUS antibody, and anti-tubulin anti-body and Weiwei Zhang (College of Life Science, Capital Normal University)for linguistic assistance during the preparation of this article.

Received November 8, 2016; accepted January 8, 2017; published January 10,2017.

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