The Protein Phosphatase RCF2 and Its Interacting PartnerNAC019 Are Critical for Heat Stress–Responsive GeneRegulation and Thermotolerance in ArabidopsisW

Qingmei Guan, Xiule Yue, Haitao Zeng, and Jianhua Zhu1

Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland 20742

ORCID ID: 0000-0003-3258-3071 (J.Z.)

Heat stress is a major environmental constraint for crop production worldwide. To respond to and cope with heat stress, plantssynthesize heat shock proteins (HSPs), which are often molecular chaperones and are under the control of heat stresstranscription factors (HSFs). Very little is known about the upstream regulators of HSFs. In a forward genetic screen forregulators of C-REPEAT BINDING FACTOR (CBF) gene expression (RCFs), we identified RCF2 and found that it is allelic toCPL1/FIERY2, which encodes a homolog of C-terminal domain phosphatase. Our results also showed that, in addition to beingcritical for cold stress tolerance, RCF2 is required for heat stress–responsive gene regulation and thermotolerance, because,compared with the wild type, the rcf2-1 mutant is hypersensitive to heat stress and because the reduced thermotolerance iscorrelated with lower expression of most of the 21 HSFs and some of the HSPs in the mutant plants. We found that RCF2interacts with the NAC transcription factor NAC019 and that RCF2 dephosphorylates NAC019 in vivo. The nac019 mutant ismore sensitive to heat stress than the wild type, and chromatin immunoprecipitation followed by quantitative PCR analysisrevealed that NAC019 binds to the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. Overexpression of RCF2 or NAC019 inArabidopsis thaliana increases thermotolerance. Together, our results suggest that, through dephosphorylation of NAC019,RCF2 is an integrator of high-temperature signal transduction and a mechanism for HSF and HSP activation.

INTRODUCTION

Land plants are frequently challenged by the changing physicalenvironment that often generates various biotic and abioticstresses, including heat and drought and sometimes a combina-tion of heat and drought. Heat stress is usually defined as acondition in which temperatures are sufficiently high for enoughtime to irreversibly damage plant function or development. Heattolerance is generally defined as the ability of the plant to growand produce economic yield under high temperatures. Heatstress reduces crop production worldwide. The detrimental ef-fects of heat stress can be alleviated by developing heat-tolerantcrop plants by various genetic strategies, including traditional andcontemporary molecular breeding protocols and transgenic ap-proaches. Although a few plants with improved thermotolerancehave been developed through the use of traditional breedingpractices, the success of genetic transformation has been limitedbecause of limited knowledge and availability of genes withknown effects on plant thermotolerance. Overcoming these limi-tations and developing strategies for improving crop tolerance willrequire a comprehensive understanding of the physiologicalresponses of plants to high temperature and of the molecularmechanisms of heat tolerance.

Heat stress can adversely impact almost all aspects of plantgrowth, development, reproduction, and yield. Although everyplant tissue is vulnerable to heat stress, the reproductive tissuesare particularly susceptible (Zinn et al., 2010). At very high tem-peratures, severe cellular injury and even cell death can occurwithin a short time (e.g., within minutes), which may be due to acatastrophic collapse of cellular organization (Schöffl et al., 1999).At moderately high temperatures, injury or cell death may occuronly after a relatively long time (e.g., hours to days). Direct injuriesdue to high temperature include protein denaturation and ag-gregation and increased fluidity of membrane lipids, while indirectinjuries include inactivation of enzymes in chloroplasts and mi-tochondria, inhibition of protein synthesis, protein degradation,loss of membrane integrity, and disruption of cytoskeletonstructures (Smertenko et al., 1997; Howarth, 2005). These injuriescan eventually result in starvation, reduced ion flux, accumulation oftoxic by-products including reactive oxygen species, and disruptedgrowth and development (Schöffl et al., 1999; Howarth, 2005;McClung and Davis, 2010; Ruelland and Zachowski, 2010; Suzukiet al., 2012). Exposure to heat stress for prolonged periods caneven result in plant death, as exemplified by the huge loss in maize(Zea mays) and soybean (Glycine max) fields caused by the de-vastating heat waves in the summer of 2012.To resist heat stress, plants use a variety of mechanisms, in-

cluding the maintenance of membrane stability, scavenging of re-active oxygen species, production of antioxidants and compatibleorganic compounds, induction of mitogen-activated protein kinaseand calcium-dependent protein kinase signaling events, and, mostimportantly, induction of molecular chaperone signaling and tran-scriptional activation (Wahid et al., 2007). A central component ofresponses to heat stress in all living organisms including plants is

1 Address correspondence to jhzhu@umd.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Jianhua Zhu (jhzhu@umd.edu).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.113.118927

The Plant Cell, Vol. 26: 438–453, January 2014, www.plantcell.org ã 2014 American Society of Plant Biologists. All rights reserved.

the induction of heat shock proteins (HSPs) through the action ofheat stress transcription factors (HSFs). HSPs are categorized intofive classes based on their approximate molecular masses in kD:HSP100, HSP90, HSP70, HSP60, and small HSPs (15 to 30 kD;Vierling, 1991; Trent, 1996). HSPs function as molecular chaper-ones and are essential for the maintenance and/or restoration ofprotein homeostasis. HSFs are important components of the re-sponse to heat stress. More than 20 HSFs are encoded by plantgenomes, and these are grouped into three major classes (A, B,and C) based on structural characteristics and phylogenetic rela-tionships (Nover et al., 2001; Baniwal et al., 2004; Scharf et al., 2012).By recognizing and binding to heat stress elements (59-GAAnnTTC-39)conserved in promoters of heat stress–responsive genes, HSFsmediate the synthesis of HSPs (Busch et al., 2005).

The complexity of plant HSFs and their interactions are cur-rently being studied, and researchers have established thatHSFA1s act as master regulators of other HSFs and, therefore, asmaster regulators of heat stress responses in tomato (Solanumlycopersicum) and Arabidopsis thaliana (Mishra et al., 2002; Liuet al., 2011; Nishizawa-Yokoi et al., 2011; Yoshida et al., 2011).However, very little is known about the more upstream regulatorsof these HSFA1s or of the other members of the HSF family. Intomato, HSFB1 functions as a synergistic coactivator of HSFA1a(Bharti et al., 2004). Because of sequence differences in the Cterminus, the Arabidopsis HSFB1 is inactive as a coactivator oftomato HSFA1a (Bharti et al., 2004). In Arabidopsis, HSFB1 andHSFB2b repress the expression of heat-inducible HSF genes,such as HSFA2 and HSFA7a (Ikeda et al., 2011). All members ofthe DREB2 family (DREB2A, DREB2B, and DREB2C) are involvedin the regulation of heat-responsive genes including HSFA3(Sakuma et al., 2006; Schramm et al., 2008; Chen et al., 2010). Liuet al. (2008) reported that a calmodulin binding protein kinase 3functions as an upstream activator for HSFA1a in Arabidopsis.AtCAM3 plays a role in the Ca2+-calmodulin heat stress signaltransduction pathway in Arabidopsis (Zhang et al., 2009). Fur-thermore, AtHSBP interacts with HSFA1a, HSFA1b, and HSFA2and negatively regulates the binding of HSFA1b to a heat stresselement in vitro, thereby functioning as a negative regulator forheat stress responses in Arabidopsis (Hsu et al., 2010). A KHdomain–containing putative RNA binding protein, RCF3 (for reg-ulator of CBF gene expression 3), acts as a negative regulator ofheat stress–responsive gene expression and thermotolerance inArabidopsis (Guan et al., 2013a). Additional critical components inthe signal transduction pathway for heat-responsive gene regu-lation remain to be identified.

In this study, we identify RCF2 in a genetic screen for proteinscritical in cold-responsive gene expression. RCF2 is allelic toCPL1/FIERY2 (FRY2), which encodes C-terminal domain (CTD)phosphatase-like 1. We show that RCF2 physically interacts witha NAC transcription factor, NAC019, and that RCF2 dephosphor-ylates NAC019 under heat stress. Mutations in RCF2/CPL1/FRY2and NAC019 result in severely reduced thermotolerance andreduced expression of heat stress–responsive genes. NAC019 isable to bind to NAC019-specific cis-promoter elements in fourHSFs. Together, our results suggest a positive regulatory pathwaythat is mediated by RCF2/CPL1/FRY2–NAC019 protein pairs andthat is essential for the activation of heat stress–responsive genesand for thermotolerance in plants.

RESULTS

RCF2 Is a Positive Regulator for Freezing Tolerance

To identify important genes involved in cold stress tolerance inplants, we screened for mutants with altered expression of a fireflyluciferase reporter gene under the control of the cold-inducibleC-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC) inan ethyl methanesulfonate–mutagenized Arabidopsis population(Guan et al., 2013a, 2013c). These mutants were designated asrcfs. One of these mutants, rcf2-1, which displays enhancedCBF2:LUC expression upon cold treatment, was chosen for in-depth characterization (Supplemental Figure 1A). Consistent withincreased CBF2:LUC expression, the transcript level of the en-dogenous CBF2 is elevated in the rcf2-1 plants under cold stress(Supplemental Figure 1B). The rcf2-1 mutation also increases thecold induction of two other cold-specific members of the CBFgene family: CBF1 and CBF3 (Supplemental Figure 1B). We de-termined the effect of the rcf2-1 mutation on freezing tolerancewith electrolyte leakage assays. Without cold acclimation, rcf2-1and wild-type plants are essentially equally sensitive to freezingtemperatures (Supplemental Figure 1C). Although tolerance to thefreezing temperatures increased in both wild-type and rcf2-1plants after a 1-week cold acclimation process, the ability to ac-climate was substantially less in rcf2-1 than in wild-type plants(Supplemental Figure 1C). Thus, RCF2 is a positive regulator forfreezing tolerance.PSEUDO-RESPONSE REGULATOR5 (PRR5) is a negative reg-

ulator of CBF genes (Nakamichi et al., 2009; Guan et al., 2013c).Therefore, we determined the levels of PRR5 expression in thercf2-1mutant plants and found that PRR5 transcripts are markedlylower in rcf2-1 plants than in wild-type plants (Supplemental Figure1D). We also found that transcript levels of AGAMOUS-LIKE8 aresubstantially lower in rcf2-1 than in the wild type with or withoutcold stress (Supplemental Figure 1E). AGAMOUS-LIKE8 is knownas a MADS box transcription factor and is required for cellulardifferentiation during leaf and fruit development, including theelongation of siliques (Gu et al., 1998). In addition, the coldinduction of RING-H2 Finger A1A is reduced in rcf2-1 plants(Supplemental Figure 1E). RING-H2 Finger A1A is one of the RINGfinger domain–containing proteins, and the function of RING-H2Finger A1A is currently unknown (Kosarev et al., 2002). These re-sults suggest that RCF2 controls cold-responsive genes in bothpositive and negative manners.We backcrossed rcf2-1 with wild-type plants. All F1 plants

showed the wild-type phenotype, and F2 plants from the self-pollinated F1 plants displayed a segregation ratio of ;3:1 (thewild type versus rcf2-1; Supplemental Table 1). These resultsindicate that the rcf2-1 mutation is recessive and is caused bya mutation in a single nuclear gene. We also crossed rcf2-1 withwild-type plants (ecotype Landsberg erecta) to generate a map-ping population. Positional cloning revealed that RCF2 is allelic toa gene encoding a phosphatase homolog termed CPL1/FRY2(Supplemental Figure 1F; Koiwa et al., 2002; Xiong et al., 2002).The rcf2-1 mutation abolishes the splicing acceptor site of the

fifth intron and results in two abnormal transcripts of RCF2/CPL1/FRY2 in rcf2-1 (Supplemental Figures 2A and 2B). The first ab-normal transcript detected in rcf2-1 contains a one-nucleotide

Role of RCF2 in Plant Thermotolerance 439

deletion of G at nucleotide 1232 in the full-length wild-type RCF2coding sequence, corresponding to the last nucleotide G of thefifth exon (Supplemental Figure 2C). The predicted RCF2 proteinencoded by the first abnormal transcript in rcf2-1 consists of anN-terminal 411–amino acid fragment of the wild-type protein plusa C-terminal tail containing an extra 40 amino acids encodedby the frame-shifted 120 nucleotides (1234 to 1353 in the firstabnormal transcript). The truncated mutant protein lacks theC-terminal 556–amino acid segment of the wild-type RCF2 thatcontains two putative double-strand RNA binding motifs(dsRBMs) and the nuclear localization signal (NLS; SupplementalFigure 2C). The second abnormal transcript in rcf2-1 includes theentire fifth intron (Supplemental Figure 2B). The fifth intron ofRCF2 is 110 nucleotides and contains four in-frame stop codons.The predicted RCF2 protein encoded by the second abnormaltranscript in rcf2-1 consists of an N-terminal 411–amino acidfragment of the wild-type protein. The truncated mutant proteinlacks the C-terminal 556–amino acid segment of wild-type RCF2that contains two putative dsRBMs and an NLS (SupplementalFigure 2D). The N-terminal portion (amino acids 47 to 638) ofRCF2 is essential for its phosphatase activity (Koiwa et al., 2004).Therefore, both abnormal transcripts in rcf2-1 would encodetruncated proteins with no or significantly reduced phosphataseactivity. The C-terminal portion of RCF2 including the two puta-tive dsRBMs and NLS is critical for protein–protein interactionsand nuclear localization (Koiwa et al., 2004; Bang et al., 2008).Because normal RCF2 transcript was not detected in the rcf2-1plants and because rcf2-1 is a recessive mutant, our data sug-gest that rcf2-1 is a loss-of-function allele of RCF2/CPL1/FRY2.The wild-type RCF2/CPL1/FRY2 gene under the control of itsnative promoter (RCF2:RCF2-FLAG) is able to complement thephenotype of rcf2-1 (e.g., restoration of the cold induction ofCBF2:LUC and endogenous CBF genes to the levels in the wildtype; Supplemental Figure 3).

The fry2-1 mutation is a point mutation at a splicing junctionbetween the eighth exon and the eighth intron of RCF2/CPL1/FRY2 (Xiong et al., 2002). The fry2-1 mutation causes missplicing(intron retention) of the eighth intron in the abnormal transcriptdetected in fry2-1 (Koiwa et al., 2004). The fry2-1 mutant pro-duces a truncated RCF2 protein consisting of an N-terminal 676amino acids of wild-type protein plus a C-terminal tail containing8 extra amino acids encoded by the included eighth intron (Koiwaet al., 2004). The truncated mutant protein generated in fry2-1lacks the C-terminal 292–amino acid segment of the wild-typeRCF2 that contains the two dsRBMs and the NLS. As suggestedby in vitro phosphatase activity assays using recombinant RCF2/CPL1 truncated proteins (Koiwa et al., 2004), the truncated mu-tant protein produced in fry2-1 might retain phosphatase activityin vivo. However, because the C-terminal portion of RCF2 isimportant for protein–protein interactions and nuclear localization,which is missing in fry2-1 (Koiwa et al., 2004; Bang et al., 2008),the mutant protein encoded by the eighth intron-retained ab-normal transcript in fry2-1may not be properly localized in the cellto interact with RCF2 targets that are potentially dephosphory-lated by RCF2. Because normal RCF2/CPL1/FRY2 transcript wasnot detected in fry2-1, and because fry2-1 is a recessive mutant,we believe that fry2-1 is another loss-of-function allele of RCF2/CPL1/FRY2.

Xiong et al. (2002) reported that fry2-1 is more tolerant to NaCland abscisic acid (ABA) during seed germination but more sen-sitive to ABA at the seedling stage than the wild type. We foundthat, like fry2-1, rcf2-1 displays increased tolerance to NaCl andABA during seed germination but shows reduced tolerance toABA at the seedling stage (Supplemental Figures 4A to 4C).Consistent with the altered expression of AGAMOUS-LIKE8 in thercf2-1 plants, we observed that fertility is slightly reduced in rcf2-1plants compared with the wild type (Supplemental Figures 4D and4E). This reduced fertility is also evident in fry2-1 (SupplementalFigures 4D and 4E). Koiwa et al. (2002) showed that cpl1 plantsflower later than the wild type, and we found that flowering time isslightly later for rcf2-1 plants than for the wild type (SupplementalFigure 4F).

RCF2 Is a Positive Regulator for Heat Stress–ResponsiveGene Expression and Thermotolerance

The expression level of RCF2 is relatively low (Xiong et al., 2002),and there are no publicly available expression profiling data col-lected through microarray analyses for RCF2 (such as the in-formation in the e-FP browser database as described by Kilianet al. [2007]). Our quantitative RT-PCR (qPCR) analyses revealedthat RCF2 reaches its peak level at the reproductive stage(Supplemental Figure 5A). We also noticed that RCF2 is slightlyupregulated after 1 h of heat stress at 37°C and then down-regulated after longer exposure to heat stress (SupplementalFigure 5B). This finding prompted us to speculate that the rcf2-1mutation may affect heat stress responses. Indeed, the soil-grownrcf2-1 plants displayed a hypersensitive phenotype to heat stress,as indicated by dramatically reduced survival rates under heatstress (Figures 1A and 1B). Consistent with the peak expressionlevel of RCF2 occurring at the reproductive stage, the flowers ofthe rcf2-1 plants were also more susceptible to heat stress thanthose from wild-type plants (Figure 1B). We then determined theexpression levels of most of the 21 HSFs in rcf2-1. The expressionof HSFC1, HSFA4a, and HSFA6b is substantially reduced in rcf2-1plants under control conditions (Figures 1C and 1F). Transcriptlevels of most HSFs (including HSFA1b) examined in this study aresignificantly lower in rcf2-1 plants than in the wild type under heatstress (Figures 1C to 1G). In contrast, transcript levels of HSFA7aand HSFB2b are slightly higher in the rcf2-1 plants than in wild-type plants (Figures 1D and 1G). DREB2A and DREB2C controlthe expression of HSFA3 under heat stress (Sakuma et al., 2006;Schramm et al., 2008; Chen et al., 2010). Correlated with thereduced heat induction of HSFA3 in rcf2-1, the expression ofDREB2A and DREB2C is substantially reduced in rcf2-1 upon heatstress (Figures 1E, 1H, and 1I). Consistent with the reduced basalthermotolerance of rcf2-1 and the overall reduced accumulation ofHSFs in rcf2-1, the heat induction of four HSPs (HSP18, HSP26.5,HSP70B, and HSP101) in whole seedlings or in flowers from soil-grown plants is significantly reduced in rcf2-1 (Figures 1J and 1K).In addition, we examined the expression levels of HSFA7b andHSP101 in rcf2-1 plants transformed with wild-type RCF2 underthe control of its native promoter (the rcf2-1 complementationlines mentioned earlier). The expression of HSFA7b and HSP101in rcf2-1 complementation lines is similar to that in the wild type(Supplemental Figures 5C and 5D). Furthermore, thermotolerance

440 The Plant Cell

in fry2-1 plants is significantly reduced, and this is correlated withthe overall decreased expression levels of heat stress–responsivegenes (Supplemental Figure 6). Together, these results indicatethat RCF2 is an important positive regulator of heat stress–responsive gene expression and themotolerance in plants.

RCF2 Interacts with a NAC Transcription Factor, NAC019, inVivo, and NAC019 Is Required for Heat Stress–ResponsiveGene Regulation and Thermotolerance

As indicated by yeast two-hybrid assays, RCF2/CPL1 interactswith a NAC transcription factor, NAC019, through the C-terminalregion (amino acids 640 to 967) of RCF2/CPL1 (Bang et al., 2008).

Because both RCF2/CPL1 and NAC019 are localized in the nu-cleus (Koiwa et al., 2004; Bang et al., 2008; Bu et al., 2008), weshowed that RCF2/CPL1 interacts with NAC019 in vivo by bi-molecular fluorescence complementation (BiFC) and split lucif-erase complementation (Split-LUC) assays in tobacco (Nicotianatabacum) leaves (Figures 2A to 2C). Coimmunoprecipitation as-says further confirmed that RCF2/CPL1 interacts with NAC019 invivo (Figure 2D).CPL1 has predicted phosphatase activity that should result in

the dephosphorylation of Ser-5-PO4 in the heptad repeat se-quence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) of the CTD of the largestsubunit of RNA polymerase II (pol II). Koiwa et al. (2004) showedthat recombinant CPL1 is able to dephosphorylate synthetic

Figure 1. Effect of the rcf2-1 Mutation on the Thermotolerance and Expression of Heat Stress–Responsive Genes.

(A) Thermotolerance of wild-type and rcf2-1 plants. Three-week-old soil-grown plants were transferred to incubators at 21 or 37°C and allowed to growfor an additional 7 d.(B) Survival rates of wild-type and rcf2-1 plants as shown in (A) and flowers of separate batches of 1-month-old wild-type and rcf2-1 plants subjected toheat stress at 37°C for 0 or 4 d. Flowers that failed to produce viable siliques were considered dead. Values are means of survival rates relative to thenonstressed conditions.(C) to (K) Expression profiles of heat stress–responsive genes in 14-d-old or 1-month-old (for HSPs in flowers) wild-type and rcf2-1 plants subjected toheat stress at 37°C for 0 or 1 h.Error bars represent SD (n = 80 [individual pots where wild-type and rcf2-1 plants were grown side by side] in [B]; n = 6 in [C] to [K]). One-way ANOVA(Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (P < 0.01). Values shown arederived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

Role of RCF2 in Plant Thermotolerance 441

phosphopeptide substrates resembling the phosphorylated CTDof the largest subunit of pol II. RCF2/CPL1 can dephosphorylateHYPONASTIC LEAVES1 (HYL1), which lacks a typical heptadrepeat sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) in its C-terminalregion or anywhere else (Manavella et al., 2012). We performeda protein gel blot analysis using Phos-tag (a chemical that bindsspecifically to the phosphorylated residues in proteins and causesa mobility shift of the phosphorylated proteins) to investigatewhether NAC019 is phosphorylated, and if NAC019 is phos-phorylated, whether RCF2/CPL1 dephosphorylates it. As shownin Figure 2E, the proportion of the unphosphorylated NAC019 isdecreased in rcf2-1 mutant plants under heat stress, suggestingthat RCF2 dephosphorylates the phosphorylated NAC019.

The expression of NAC019 is strongest at the reproductivestage, and NAC019 is upregulated by heat stress (Supplemental

Figures 7A and 7B). We observed that the expression of NAC019is reduced in rcf2-1 mutant plants with or without heat stress(Supplemental Figure 7C). These results indicate that RCF2 is notonly important for posttranslational modification of NAC019 but isalso required for NAC019 expression at the transcriptional orposttranscriptional level. We obtained a T-DNA knockout ofNAC019 (Supplemental Figure 7D). Soil-grown nac019 mutantplants are more sensitive to heat stress than the wild type (Fig-ures 3A and 3C). Flowers of the nac019 mutant plants are hy-persensitive to heat stress (Figures 3B and 3C). Heat induction ofHSFA1b, HSFA6b, HSFB1, and HSFC1 is significantly lower innac019 mutant plants than in wild-type plants (Figures 3D to 3F),while the heat-induced transcript level of HSFA7a is moderatelyelevated in nac019 (Figure 3G). Correlated with the overall re-duced heat induction of HSFs, transcripts of HSP18, HSP26.5,

Figure 2. Interaction between RCF2 and NAC019 in Vivo, and Dephosphorylation of NAC019 by RCF2.

(A) Interaction between RCF2 and NAC019 in tobacco leaves as determined by BiFC analysis. Yellow fluorescent protein (YFP) images were detected atan approximate frequency of 4.23% (110 of 2600 tobacco leaf epidermal cells analyzed exhibited BiFC events). Bars = 25 mm.(B) Interaction of RCF2 and NAC019 as determined by Split-LUC assay.(C) Quantification of LUC expression as shown in (B). Error bars indicate SD (n = 16). One-way ANOVA (Tukey-Kramer test) was performed, andstatistically significant differences are indicated by different lowercase letters (P < 0.01).(D) In vivo pull-down analysis of RCF2 and NAC019 interaction in tobacco leaves. IP, immunoprecipitation.(E) Phosphorylation status of NAC019 in wild-type and rcf2-1 plants expressing 35S:HA-NAC019. Plants were subjected to heat stress at 37°C for 0 or1 h. Proteins were extracted and immunoprecipitated before being separated on an SDS-PAGE gel containing 50 mM Phos-tag. After being transferredto a polyvinylidene difluoride membrane, the phosphorylated (indicated as NAC019-P next to an arrow) and unphosphorylated (indicated as NAC019next to an arrow) forms of NAC019 were detected by anti-HA antibody (top panels). Protein gel blots with immunoprecipitated proteins, which wereseparated on a regular SDS-PAGE gel, blotted with anti-HA antibody, were used as loading controls (bottom panels). WT-CIP, Wild-type plantsexpressing 35S:HA-NAC019 treated with alkaline phosphatase, calf intestinal (New England Biolabs), for 0, 1, or 3 h. Blots were quantified with ImageJsoftware, and values are ratios of phosphorylated (above top panels) or unphosphorylated (below top panels) NAC019 to the corresponding loadingcontrol (bottom panels).Values shown are derived from experiments that were performed at least three times with similar results, and representative data from one repetition arepresented.

442 The Plant Cell

HSP70B, and HSP101 are less abundant in whole seedlings ofnac019 than in the wild type under heat stress (Figures 3H and 3I).Furthermore, relative to flowers of soil-grown wild-type plants,flowers of soil-grown nac019 plants have a substantially reducedaccumulation of HSP18 and HSP101 transcripts under heat stress(Figures 3H and 3I). We also examined the expression levels ofHSFA6b and HSP18 in nac019 plants transformed with wild-typeNAC019 driven by its native promoter (NAC019:NAC019-HA[nac019 complementation lines]; Supplemental Figure 7E). Theexpression of HSFA6b and HSP18 in nac019 complementation

lines is similar to that in the wild type (Supplemental Figures 7F and7G). Thus, the in vivo target of RCF2, NAC019, is required forthermotolerance and heat stress–responsive gene expression.

NAC019 Binds to cis-Elements in Promoters of HSFA1b,HSFA6b, HSFA7a, and HSFC1

NAC019 can potentially bind to the NAC recognition cis-promoterelement CATGT when that element is immediately followed by thecore binding site CACG or CACG flanked by one or more spacer

Figure 3. Effect of the nac019 Mutation on the Thermotolerance and Expression of Heat Stress–Responsive Genes.

(A) Thermotolerance of wild-type and nac019 plants. Three-week-old soil-grown wild-type and nac019 plants were subjected to 37°C for 0 or 7 d.(B) Survival of flowers of 1-month-old wild-type and nac019 plants under heat stress (37°C for 0 d [top panel] or 4 d [bottom panel]). Flowers that failedto produce viable siliques were considered dead.(C) Survival rates of plants in (A) and flowers in (B). Values are means of survival rates relative to the nonstressed conditions.(D) to (I) Expression profiles of heat stress–responsive genes in 14-d-old or 1-month-old (for HSPs in flowers) wild-type and nac019 plants subjected toheat stress at 37°C for 0 or 1 h.Error bars represent SD (n = 80 [individual pots where wild-type and nac019 plants were grown side by side] in [C]; n = 6 in [D] to [I]). One-way ANOVA(Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (P < 0.01). Values shown arederived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.

Role of RCF2 in Plant Thermotolerance 443

DNA sequences (Tran et al., 2004; Bu et al., 2008). Such con-sensus cis-elements that NAC019 can potentially bind to arefound in the promoter regions of HSFA1b, HSFA6b, HSFA7a,HSFB1, and HSFC1 (Supplemental Figures 8 and 9). We firstperformed electrophoretic mobility shift assays (EMSAs) to de-termine whether NAC019 is able to bind to cis-elements in theHSFA6b promoter in vitro. As shown in Supplemental Figure 10A,NAC019 can bind to cis-promoter elements in HSFA6b. TheEMSA experiments also showed that RCF2 cannot bind to theNAC019 binding sites in the promoter of HSFA6b (SupplementalFigure 10B). Under our EMSA experimental conditions, we did notobserve RCF2 supershifts NAC019 through its binding in vitroto NAC019 on specific cis-elements in the HSFA6b promoter(Supplemental Figure 10B). We then performed chromatinimmunoprecipitation (ChIP) assays followed by qPCR analysesusing the nac019 plants expressing NAC019:NAC019-HA(Supplemental Figure 7E) to determine whether NAC019 is ableto bind to those cis-promoter elements in vivo. ChIP-qPCRexperiments showed that NAC019 can bind to promoter regions

of HSFA1b, HSFA6b, HSFA7a, and HSFC1 but not of HSFB1,where the NAC019 binding sites are located (Figures 4A to 4E).Because RCF2 physically interacts with NAC019, we performedChIP-qPCR analysis with rcf2-1 plants expressing RCF2:RCF2-FLAG (Supplemental Figure 3A) to investigate whether RCF2 isenriched in promoter regions where NAC019 is bound. The datapresented in Supplemental Figure 11 indicate that RCF2 is en-riched in promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1but not of HSFB1. These results suggest that, although RCF2 isunable to bind to cis-promoter elements of HSFs, as indicated inSupplemental Figure 10B, NAC019 binds to specific HSF pro-moters and RCF2 interacts with NAC019 on these promoters.We subsequently performed a dual luciferase reporter assay in

tobacco leaves to examine the effect of RCF2 and NAC019 onHSFA6b promoter activity in vivo. RCF2 enhances the trans-activation of NAC019 on the HSFA6b promoter under both normaland heat stress conditions (Figure 4F). The phosphatase-inactiveform of RCF2 (RCF2[D161A]; Hausmann et al., 2005) fails to en-hance NAC019 transactivation activity on the HSFA6b promoter

Figure 4. NAC019 Directly Binds to Promoters of HSFs.

(A) to (E) NAC019 binds to the promoter regions of HSFA1b, HSFA6b, HSFA7a, and HSFC1 but not of HSFB1, as determined by ChIP-qPCR analyseswith 15-d-old nac019 plants expressing NAC019:NAC019-HA (Supplemental Figure 5D) subjected to 37°C for 1 h. Regions of amplification arespecified (positions are relative to the translation start site). Amplified regions B in (A) to (D) and C in (E) served as negative controls. Primers used forthe amplifications are listed in Supplemental Table 2.(F) Relative luciferase activity from the dual luciferase reporter assays in tobacco leaves. RCF2(D161A) is the phosphatase-inactive form of RCF2.Error bars represent SD (n = 6 in [A] to [E]; n = 15 in [F]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences areindicated by different lowercase letters (P < 0.01). Values shown are derived from experiments that were performed at least three times with similarresults, and representative data from one repetition are presented.

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(Figure 4F). In contrast to the wild-type RCF2, the phosphatase-inactive form of RCF2 (RCF2[D161A]) is unable to restore the heatinduction of HSFA7b and HSP101 in rcf2-1 to the level in the wildtype (Supplemental Figures 12A and 12B). These data suggestthat phosphatase activity of RCF2 is required for heat stress–responsive gene expression in planta.

Overexpression of RCF2 or NAC019 in ArabidopsisIncreases Thermotolerance

Because both RCF2 and NAC019 are required for heat stress–responsive gene expression and thermotolerance, we generatedArabidopsis transgenic plants expressing RCF2 or NAC019 underthe control of the 35S promoter (35S:FLAG-RCF2 or 35S:HA-NAC019) to investigate whether the overexpression of RCF2 orNAC019 can improve plant performance under heat stress(Supplemental Figures 13A and 13B). Overexpression of RCF2increases the thermotolerance of transgenic plants, as indicatedby the reduced damage to whole plants and flowers (Figures 5Ato 5C). Correlated with the increased thermotolerance, the RCF2overexpression plants have overall increased expression levels ofheat stress–responsive genes (Figures 5D to 5J). The 35S:FLAG-RCF2 transgene is able to restore the heat induction of HSFA7band HSP101 in rcf2-1 to the expression level in the wild type(Supplemental Figures 13C and 13D), suggesting that the 35S:FLAG-RCF2 transgene is functional in vivo. The same is true fortransgenic plants overexpressing NAC019. The NAC019 over-expression plants are more heat tolerant than the wild type, asindicated by reduced damage to whole plants and flowers (Fig-ures 6A to 6C). Except for HSFA7a, whose transcript is reduced,the expression levels of heat stress–responsive genes are ele-vated in the NAC019 overexpression plants (Figures 6D to 6I). The35S:HA-NAC019 transgene can complement the nac019 mutantphenotype (e.g., the transgene can restore the heat induction ofHSFA6b and HSP18 in nac019 to the expression level in the wildtype; Supplemental Figures 13E and 13F), suggesting that the35S:HA-NAC019 transgene is functional in vivo.

DISCUSSION

In a genetic screen for regulators of cold-responsive gene ex-pression, we have identified RCF2, which is a new allele of CPL1/FRY2 (Supplemental Figure 1). Previous reports showed thatCPL1/FRY2 is involved in the repression of genes that are re-sponsive to cold, ABA, and NaCl (Koiwa et al., 2002; Xiong et al.,2002). The upregulation of stress-responsive genes in cpl1 orfry2-1 mutants might be due to the increased expression of CBFgenes (Xiong et al., 2002). Similar to the observations of Koiwaet al. (2002) and Xiong et al. (2002), our results indicate that CBFgenes are upregulated in rcf2-1 plants under cold stress(Supplemental Figure 1B) and that the upregulation of CBFgenes is very likely a compensatory response to the reduced coldtolerance of rcf2-1 mutant plants (Supplemental Figure 1C). Thiscompensatory response may be partially caused by the reducedexpression of PRR5, the negative regulator of CBF genes(Nakamichi et al., 2009; Guan et al., 2013c). PRR5 may directly orindirectly repress CBF genes (Nakamichi et al., 2009). We haveobserved a similar phenomenon in rcf1-1 mutant plants, which

accumulate increased levels of cold-responsive genes, includingCBFs, but are hypersensitive to cold stress (Guan et al., 2013c).RCF1 encodes a DEAD box RNA helicase critical for pre-mRNAsplicing (Guan et al., 2013c). In rcf1-1, the function of PRR5 andSK12 (a shaggy-like Ser/Thr kinase, another negative regulator ofCBF genes) is compromised, because PRR5 and SK12 aremisspliced. Reduced activities of PRR5 and SK12 in rcf1-1 maypartially explain the increased expression of cold-responsivegenes, including CBFs. Our results in this study further suggestthat additional important positive and/or negative regulatorsof cold tolerance, which may function in the CBF-independentpathways, may not function properly in the rcf2-1mutant. Matsudaet al. (2009) reported that CPL1 represses the wound-inducedtranscription of jasmonic acid biosynthetic genes, probably byrepressing the transcriptional activity of RNA pol II transcriptionmachinery via the CPL1-mediated phosphorylation status of theCTD of the largest subunit of RNA pol II. The precise mechanismsby which RCF2/CPL1/FRY2 preferentially affects cold, ABA, NaCl,and wounding responses remain unknown. From microarrayanalysis, Aksoy et al. (2013) showed that 114 genes were upre-gulated in the cpl1-2/fry2-1 mutant plants under unstressed con-ditions and that CPL1 appears to repress the expression of genesencoding the signaling components downstream of iron deficiencyby an unknown mechanism.In this study, we observed that RCF2/CPL1/FRY2 can posi-

tively control the expression of genes under either cold stress orheat stress (Figure 1; Supplemental Figures 1D, 1E, and 6).Aksoy et al. (2013) also reported in a microarray study that 132genes are downregulated in the cpl1-2/fry2-1 mutant plantsunder unstressed conditions, although further investigation wasneeded to elucidate how CPL1/FRY2 controls the expression ofthese genes in a positive manner. The observation that the ex-pression of RCF2/CPL1/FRY2 is upregulated by heat stressmotivated us to investigate the role of RCF2 in heat stress re-sponses (Supplemental Figure 5B). We observed that rcf2-1 andfry2-1 mutant plants are hypersensitive to heat stress (Figure 1;Supplemental Figure 6). The reduced thermotolerance of rcf2-1and fry2-1 plants is correlated with the overall reduced expres-sion of many HSFs and HSPs in heat-stressed rcf2-1 and fry2-1plants (Figure 1; Supplemental Figure 6). Reduced expression ofone of the master regulators of HSFs, HSFA1b, in rcf2-1 andfry2-1 may contribute to the overall reduced expression of theHSFs examined in rcf2-1 and fry2-1 (Figure 1; SupplementalFigure 6). In addition, lower heat induction of HSFA3 in rcf2-1and fry2-1 may result from the reduced expression of DREB2Aand DREB2C, because these two proteins control the expres-sion of HSFA3 under heat stress (Sakuma et al., 2006; Schrammet al., 2008; Chen et al., 2010). HSFB1 and HSFB2b act as re-pressors of the expression of heat-inducible HSF genes, such asHSFA2 and HSFA7a (Ikeda et al., 2011). In rcf2-1 and fry2-1under heat stress, the expression of HSFB1 is reduced whilethat of HSFB2b is slightly upregulated. Therefore, the reducedexpression of HSFA2 in rcf2-1 and fry2-1 and the elevated levelof HSFA7a transcripts might reflect the collective effects of thealtered expression of HSFB1 and HSFB2b in rcf2-1 and fry2-1.We noticed that the expression levels of HSFA1b and HSFC1

are reduced in wild-type plants after 1 h of heat treatment(Figures 1C and 3D). HSFA1b expression is induced slightly after

Role of RCF2 in Plant Thermotolerance 445

a 6-h heat treatment (Kilian et al., 2007), indicating that the ex-pression level of HSFA1b under heat stress fluctuates and thatthe downregulation by 1 h of heat stress is an early response ofplants to high temperature. The upregulation of HSFA1b at 6 hafter heat stress might be required for the activation of late-response genes under heat stress. HSFA1b is a positive regulator

of heat stress (Liu et al., 2011; Yoshida et al., 2011), and over-expression of HSFA1b increases the thermotolerance of Arabi-dopsis (Prändl et al., 1998). In contrast, the expression levels ofHSFC1 in Arabidopsis shoot tissues after 6 h of heat treatmentare similar as those after 1 h of heat treatment (Kilian et al., 2007).HSFC1 may function as a coactivator when combining with class

Figure 5. Overexpression of RCF2 in Arabidopsis Increases Thermotolerance.

(A) Thermotolerance of RCF2 overexpression (OE ) lines. Five-week-old wild-type and RCF2 OE plants grown at 21°C were subjected to 37°C for0 weeks (top row) or 2 weeks (bottom row).(B) Shoot fresh weight and chlorophyll content of plants shown in (A).(C) Survival rates of flowers of 5-week-old wild-type and RCF2 OE plants subjected to 37°C for 0 or 4 d. Values are means of survival rates relative to thenonstressed conditions.(D) to (J) Expression of heat-responsive genes in 15-d-old or 1-month-old (for HSPs in flowers) wild-type and RCF2 OE plants subjected to 37°C for 0 or1 h.Error bars indicate SD (n = 60 for shoot fresh weight, chlorophyll content, and survival rate of flowers in [B] and [C]; n = 6 for heat-responsive geneexpression in [D] to [J]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by differentlowercase letters (P < 0.01). Values shown are derived from experiments that were performed at least three times with similar results, and representativedata from one repetition are presented.

446 The Plant Cell

A HSFs, because HSFC1 has no activation function (Kotak et al.,2004), suggesting that the downregulation of HSFC1 may becorrelated with the downregulation of HSFA1b after a 1-h heatstress treatment (Kilian et al., 2007). The biological function ofHSFC1 in plants under heat stress has not been experimentallydetermined.

Using BiFC, Split-LUC, and coimmunoprecipitation analyses,we showed that RCF2 interacts with the NAC transcription factor,NAC019, in vivo (Figures 2A to 2D). NAC019 is involved in jas-monic acid–signaled defense responses by directly binding to thepromoter of VEGETATIVE STORAGE PROTEIN1 and is also in-volved in drought stress and ABA signaling (Tran et al., 2004; Bu

Figure 6. Overexpression of NAC019 in Arabidopsis Increases Thermotolerance.

(A) Thermotolerance of NAC019 overexpression (OE ) lines. The top row shows 3-week-old wild-type and NAC019 OE plants grown at 21°C with normalmorphology; the bottom row shows 5-week-old wild-type and NAC019 OE plants that were subjected to heat stress at 37°C for 2 weeks.(B) Shoot fresh weight and chlorophyll content of plants shown in (A).(C) Survival rates of flowers of 5-week-old wild-type and NAC019 OE plants subjected to 37°C for 0 or 4 d. Values are mean survival rates relative to thenonstressed conditions.(D) to (I) Expression of heat-responsive genes in 15-d-old or 1-month-old (for HSPs in flowers) wild-type and NAC019 OE plants subjected to 37°C for0 or 1 h.Error bars indicate SD (n = 60 for shoot fresh weight, chlorophyll content, and survival rate of flowers in [B] and [C]; n = 6 for heat-responsive geneexpression in [D] to [I]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different low-ercase letters (P < 0.01). Values shown are derived from experiments that were performed at least three times with similar results, and representativedata from one repetition are presented.

Role of RCF2 in Plant Thermotolerance 447

et al., 2008; Jensen et al., 2010). RCF2/CPL1/FRY2 is predictedto dephosphorylate Ser-5-PO4 in the heptad repeat sequence ofthe CTD of the largest subunit of RNA pol II. Koiwa et al. (2004)reported that recombinant CPL1 is able to dephosphorylatesynthetic phosphopeptide substrates resembling the phosphor-ylated CTD domain of the largest subunit of RNA pol II. To date,no catalytic activity of CPL1 toward native RNA pol II has beendemonstrated. HYL1 is also probably dephosphorylated by CPL1(Manavella et al., 2012). Based on the above evidence, we de-termined the phosphorylation status of NAC019 in rcf2-1 plantsand found that the proportion of the unphosphorylated form ofNAC019 is reduced in rcf2-1 plants under heat stress, suggestingthat NAC019 might be dephosphorylated by RCF2 (Figure 2E). Inaddition, we found that the expression of NAC019 is reduced inheat-stressed or unstressed rcf2-1 mutant plants (SupplementalFigure 7C). These results indicate that RCF2 is not only importantfor posttranslational modification of NAC019 but is also requiredfor NAC019 expression at the transcriptional or posttranscriptionallevel. We subsequently investigated the role of NAC019 in theheat stress responses. The nac019 mutant plants show increasedsensitivity to heat stress, especially in the reproductive stage,when NAC019 reaches its peak expression level (Figures 3A to3C; Supplemental Figure 7A). The reduced thermotolerance ofnac019 is correlated with the reduced accumulation of five HSFs(HSFA1b, HSFA6b, HSFA7a, HSFB1, and HSFC1) and four HSPs(HSP18, HSP26.5, HSP70B, and HSP101) in heat-stressed nac019plants (Figures 3D to 3I).

The NAC transcription factor complete recognition sequencewas determined with a yeast one-hybrid system (Tran et al.,2004), and the recognition sequence contains CATGT and har-bors CACG as a core binding site. Bu et al. (2008) reported thatNAC019 shows high affinity with site CATGTCCACG (CATGT andCACG are spaced by one nucleotide), and NAC019 can bind toeither site CATGT or site CACG. Because consensus NAC019binding sites were found in the promoter regions of the five HSFsthat were differentially expressed in nac019 mutant plants, wethen performed EMSA, and a high-affinity DNA–protein complex

was detected and the DNA binding activity was reduced with theaddition of competitor (Supplemental Figure 10A), indicating thatNAC019 binds to the HSFA6b promoter in vitro. The cis-promoterelement (a 28-bp segment) in HSFA6b contains a NAC recogni-tion site, CATGT, and a core binding site, CACG, which is fol-lowed by a 19-bp spacer DNA sequence; cis-promoter elementsin promoter regions of three other HSFs (HSFA1b, HSFA7a, andHSFC1) contain CATGT and CACG spaced by 18-bp (HSFA1band HSFC1) and 51-bp (HSFA7a) DNA sequences (SupplementalFigures 8 and 9). ChIP-qPCR analyses further confirmed thatNAC019 binds to cis-elements in the promoters of HSFA1b,HSFA6b, HSFA7a, andHSFC1 but not of HSFB1 in vivo (Figure 4).RCF2 does not bind to cis-promoter elements in vitro(Supplemental Figure 10B). We observed that RCF2 is enrichedin promoter regions of HSFA1b, HSFA6b, HSFA7a, and HSFC1where NAC019 is bound. Because RCF2 interacts with NAC019in vivo, our data suggest that RCF2 interacts with NAC019 onthe promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1(Supplemental Figure 11). Our data also suggest that HSFB1 isan indirect target of RCF2-NAC019. Furthermore, we showedthrough dual luciferase reporter assays that RCF2 is able toenhance the transactivation of NAC019 on the HSFA6b pro-moter and that RCF2(D161A), the phosphatase-inactive form ofRCF2 (Hausmann et al., 2005), eliminates the enhanced trans-activation of RCF2 and NAC019 on the HSFA6b promoter(Figure 4F). Finally, we showed that RCF2(D161A) driven by theRCF2 native promoter is not functional in vivo to control heatstress–responsive gene expression (Supplemental Figures 12Aand 12B). Our data suggest that the phosphatase activity ofRCF2 is required for heat stress–responsive gene expression.Because of the essential role of RNA pol II in gene transcription,RCF2 may dephosphorylate both NAC019 and the CTD of thelargest subunit of RNA pol II under heat stress. Since we lacka mutant form of RCF2 that fails to dephosphorylate NAC019but is still able to dephosphorylate the CTD of the largest sub-unit of RNA pol II, we do not know whether RCF2 phosphataseactivity on the CTD of the largest subunit of RNA pol II is criticalfor heat stress responses. The importance of RCF2 and NAC019in thermotolerance is further supported by gain-of-functionstudies. Transgenic Arabidopsis plants constitutively expressingRCF2 or NAC019 are heat tolerant (Figures 5 and 6). The in-creased thermotolerance in RCF2 overexpression or NAC019overexpression plants is correlated with the elevated accumu-lation of heat-responsive genes (Figures 5 and 6). These resultsfurther support the inference that both RCF2 and NAC019 arepositive regulators for heat-responsive gene expression andthermotolerance in plants (Figure 7). It is clear that, althoughevery plant tissue is vulnerable to heat stress, the reproductivetissues are particularly susceptible (Zinn et al., 2010). Becausethe peak expression levels of RCF2 and NAC019 occur at thereproductive stage and RCF2 and NAC019 protect plant re-productive tissues (flowers) from heat stress (Figures 1B and 3B;Supplemental Figures 5A and 7A), and because RCF2 andNAC019 are highly conserved across plant species (SupplementalFigure 14), the manipulation of RCF2 and NAC019 levels in heat-sensitive crops such as maize and soybean may increase theirthermotolerance and especially the thermotolerance of theirreproductive tissues.

Figure 7. Working Model for RCF2 and NAC019 Function in HeatStress–Responsive Gene Expression and Thermotolerance.

Heat stress induces the expression of HSFs, which are responsible forthe accumulation of HSPs. Heat stress also regulates RCF2 expression.RCF2 interacts with and dephosphorylates NAC019, and both proteinsare required for the heat induction of HSFs and thermotolerance.

448 The Plant Cell

METHODS

Plant Materials and Growth Conditions

A firefly luciferase reporter gene driven by the cold stress–responsiveCBF2promoter (21500 to 21 bp upstream of the transcription start site) wasintroduced into Arabidopsis thaliana plants in the Columbia glabrous1background. Seeds from one homozygous line expressing a singlefunctional copy of the CBF2:LUC gene (referred to as the wild type) weremutagenized with ethyl methanesulfonate. The rcf2-1 mutant with alteredCBF2:LUC gene expression was isolated fromM2 seedlings with a charge-coupled device camera imaging system (Ishitani et al. 1997; Guan et al.,2013a, 2013c).

Seeds of the following T-DNA insertion mutants were obtained from theABRC: SALK_096295 (nac019) and CS24934 (fry2-1). Arabidopsis seed-lings on Murashige and Skoog (MS) medium agar plates (13MS salts, 2%Suc, and 0.6% agar, pH 5.7) were routinely grown under cool-white light(;120 mmol m22 s21) at 216 1°C with a 16-h-light/8-h-dark photoperiod.Soil-grown plants were kept under cool-white light (;100 mmol m22 s21)with a 16-h-light/8-h-dark photoperiod at 21 6 1°C and with a 1:1 ratio ofpotting soil Metro Mix 360 and LC1 (Sun Gro Horticulture).

Genetic Mapping and Complementation of rcf2-1

The rcf2-1 mutant was crossed with the Landsberg erecta accession, and1632 mutant plants were chosen from the F2 generation based on alteredCBF2:LUC phenotype. Simple sequence length polymorphism markerswere designed according to the information in the Cereon ArabidopsisPolymorphism Collection and were used to analyze recombination events(Jander et al., 2002). Initial mapping revealed that the rcf2-1 mutation islocated on the upper arm of chromosome 4 between F9F13 and F7K2. Finemapping within this chromosomal interval narrowed the RCF2 locus to theBAC clone F17L22. All candidate genes in this BAC were sequenced fromthe rcf2-1 mutant and compared with those in GenBank to find the rcf2-1mutation.

For complementation of the rcf2-1mutant, a 7104-bp genomic fragmentof At4g21670 that included 1695 bp upstream of the translation initiationcodon was amplified with F17L22 as a template (for primer sequences, seeSupplemental Table 2). The amplified fragment was first cloned throughGateway technology (Invitrogen) into the binary vector pEarleyGate302.The resulting construct (RCF2:RCF2-FLAG) was transferred into Agro-bacterium tumefaciens (strain GV3101), and rcf2-1 plants were transformedby the floral dip method (Clough and Bent, 1998).

Gene Complementation of nac019

For complementation of the nac019 mutant, a genomic fragment of theNAC019 gene including its promoter, introns, and exons (39-untranslatedregion excluded) was amplified with F14G24 as a template (for primersequences, see Supplemental Table 2). The amplified fragment was firstcloned through Gateway technology (Invitrogen) into the binary vectorpEarleyGate301. The resulting construct (NAC019:NAC019-HA) wastransferred into Agrobacterium (strain GV3101), and nac019 plants weretransformed by the floral dip method.

Overexpression of RCF2 and NAC019

Coding regions of RCF2 and NAC019 were amplified by PCR and clonedinto pEarleyGate202 andpEarleyGate201, respectively. The resulting construct(35S:FLAG-RCF2 or 35S:HA-NAC019) was transferred into Agrobacterium(strain GV3101), and wild-type plants were transformed by the floral dipmethod. The construct (35S:FLAG-RCF2 or 35S:HA-NAC019) was alsotransferred to the rcf2-1 or nac019 plant to determine whether 35S:FLAG-RCF2 or 35S:HA-NAC019 is functional in vivo.

Detection of Abnormal RCF2 Transcripts in rcf2-1 and Real-TimeRT-PCR Analysis

Fourteen-day-old seedlings grown onMSmedium (13MS salts, 2% Suc,and 0.6% agar, pH 5.7) were used for RNA isolation. Total RNA wasextracted from wild-type, mutant, and/or transgenic plants with Trizolreagent (Invitrogen), and the extracted RNA was treated with DNase I(New England Biolabs) to remove potential genomic DNA contaminations.

For the detection of abnormal RCF2 transcripts in the wild type andrcf2-1, 5 mg of total RNA was used for the synthesis of the first-strandcDNA with the Maxima First-Strand cDNA Synthesis kit in a total volumeof 20 mL (Fermentas). RCF2 fragments were amplified with a pair ofprimers (RCF2 RT F and RCF2 RT R) with cDNA templates using Taq DNApolymerase (Thermo Scientific Fermentas). The RT-PCR products weresubcloned into the pGEM-T Easy vector (Promega) and sequenced.Sequence analysis was done with BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html).

For real-time RT-PCR analysis, 5 mg of total RNA was used for syn-thesis of the first-strand cDNA with the Maxima First-Strand cDNASynthesis kit in a total volume of 20 mL (Fermentas). The cDNA reactionmixture was diluted two times, and 5mLwas used as a template in a 20-mLPCR. PCRs included a preincubation at 95°C for 2 min followed by 45cycles of denaturation at 95°C for 15 s, annealing at 56°C for 40 s, andextension at 72°C for 45 s. All reactions were performed in the CFX96Real-Time PCR Detection system (Bio-Rad) using Maxima SYBR Green/Fluorescein qPCR Master Mix (Fermentas). Each experiment had sixbiological replicates (three technical replicates for each biological repli-cate). The comparative threshold cycle method was applied. TUB8 wasused as a reference gene. The primers used in this study are listed inSupplemental Table 2.

BiFC, Split-LUC, and Coimmunoprecipitation Assays

BiFC assays were conducted as described (Walter et al., 2004). Briefly,coding regions of RCF2 and NAC019 were amplified by PCR and clonedinto pSPYNE-35S and pSPYCE-35S, respectively. The resulting constructs(RCF2-nYFP and NAC019-cYFP) were transformed into Agrobacteriumstrain C58C1 and coinfiltrated with 35S:p19 (p19 is an RNA-silencing re-pressor protein from Tomato bushy stunt virus [Voinnet et al., 2003]) inAgrobacterium strain C58C1 into the 3-week-old leaves of tobacco(Nicotiana tabacum) plants. The infiltrated tobacco plantswere grown for anadditional 3 d in a growth chamber under a 16-h-light/8-h-dark photoperiodat 21°C. Yellow fluorescent protein signals in transformed tobacco leaveswere then detected with a Leica SP5X confocal microscope (Leica Mi-crosystems).

For Split-LUC assay, coding regions of RCF2 and NAC019 were am-plified by PCR and cloned into the Gateway-compatible firefly luciferasecomplementation imaging vectors (modified from original plasmids de-scribed by Chen et al. [2008]). The resulting constructs (RCF2-nLUC andNAC019-cLUC) were transformed into Agrobacterium strain C58C1 andcoinfiltrated with 35S:p19 in Agrobacterium strain C58C1 into the 3-week-old leaves of tobacco plants. The infiltrated tobacco plants were grown foran additional 3 d in a growth chamber under a 16-h-light/8-h-dark pho-toperiod at 21°C. Luciferase expression was observed with a charge-coupled device camera as described (Ishitani et al., 1997).

Coimmunoprecipitation analyses were performed as described (Leisteret al., 2005; Choi et al., 2012) with minor modifications. Briefly, codingregions of RCF2 and NAC019 were amplified by PCR and cloned intopEarleyGate 202 and pEarleyGate 201, respectively. The resulting con-structs (FLAG-RCF2 and HA-NAC019) were transformed into Agro-bacterium strain C58C1 and coinfiltrated with 35S:p19 in Agrobacteriumstrain C58C1 into the 3-week-old leaves of tobacco plants. The infiltratedtobacco plants were grown for an additional 3 d in a growth chamber undera 16-h-light/8-h-dark photoperiod at 21°C. Proteins were extracted from

Role of RCF2 in Plant Thermotolerance 449

leaf samples with extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mMNaCl,2 mM EDTA, 1 mM DTT, 10% glycerol, 1% Triton X-100, 1 mM phenyl-methylsulfonyl fluoride [PMSF], and 13 Halt protease inhibitor cocktail[Fisher Scientific]). The protein extracts were incubated overnight with anti-hemagglutinin (HA) antibody (Sigma-Aldrich). The immunocomplexes werecollected by adding protein A agarose beads and were washed with im-munoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mMEDTA, 1 mM DTT, 10% glycerol, 0.15% Triton X-100, 1 mM PMSF, and13 Halt protease inhibitor cocktail [Fisher Scientific]). The pellet (im-munocomplexes with beads) was resuspended in 23 SDS-PAGE loadingbuffer. Eluted proteins were analyzed by immunoblotting using anti-FLAGantibody (Sigma-Aldrich) or anti-HA antibody (Sigma-Aldrich). Chemi-luminescence signals were detected by autoradiography.

Mobility Shift Assay to Detect Phosphorylated NAC019 in Vivo

The 35S:HA-NAC019 construct generated for the overexpression ofNAC019 was also transferred into Agrobacterium (strain GV3101), andrcf2-1mutant plants were transformed by the floral dip method. Proteinswere extracted from 3-week-old wild-type and rcf2-1 transgenic plantsexpressing 35S:HA-NAC019 subjected to heat stress at 37°C for 0 or 1 hwith extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mMNaCl, 2 mM EDTA,1 mM DTT, 10% glycerol, 1% Triton X-100, 1 mM PMSF, and 13 Haltprotease inhibitor cocktail [Fisher Scientific]) and separated on a 12%SDS-PAGE gel containing 50 mM Phos-tag (NARD Institute) and 100 mMMnCl2.Separated proteins were then transferred to a polyvinylidene difluoridemembrane and blotted with anti-HA antibody (Sigma-Aldrich). Proteinsextracted from 3-week-old wild-type plants expressing 35S:HA-NAC019were treated with alkaline phosphatase, calf intestinal (New England Biolabs),at 37°C for 0, 1, or 3 h and were used as an indication of phosphorylated andunphosphorylated NAC019. All of the proteins tested were also separated ona regular SDS-PAGE gel and blotted with anti-HA antibody as a loadingcontrol.

ChIP Assays

The construct (RCF2:RCF2-FLAG or NAC019:NAC019-HA) used for genecomplementation assays described above was also transformed into wild-type plants through Agrobacterium (strain GV3101)–mediated trans-formation by the floral dip method. ChIP assays were performed with15-d-old seedlings expressing RCF2:RCF2-FLAG or NAC019:NAC019-HAas described (Guan et al., 2013b). Briefly, seedlings were cross-linked with1% formaldehyde, and chromatin was isolated, sonicated (FisherBiodismembrator; model 120), and precleared with salmon sperm DNA/protein G or A agarose beads for 1 h (protein G was used for anti-FLAG;protein A was used for anti-HA). Samples were then immunoprecipitatedwith anti-FLAG (Sigma-Aldrich; F1804) or anti-HA (Sigma-Aldrich; H6908)antibody at 4°C overnight. The chromatin antibody complex was pre-cipitated with salmon sperm DNA/protein G or A agarose beads, washedwith four different buffers for 5 min per buffer, and reverse cross-linked inelution buffer (1%SDS and 0.1MNaHCO3) containing 200mMNaCl for 6 hat 65°C. Proteins in the complexwere removed by proteinaseK at 45°C for 1h. DNA was precipitated in the presence of 2 volumes of ethanol, one-tenthvolumeof 3Msodiumacetate, pH5.2, and2mgof glycogen. Real-timePCRanalysis was performed with immunoprecipitated DNA using a Bio-RadCFX96 real-time system. All primers are listed in Supplemental Table 2.

Dual Luciferase Reporter Assays

The putative HSFA6b promoter fragment (;1.8 kb upstream of thetranslation start site) containing the cis-element CATGT and CACG thatare recognized by NAC019 (as determined by ChIP-qPCR analysis) wasamplified by PCR with BAC clone MWI23 as a template. The PCR productwas cloned into the transient expression vector pGreenII 0800-LUC to

serve as a reporter plasmid (HSFA6b:LUC). The coding regions ofNAC019 and RCF2 (one copy of FLAG-encoding sequence was includedin the forward PCR primer before the translation start site of RCF2) wereamplified by PCR and cloned into the transient expression vector pGreenII62-SK to serve as effect plasmids (pGreenII 62Sk-NAC019 and pGreenII-FLAG-RCF2, abbreviated as NAC019 and RCF2, respectively). The RCF2coding region (one copy of FLAG-encoding sequence was included in theforward PCR primer before the translation start site of RCF2) with oneamino acid substitution in the phosphatase catalytic domain (D161A) wasamplified by PCR and cloned into the transient expression vector pGreenII62-SK to serve as an effect plasmid (pGreenII-FLAG-RCF2[D161A],abbreviated as RCF2[D161A]). The resulting plasmids (HSFA6b:LUC,NAC019, RCF2, and RCF2[D161A]) were transformed into Agrobacterium(strain C58C1). Four-week-old tobacco leaves were coinfiltrated withAgrobacterium (strain C58C1) harboring the above plasmids in the fol-lowing combinations and ratios: HSFA6b:LUC (100%); HSFA6b:LUC +NAC019 (1:9); HSFA6b:LUC + RCF2 + NAC019 (1:4.5:4.5); and HSFA6b:LUC + RCF2 (D161A) + NAC019 (1:4.5:4.5). The infiltrated tobacco plantswere grown for an additional 3 d in a growth chamber under a 16-h-light/8-h-dark photoperiod at 21°C and were subjected to 0 or 37°C for 1 h. Theactivities of firefly luciferase under the control of the HSFA6b promoter(HSFA6b:LUC ) and Renilla luciferase under the control of the 35S pro-moter (35S:LUC; both the HSFA6b:LUC and 35S:LUC transgenes arepresent on theHSFA6b:LUC reporter plasmid, and 35S:LUC served as aninternal control) were measured using the reagents contained in the Dual-Luciferase Reporter Assay system (Promega) with the Modulus Micro-plate Multimode Reader (Turner BioSystem) as described (Hellens et al.,2005). Normalized data (the ratio of luminescent signal intensity from theHSFA6b:LUC reporter and luminescent signal intensity from the internalcontrol reporter, 35S:LUC ) from 15 independent biological samples arepresented.

EMSAs to Detect the Binding of NAC019 to the HSFA6b Promoterin Vitro

The coding regions of NAC019 and RCF2 were amplified by PCR andcloned into the pMAL-c5E vector (New England Biolabs). The resultingconstructs (MBP-NAC019 and MBP-RCF2) were transformed intoEscherichia coli strain Rosetta (DE3) pLysS (EMD Millipore). The pro-duction and purification of MBP-NAC019 and MBP-RCF2 fusion proteinswere performed following the manufacturer’s instructions (New EnglandBiolabs). Complementary pairs of 59-end biotin-labeled and unlabeledoligonucleotides that contain 33 tandem repeats of NAC019 recognitionsites (for sequences, see Supplemental Table 2) were annealed and usedas probes for the EMSA studies. The EMSA reactions were performedwith a LightShift Chemiluminescent EMSA kit (Pierce). In the reaction,purified MBP-NAC019 or MBP-RCF2 protein was incubated with bindingbuffer (12 mMTris-HCl, 60mMKCl, 2.5% glycerol, 5 mMMgCl2, 50 ng/mLpoly[dI.dC], 0.05% Nonidet P-40, 0.1 mM EDTA, and unlabeled probe) for20 min on ice before labeled probe was added and incubated for anadditional 20 min at room temperature. After incubation, the reactionmixture was loaded on a 4%polyacrylamide gel (acrylamide:bisacrylamide,37.5:1; Bio-Rad) and run in 0.53 Tris-borate-EDTA buffer at 4°C. The DNA–protein complex was transferred to a Hybond-N+ membrane (Amersham),and the membrane was cross-linked. Detection was performed accordingto the manufacturer’s instructions (Pierce).

Determination of Chlorophyll Content

Chlorophyll content was determined as described (Lichtenthaler andWellburn, 1983) with minor modifications. Chlorophyll was extracted byincubating ground seedlings in 80% acetone overnight at 4°C in darknessand with continuous shaking. The contents of chlorophyll a and b werecalculated as 7.49A664.9 + 20.3A648.2.

450 The Plant Cell

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative database under the following accession numbers: RCF2 (At4g21670),NAC019 (At5g52890), CBF1 (At4g25490), CBF2 (At4g25470), CBF3(At4g25480), PRR5 (At5g24470), RING-H2 Finger A1A (At4g11370),AGAMOUS-LIKE8 (At5g60910), HSFA1b (At5g16820), HSFA2 (At2g26150),HSFA3 (At5g03720), HSFA4a (At4g18880), HSFA6a (At5g43840), HSFA6b(At3g22830),HSFA7a (At3g51910),HSFA7b (At3g63350),HSFB1 (At4g36990),HSFB2a (At5g62020), HSFB2b (At4g11660), HSFC1 (At3g24520),DREB2A (At5g05410), DREB2C (At2g40340), HSP18 (At5g59720),HSP26.5 (At1g52560), HSP70B (At1g16030), and HSP101 (At1g74310).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Isolation of the rcf2-1 Mutant and Map-Based Cloning of RCF2.

Supplemental Figure 2. The Nature of Two Abnormal RCF2 Tran-scripts in rcf2-1 Mutant Plants.

Supplemental Figure 3. Gene Complementation of the rcf2-1 Mutant.

Supplemental Figure 4. Sensitivity of rcf2-1 to NaCl and ABA, andDevelopmental Defects of rcf2-1 and fry2-1 Plants.

Supplemental Figure 5. RCF2 Expression Profiles, and Heat Stress–Responsive Gene Expression in rcf2-1 Complementation Lines.

Supplemental Figure 6. Effect of the fry2-1 Mutation on theThermotolerance and Expression of Heat Stress–Responsive Genes.

Supplemental Figure 7. NAC019 Expression Profiles, and HeatStress–Responsive Genes in nac019 Complementation Lines.

Supplemental Figure 8. NAC019 Recognition Sites (CATGT) andCore Binding Sites (CACG) in the Promoter Regions of HSFA1b,HSFA6b, and HSFA7a.

Supplemental Figure 9. NAC019 Recognition Sites (CATGT) and CoreBinding Sites (CACG) in the Promoter Regions of HSFB1 and HSFC1.

Supplemental Figure 10. EMSA of the Binding of NAC019 to the cis-Promoter Element in HSFA6b.

Supplemental Figure 11. RCF2 Is Enriched in the Promoters ofHSFA1b, HSFA6b, HSFA7a, and HSFC1.

Supplemental Figure 12. Expression Levels of HSFA7b and HSP101in rcf2-1 Plants Expressing RCF2:RCF2(D161A).

Supplemental Figure 13. Complementation of rcf2-1 with 35S:FLAG-RCF2 and Complementation of nac019 with 35S:HA-NAC019.

Supplemental Figure 14. Comparison of AtRCF2 and AtNAC019 withTheir Close Homologs.

Supplemental Table 1. Genetic Analysis of the rcf2-1 Mutant (WildType [Female] 3 rcf2-1 [Male] Cross).

Supplemental Table 2. Primers Used in This Study.

Supplemental References.

ACKNOWLEDGMENTS

We thank Hisashi Koiwa for providing RCF2 full-length cDNA and DongLiu for providing the Gateway-compatible Split-LUC vectors. We thankJheesoo Ahn, Grace Wang, Xiaohui Hu, and Rongrong Wang fortechnical assistance. This work was supported by the National ScienceFoundation (Grant MCB0950242 to J.Z.).

AUTHOR CONTRIBUTIONS

Q.G. and J.Z. designed the research. Q.G., X.Y., H.Z., and J.Z. performedthe research (J.Z. isolated the rcf2-1 mutant; Q.G. and X.Y. performedmap-based cloning of rcf2-1; H.Z. made the RCF2:RCF2-FLAG andNAC019:NAC019-HA constructs; Q.G. performed the rest of the experi-ments). Q.G. and J.Z. analyzed the data. Q.G. and J.Z. wrote the article.

Received September 22, 2013; revised November 18, 2013; acceptedDecember 15, 2013; published January 10, 2014.

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Role of RCF2 in Plant Thermotolerance 453

DOI 10.1105/tpc.113.118927; originally published online January 10, 2014; 2014;26;438-453Plant Cell

Qingmei Guan, Xiule Yue, Haitao Zeng and Jianhua ZhuArabidopsisResponsive Gene Regulation and Thermotolerance in

−The Protein Phosphatase RCF2 and Its Interacting Partner NAC019 Are Critical for Heat Stress

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