- © 2014 American Society of Plant Biologists. All rights reserved.
Abstract
Heat stress is a major environmental constraint for crop production worldwide. To respond to and cope with heat stress, plants synthesize heat shock proteins (HSPs), which are often molecular chaperones and are under the control of heat stress transcription factors (HSFs). Very little is known about the upstream regulators of HSFs. In a forward genetic screen for regulators of C-REPEAT BINDING FACTOR (CBF) gene expression (RCFs), we identified RCF2 and found that it is allelic to CPL1/FIERY2, which encodes a homolog of C-terminal domain phosphatase. Our results also showed that, in addition to being critical 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 is correlated with lower expression of most of the 21 HSFs and some of the HSPs in the mutant plants. We found that RCF2 interacts with the NAC transcription factor NAC019 and that RCF2 dephosphorylates NAC019 in vivo. The nac019 mutant is more sensitive to heat stress than the wild type, and chromatin immunoprecipitation followed by quantitative PCR analysis revealed that NAC019 binds to the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. Overexpression of RCF2 or NAC019 in Arabidopsis 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 physical environment that often generates various biotic and abiotic stresses, including heat and drought and sometimes a combination of heat and drought. Heat stress is usually defined as a condition in which temperatures are sufficiently high for enough time to irreversibly damage plant function or development. Heat tolerance is generally defined as the ability of the plant to grow and produce economic yield under high temperatures. Heat stress reduces crop production worldwide. The detrimental effects of heat stress can be alleviated by developing heat-tolerant crop plants by various genetic strategies, including traditional and contemporary molecular breeding protocols and transgenic approaches. Although a few plants with improved thermotolerance have been developed through the use of traditional breeding practices, the success of genetic transformation has been limited because of limited knowledge and availability of genes with known effects on plant thermotolerance. Overcoming these limitations and developing strategies for improving crop tolerance will require a comprehensive understanding of the physiological responses of plants to high temperature and of the molecular mechanisms of heat tolerance.
Heat stress can adversely impact almost all aspects of plant growth, development, reproduction, and yield. Although every plant tissue is vulnerable to heat stress, the reproductive tissues are particularly susceptible (Zinn et al., 2010). At very high temperatures, severe cellular injury and even cell death can occur within a short time (e.g., within minutes), which may be due to a catastrophic collapse of cellular organization (Schöffl et al., 1999). At moderately high temperatures, injury or cell death may occur only after a relatively long time (e.g., hours to days). Direct injuries due to high temperature include protein denaturation and aggregation and increased fluidity of membrane lipids, while indirect injuries include inactivation of enzymes in chloroplasts and mitochondria, inhibition of protein synthesis, protein degradation, loss of membrane integrity, and disruption of cytoskeleton structures (Smertenko et al., 1997; Howarth, 2005). These injuries can eventually result in starvation, reduced ion flux, accumulation of toxic by-products including reactive oxygen species, and disrupted growth and development (Schöffl et al., 1999; Howarth, 2005; McClung and Davis, 2010; Ruelland and Zachowski, 2010; Suzuki et al., 2012). Exposure to heat stress for prolonged periods can even result in plant death, as exemplified by the huge loss in maize (Zea mays) and soybean (Glycine max) fields caused by the devastating heat waves in the summer of 2012.
To resist heat stress, plants use a variety of mechanisms, including the maintenance of membrane stability, scavenging of reactive oxygen species, production of antioxidants and compatible organic compounds, induction of mitogen-activated protein kinase and calcium-dependent protein kinase signaling events, and, most importantly, induction of molecular chaperone signaling and transcriptional activation (Wahid et al., 2007). A central component of responses to heat stress in all living organisms including plants is the induction of heat shock proteins (HSPs) through the action of heat stress transcription factors (HSFs). HSPs are categorized into five 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 chaperones and are essential for the maintenance and/or restoration of protein homeostasis. HSFs are important components of the response to heat stress. More than 20 HSFs are encoded by plant genomes, and these are grouped into three major classes (A, B, and C) based on structural characteristics and phylogenetic relationships (Nover et al., 2001; Baniwal et al., 2004; Scharf et al., 2012). By recognizing and binding to heat stress elements (5′-GAAnnTTC-3′) conserved in promoters of heat stress–responsive genes, HSFs mediate the synthesis of HSPs (Busch et al., 2005).
The complexity of plant HSFs and their interactions are currently being studied, and researchers have established that HSFA1s act as master regulators of other HSFs and, therefore, as master regulators of heat stress responses in tomato (Solanum lycopersicum) and Arabidopsis thaliana (Mishra et al., 2002; Liu et al., 2011; Nishizawa-Yokoi et al., 2011; Yoshida et al., 2011). However, very little is known about the more upstream regulators of these HSFA1s or of the other members of the HSF family. In tomato, HSFB1 functions as a synergistic coactivator of HSFA1a (Bharti et al., 2004). Because of sequence differences in the C terminus, the Arabidopsis HSFB1 is inactive as a coactivator of tomato HSFA1a (Bharti et al., 2004). In Arabidopsis, HSFB1 and HSFB2b repress the expression of heat-inducible HSF genes, such as HSFA2 and HSFA7a (Ikeda et al., 2011). All members of the DREB2 family (DREB2A, DREB2B, and DREB2C) are involved in the regulation of heat-responsive genes including HSFA3 (Sakuma et al., 2006; Schramm et al., 2008; Chen et al., 2010). Liu et al. (2008) reported that a calmodulin binding protein kinase 3 functions as an upstream activator for HSFA1a in Arabidopsis. AtCAM3 plays a role in the Ca2+-calmodulin heat stress signal transduction pathway in Arabidopsis (Zhang et al., 2009). Furthermore, AtHSBP interacts with HSFA1a, HSFA1b, and HSFA2 and negatively regulates the binding of HSFA1b to a heat stress element in vitro, thereby functioning as a negative regulator for heat stress responses in Arabidopsis (Hsu et al., 2010). A KH domain–containing putative RNA binding protein, RCF3 (for regulator of CBF gene expression 3), acts as a negative regulator of heat stress–responsive gene expression and thermotolerance in Arabidopsis (Guan et al., 2013a). Additional critical components in the signal transduction pathway for heat-responsive gene regulation remain to be identified.
In this study, we identify RCF2 in a genetic screen for proteins critical in cold-responsive gene expression. RCF2 is allelic to CPL1/FIERY2 (FRY2), which encodes C-terminal domain (CTD) phosphatase-like 1. We show that RCF2 physically interacts with a NAC transcription factor, NAC019, and that RCF2 dephosphorylates NAC019 under heat stress. Mutations in RCF2/CPL1/FRY2 and NAC019 result in severely reduced thermotolerance and reduced expression of heat stress–responsive genes. NAC019 is able to bind to NAC019-specific cis-promoter elements in four HSFs. Together, our results suggest a positive regulatory pathway that is mediated by RCF2/CPL1/FRY2–NAC019 protein pairs and that is essential for the activation of heat stress–responsive genes and for thermotolerance in plants.
RESULTS
RCF2 Is a Positive Regulator for Freezing Tolerance
To identify important genes involved in cold stress tolerance in plants, we screened for mutants with altered expression of a firefly luciferase reporter gene under the control of the cold-inducible C-REPEAT BINDING FACTOR2 (CBF2) promoter (CBF2:LUC) in an ethyl methanesulfonate–mutagenized Arabidopsis population (Guan et al., 2013a, 2013c). These mutants were designated as rcfs. One of these mutants, rcf2-1, which displays enhanced CBF2:LUC expression upon cold treatment, was chosen for in-depth characterization (Supplemental Figure 1A). Consistent with increased CBF2:LUC expression, the transcript level of the endogenous CBF2 is elevated in the rcf2-1 plants under cold stress (Supplemental Figure 1B). The rcf2-1 mutation also increases the cold induction of two other cold-specific members of the CBF gene family: CBF1 and CBF3 (Supplemental Figure 1B). We determined the effect of the rcf2-1 mutation on freezing tolerance with electrolyte leakage assays. Without cold acclimation, rcf2-1 and wild-type plants are essentially equally sensitive to freezing temperatures (Supplemental Figure 1C). Although tolerance to the freezing temperatures increased in both wild-type and rcf2-1 plants after a 1-week cold acclimation process, the ability to acclimate was substantially less in rcf2-1 than in wild-type plants (Supplemental Figure 1C). Thus, RCF2 is a positive regulator for freezing tolerance.
PSEUDO-RESPONSE REGULATOR5 (PRR5) is a negative regulator of CBF genes (Nakamichi et al., 2009; Guan et al., 2013c). Therefore, we determined the levels of PRR5 expression in the rcf2-1 mutant plants and found that PRR5 transcripts are markedly lower in rcf2-1 plants than in wild-type plants (Supplemental Figure 1D). We also found that transcript levels of AGAMOUS-LIKE8 are substantially lower in rcf2-1 than in the wild type with or without cold stress (Supplemental Figure 1E). AGAMOUS-LIKE8 is known as a MADS box transcription factor and is required for cellular differentiation during leaf and fruit development, including the elongation of siliques (Gu et al., 1998). In addition, the cold induction of RING-H2 Finger A1A is reduced in rcf2-1 plants (Supplemental Figure 1E). RING-H2 Finger A1A is one of the RING finger domain–containing proteins, and the function of RING-H2 Finger A1A is currently unknown (Kosarev et al., 2002). These results suggest that RCF2 controls cold-responsive genes in both positive 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 (the wild type versus rcf2-1; Supplemental Table 1). These results indicate that the rcf2-1 mutation is recessive and is caused by a mutation in a single nuclear gene. We also crossed rcf2-1 with wild-type plants (ecotype Landsberg erecta) to generate a mapping population. Positional cloning revealed that RCF2 is allelic to a 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 abnormal transcript detected in rcf2-1 contains a one-nucleotide deletion of G at nucleotide 1232 in the full-length wild-type RCF2 coding sequence, corresponding to the last nucleotide G of the fifth exon (Supplemental Figure 2C). The predicted RCF2 protein encoded by the first abnormal transcript in rcf2-1 consists of an N-terminal 411–amino acid fragment of the wild-type protein plus a C-terminal tail containing an extra 40 amino acids encoded by the frame-shifted 120 nucleotides (1234 to 1353 in the first abnormal transcript). The truncated mutant protein lacks the C-terminal 556–amino acid segment of the wild-type RCF2 that contains two putative double-strand RNA binding motifs (dsRBMs) and the nuclear localization signal (NLS; Supplemental Figure 2C). The second abnormal transcript in rcf2-1 includes the entire fifth intron (Supplemental Figure 2B). The fifth intron of RCF2 is 110 nucleotides and contains four in-frame stop codons. The predicted RCF2 protein encoded by the second abnormal transcript in rcf2-1 consists of an N-terminal 411–amino acid fragment of the wild-type protein. The truncated mutant protein lacks the C-terminal 556–amino acid segment of wild-type RCF2 that contains two putative dsRBMs and an NLS (Supplemental Figure 2D). The N-terminal portion (amino acids 47 to 638) of RCF2 is essential for its phosphatase activity (Koiwa et al., 2004). Therefore, both abnormal transcripts in rcf2-1 would encode truncated proteins with no or significantly reduced phosphatase activity. The C-terminal portion of RCF2 including the two putative dsRBMs and NLS is critical for protein–protein interactions and nuclear localization (Koiwa et al., 2004; Bang et al., 2008). Because normal RCF2 transcript was not detected in the rcf2-1 plants and because rcf2-1 is a recessive mutant, our data suggest that rcf2-1 is a loss-of-function allele of RCF2/CPL1/FRY2. The wild-type RCF2/CPL1/FRY2 gene under the control of its native promoter (RCF2:RCF2-FLAG) is able to complement the phenotype of rcf2-1 (e.g., restoration of the cold induction of CBF2:LUC and endogenous CBF genes to the levels in the wild type; Supplemental Figure 3).
The fry2-1 mutation is a point mutation at a splicing junction between 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 transcript detected in fry2-1 (Koiwa et al., 2004). The fry2-1 mutant produces a truncated RCF2 protein consisting of an N-terminal 676 amino acids of wild-type protein plus a C-terminal tail containing 8 extra amino acids encoded by the included eighth intron (Koiwa et al., 2004). The truncated mutant protein generated in fry2-1 lacks the C-terminal 292–amino acid segment of the wild-type RCF2 that contains the two dsRBMs and the NLS. As suggested by in vitro phosphatase activity assays using recombinant RCF2/CPL1 truncated proteins (Koiwa et al., 2004), the truncated mutant protein produced in fry2-1 might retain phosphatase activity in vivo. However, because the C-terminal portion of RCF2 is important 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 abnormal transcript in fry2-1 may not be properly localized in the cell to interact with RCF2 targets that are potentially dephosphorylated by RCF2. Because normal RCF2/CPL1/FRY2 transcript was not 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 NaCl and abscisic acid (ABA) during seed germination but more sensitive to ABA at the seedling stage than the wild type. We found that, like fry2-1, rcf2-1 displays increased tolerance to NaCl and ABA during seed germination but shows reduced tolerance to ABA at the seedling stage (Supplemental Figures 4A to 4C). Consistent with the altered expression of AGAMOUS-LIKE8 in the rcf2-1 plants, we observed that fertility is slightly reduced in rcf2-1 plants compared with the wild type (Supplemental Figures 4D and 4E). This reduced fertility is also evident in fry2-1 (Supplemental Figures 4D and 4E). Koiwa et al. (2002) showed that cpl1 plants flower later than the wild type, and we found that flowering time is slightly later for rcf2-1 plants than for the wild type (Supplemental Figure 4F).
RCF2 Is a Positive Regulator for Heat Stress–Responsive Gene Expression and Thermotolerance
The expression level of RCF2 is relatively low (Xiong et al., 2002), and there are no publicly available expression profiling data collected through microarray analyses for RCF2 (such as the information in the e-FP browser database as described by Kilian et al. [2007]). Our quantitative RT-PCR (qPCR) analyses revealed that RCF2 reaches its peak level at the reproductive stage (Supplemental Figure 5A). We also noticed that RCF2 is slightly upregulated after 1 h of heat stress at 37°C and then downregulated after longer exposure to heat stress (Supplemental Figure 5B). This finding prompted us to speculate that the rcf2-1 mutation may affect heat stress responses. Indeed, the soil-grown rcf2-1 plants displayed a hypersensitive phenotype to heat stress, as indicated by dramatically reduced survival rates under heat stress (Figures 1A and 1B). Consistent with the peak expression level of RCF2 occurring at the reproductive stage, the flowers of the rcf2-1 plants were also more susceptible to heat stress than those from wild-type plants (Figure 1B). We then determined the expression levels of most of the 21 HSFs in rcf2-1. The expression of HSFC1, HSFA4a, and HSFA6b is substantially reduced in rcf2-1 plants under control conditions (Figures 1C and 1F). Transcript levels of most HSFs (including HSFA1b) examined in this study are significantly lower in rcf2-1 plants than in the wild type under heat stress (Figures 1C to 1G). In contrast, transcript levels of HSFA7a and HSFB2b are slightly higher in the rcf2-1 plants than in wild-type plants (Figures 1D and 1G). DREB2A and DREB2C control the expression of HSFA3 under heat stress (Sakuma et al., 2006; Schramm et al., 2008; Chen et al., 2010). Correlated with the reduced heat induction of HSFA3 in rcf2-1, the expression of DREB2A and DREB2C is substantially reduced in rcf2-1 upon heat stress (Figures 1E, 1H, and 1I). Consistent with the reduced basal thermotolerance of rcf2-1 and the overall reduced accumulation of HSFs 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 and HSP101 in rcf2-1 plants transformed with wild-type RCF2 under the control of its native promoter (the rcf2-1 complementation lines mentioned earlier). The expression of HSFA7b and HSP101 in rcf2-1 complementation lines is similar to that in the wild type (Supplemental Figures 5C and 5D). Furthermore, thermotolerance in fry2-1 plants is significantly reduced, and this is correlated with the overall decreased expression levels of heat stress–responsive genes (Supplemental Figure 6). Together, these results indicate that RCF2 is an important positive regulator of heat stress–responsive gene expression and themotolerance in plants.
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 grow for 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 to heat 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 the nonstressed 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 to heat 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 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.
RCF2 Interacts with a NAC Transcription Factor, NAC019, in Vivo, and NAC019 Is Required for Heat Stress–Responsive Gene Regulation and Thermotolerance
As indicated by yeast two-hybrid assays, RCF2/CPL1 interacts with a NAC transcription factor, NAC019, through the C-terminal region (amino acids 640 to 967) of RCF2/CPL1 (Bang et al., 2008). Because both RCF2/CPL1 and NAC019 are localized in the nucleus (Koiwa et al., 2004; Bang et al., 2008; Bu et al., 2008), we showed that RCF2/CPL1 interacts with NAC019 in vivo by bimolecular fluorescence complementation (BiFC) and split luciferase complementation (Split-LUC) assays in tobacco (Nicotiana tabacum) leaves (Figures 2A to 2C). Coimmunoprecipitation assays further confirmed that RCF2/CPL1 interacts with NAC019 in vivo (Figure 2D).
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 at an approximate frequency of 4.23% (110 of 2600 tobacco leaf epidermal cells analyzed exhibited BiFC events). Bars = 25 μm.
(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, and statistically 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 or 1 h. Proteins were extracted and immunoprecipitated before being separated on an SDS-PAGE gel containing 50 μM Phos-tag. After being transferred to a polyvinylidene difluoride membrane, the phosphorylated (indicated as NAC019-P next to an arrow) and unphosphorylated (indicated as NAC019 next to an arrow) forms of NAC019 were detected by anti-HA antibody (top panels). Protein gel blots with immunoprecipitated proteins, which were separated on a regular SDS-PAGE gel, blotted with anti-HA antibody, were used as loading controls (bottom panels). WT-CIP, Wild-type plants expressing 35S:HA-NAC019 treated with alkaline phosphatase, calf intestinal (New England Biolabs), for 0, 1, or 3 h. Blots were quantified with ImageJ software, and values are ratios of phosphorylated (above top panels) or unphosphorylated (below top panels) NAC019 to the corresponding loading control (bottom panels).
Values shown are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.
CPL1 has predicted phosphatase activity that should result in the dephosphorylation of Ser-5-PO4 in the heptad repeat sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) of the CTD of the largest subunit of RNA polymerase II (pol II). Koiwa et al. (2004) showed that recombinant CPL1 is able to dephosphorylate synthetic phosphopeptide substrates resembling the phosphorylated CTD of the largest subunit of pol II. RCF2/CPL1 can dephosphorylate HYPONASTIC LEAVES1 (HYL1), which lacks a typical heptad repeat sequence (Tyr-Ser-Pro-Thr-Ser-Pro-Ser) in its C-terminal region or anywhere else (Manavella et al., 2012). We performed a protein gel blot analysis using Phos-tag (a chemical that binds specifically to the phosphorylated residues in proteins and causes a mobility shift of the phosphorylated proteins) to investigate whether NAC019 is phosphorylated, and if NAC019 is phosphorylated, whether RCF2/CPL1 dephosphorylates it. As shown in Figure 2E, the proportion of the unphosphorylated NAC019 is decreased in rcf2-1 mutant plants under heat stress, suggesting that RCF2 dephosphorylates the phosphorylated NAC019.
The expression of NAC019 is strongest at the reproductive stage, and NAC019 is upregulated by heat stress (Supplemental Figures 7A and 7B). We observed that the expression of NAC019 is reduced in rcf2-1 mutant plants with or without heat stress (Supplemental Figure 7C). These results indicate that RCF2 is not only important for posttranslational modification of NAC019 but is also required for NAC019 expression at the transcriptional or posttranscriptional level. We obtained a T-DNA knockout of NAC019 (Supplemental Figure 7D). Soil-grown nac019 mutant plants are more sensitive to heat stress than the wild type (Figures 3A and 3C). Flowers of the nac019 mutant plants are hypersensitive to heat stress (Figures 3B and 3C). Heat induction of HSFA1b, HSFA6b, HSFB1, and HSFC1 is significantly lower in nac019 mutant plants than in wild-type plants (Figures 3D to 3F), while the heat-induced transcript level of HSFA7a is moderately elevated in nac019 (Figure 3G). Correlated with the overall reduced heat induction of HSFs, transcripts of HSP18, HSP26.5, HSP70B, and HSP101 are less abundant in whole seedlings of nac019 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 reduced accumulation of HSP18 and HSP101 transcripts under heat stress (Figures 3H and 3I). We also examined the expression levels of HSFA6b and HSP18 in nac019 plants transformed with wild-type NAC019 driven by its native promoter (NAC019:NAC019-HA [nac019 complementation lines]; Supplemental Figure 7E). The expression of HSFA6b and HSP18 in nac019 complementation lines is similar to that in the wild type (Supplemental Figures 7F and 7G). Thus, the in vivo target of RCF2, NAC019, is required for thermotolerance and heat stress–responsive gene expression.
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 failed to 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 to heat 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 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.
NAC019 Binds to cis-Elements in Promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1
NAC019 can potentially bind to the NAC recognition cis-promoter element CATGT when that element is immediately followed by the core binding site CACG or CACG flanked by one or more spacer DNA sequences (Tran et al., 2004; Bu et al., 2008). Such consensus cis-elements that NAC019 can potentially bind to are found in the promoter regions of HSFA1b, HSFA6b, HSFA7a, HSFB1, and HSFC1 (Supplemental Figures 8 and 9). We first performed electrophoretic mobility shift assays (EMSAs) to determine whether NAC019 is able to bind to cis-elements in the HSFA6b promoter in vitro. As shown in Supplemental Figure 10A, NAC019 can bind to cis-promoter elements in HSFA6b. The EMSA experiments also showed that RCF2 cannot bind to the NAC019 binding sites in the promoter of HSFA6b (Supplemental Figure 10B). Under our EMSA experimental conditions, we did not observe RCF2 supershifts NAC019 through its binding in vitro to NAC019 on specific cis-elements in the HSFA6b promoter (Supplemental Figure 10B). We then performed chromatin immunoprecipitation (ChIP) assays followed by qPCR analyses using the nac019 plants expressing NAC019:NAC019-HA (Supplemental Figure 7E) to determine whether NAC019 is able to bind to those cis-promoter elements in vivo. ChIP-qPCR experiments 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 performed ChIP-qPCR analysis with rcf2-1 plants expressing RCF2:RCF2-FLAG (Supplemental Figure 3A) to investigate whether RCF2 is enriched in promoter regions where NAC019 is bound. The data presented in Supplemental Figure 11 indicate that RCF2 is enriched in promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1 but not of HSFB1. These results suggest that, although RCF2 is unable to bind to cis-promoter elements of HSFs, as indicated in Supplemental Figure 10B, NAC019 binds to specific HSF promoters and RCF2 interacts with NAC019 on these promoters.
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 analyses with 15-d-old nac019 plants expressing NAC019:NAC019-HA (Supplemental Figure 5D) subjected to 37°C for 1 h. Regions of amplification are specified (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 for the 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 are indicated by different lowercase letters (P < 0.01). Values shown are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.
We subsequently performed a dual luciferase reporter assay in tobacco leaves to examine the effect of RCF2 and NAC019 on HSFA6b promoter activity in vivo. RCF2 enhances the transactivation of NAC019 on the HSFA6b promoter under both normal and heat stress conditions (Figure 4F). The phosphatase-inactive form of RCF2 (RCF2[D161A]; Hausmann et al., 2005) fails to enhance NAC019 transactivation activity on the HSFA6b promoter (Figure 4F). In contrast to the wild-type RCF2, the phosphatase-inactive form of RCF2 (RCF2[D161A]) is unable to restore the heat induction of HSFA7b and HSP101 in rcf2-1 to the level in the wild type (Supplemental Figures 12A and 12B). These data suggest that phosphatase activity of RCF2 is required for heat stress–responsive gene expression in planta.
Overexpression of RCF2 or NAC019 in Arabidopsis Increases Thermotolerance
Because both RCF2 and NAC019 are required for heat stress–responsive gene expression and thermotolerance, we generated Arabidopsis transgenic plants expressing RCF2 or NAC019 under the control of the 35S promoter (35S:FLAG-RCF2 or 35S:HA-NAC019) to investigate whether the overexpression of RCF2 or NAC019 can improve plant performance under heat stress (Supplemental Figures 13A and 13B). Overexpression of RCF2 increases the thermotolerance of transgenic plants, as indicated by the reduced damage to whole plants and flowers (Figures 5A to 5C). Correlated with the increased thermotolerance, the RCF2 overexpression plants have overall increased expression levels of heat stress–responsive genes (Figures 5D to 5J). The 35S:FLAG-RCF2 transgene is able to restore the heat induction of HSFA7b and 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 for transgenic plants overexpressing NAC019. The NAC019 overexpression plants are more heat tolerant than the wild type, as indicated by reduced damage to whole plants and flowers (Figures 6A to 6C). Except for HSFA7a, whose transcript is reduced, the expression levels of heat stress–responsive genes are elevated in the NAC019 overexpression plants (Figures 6D to 6I). The 35S:HA-NAC019 transgene can complement the nac019 mutant phenotype (e.g., the transgene can restore the heat induction of HSFA6b and HSP18 in nac019 to the expression level in the wild type; Supplemental Figures 13E and 13F), suggesting that the 35S:HA-NAC019 transgene is functional in vivo.
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 for 0 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 the nonstressed 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 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 gene expression in [D] to [J]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (P < 0.01). Values shown are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.
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 normal morphology; 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 the nonstressed 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 for 0 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 gene expression 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 are derived from experiments that were performed at least three times with similar results, and representative data from one repetition are presented.
DISCUSSION
In a genetic screen for regulators of cold-responsive gene expression, we have identified RCF2, which is a new allele of CPL1/FRY2 (Supplemental Figure 1). Previous reports showed that CPL1/FRY2 is involved in the repression of genes that are responsive to cold, ABA, and NaCl (Koiwa et al., 2002; Xiong et al., 2002). The upregulation of stress-responsive genes in cpl1 or fry2-1 mutants might be due to the increased expression of CBF genes (Xiong et al., 2002). Similar to the observations of Koiwa et al. (2002) and Xiong et al. (2002), our results indicate that CBF genes are upregulated in rcf2-1 plants under cold stress (Supplemental Figure 1B) and that the upregulation of CBF genes is very likely a compensatory response to the reduced cold tolerance of rcf2-1 mutant plants (Supplemental Figure 1C). This compensatory response may be partially caused by the reduced expression of PRR5, the negative regulator of CBF genes (Nakamichi et al., 2009; Guan et al., 2013c). PRR5 may directly or indirectly repress CBF genes (Nakamichi et al., 2009). We have observed a similar phenomenon in rcf1-1 mutant plants, which accumulate increased levels of cold-responsive genes, including CBFs, but are hypersensitive to cold stress (Guan et al., 2013c). RCF1 encodes a DEAD box RNA helicase critical for pre-mRNA splicing (Guan et al., 2013c). In rcf1-1, the function of PRR5 and SK12 (a shaggy-like Ser/Thr kinase, another negative regulator of CBF genes) is compromised, because PRR5 and SK12 are misspliced. Reduced activities of PRR5 and SK12 in rcf1-1 may partially explain the increased expression of cold-responsive genes, including CBFs. Our results in this study further suggest that additional important positive and/or negative regulators of cold tolerance, which may function in the CBF-independent pathways, may not function properly in the rcf2-1 mutant. Matsuda et al. (2009) reported that CPL1 represses the wound-induced transcription of jasmonic acid biosynthetic genes, probably by repressing the transcriptional activity of RNA pol II transcription machinery via the CPL1-mediated phosphorylation status of the CTD of the largest subunit of RNA pol II. The precise mechanisms by which RCF2/CPL1/FRY2 preferentially affects cold, ABA, NaCl, and wounding responses remain unknown. From microarray analysis, Aksoy et al. (2013) showed that 114 genes were upregulated in the cpl1-2/fry2-1 mutant plants under unstressed conditions and that CPL1 appears to repress the expression of genes encoding the signaling components downstream of iron deficiency by an unknown mechanism.
In this study, we observed that RCF2/CPL1/FRY2 can positively control the expression of genes under either cold stress or heat stress (Figure 1; Supplemental Figures 1D, 1E, and 6). Aksoy et al. (2013) also reported in a microarray study that 132 genes are downregulated in the cpl1-2/fry2-1 mutant plants under unstressed conditions, although further investigation was needed to elucidate how CPL1/FRY2 controls the expression of these genes in a positive manner. The observation that the expression of RCF2/CPL1/FRY2 is upregulated by heat stress motivated us to investigate the role of RCF2 in heat stress responses (Supplemental Figure 5B). We observed that rcf2-1 and fry2-1 mutant plants are hypersensitive to heat stress (Figure 1; Supplemental Figure 6). The reduced thermotolerance of rcf2-1 and fry2-1 plants is correlated with the overall reduced expression of many HSFs and HSPs in heat-stressed rcf2-1 and fry2-1 plants (Figure 1; Supplemental Figure 6). Reduced expression of one of the master regulators of HSFs, HSFA1b, in rcf2-1 and fry2-1 may contribute to the overall reduced expression of the HSFs examined in rcf2-1 and fry2-1 (Figure 1; Supplemental Figure 6). In addition, lower heat induction of HSFA3 in rcf2-1 and fry2-1 may result from the reduced expression of DREB2A and DREB2C, because these two proteins control the expression of HSFA3 under heat stress (Sakuma et al., 2006; Schramm et al., 2008; Chen et al., 2010). HSFB1 and HSFB2b act as repressors of the expression of heat-inducible HSF genes, such as HSFA2 and HSFA7a (Ikeda et al., 2011). In rcf2-1 and fry2-1 under heat stress, the expression of HSFB1 is reduced while that of HSFB2b is slightly upregulated. Therefore, the reduced expression of HSFA2 in rcf2-1 and fry2-1 and the elevated level of HSFA7a transcripts might reflect the collective effects of the altered 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 a 6-h heat treatment (Kilian et al., 2007), indicating that the expression level of HSFA1b under heat stress fluctuates and that the downregulation by 1 h of heat stress is an early response of plants to high temperature. The upregulation of HSFA1b at 6 h after 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 overexpression of HSFA1b increases the thermotolerance of Arabidopsis (Prändl et al., 1998). In contrast, the expression levels of HSFC1 in Arabidopsis shoot tissues after 6 h of heat treatment are similar as those after 1 h of heat treatment (Kilian et al., 2007). HSFC1 may function as a coactivator when combining with class A HSFs, because HSFC1 has no activation function (Kotak et al., 2004), suggesting that the downregulation of HSFC1 may be correlated with the downregulation of HSFA1b after a 1-h heat stress treatment (Kilian et al., 2007). The biological function of HSFC1 in plants under heat stress has not been experimentally determined.
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 jasmonic acid–signaled defense responses by directly binding to the promoter of VEGETATIVE STORAGE PROTEIN1 and is also involved in drought stress and ABA signaling (Tran et al., 2004; Bu et al., 2008; Jensen et al., 2010). RCF2/CPL1/FRY2 is predicted to dephosphorylate Ser-5-PO4 in the heptad repeat sequence of the CTD of the largest subunit of RNA pol II. Koiwa et al. (2004) reported that recombinant CPL1 is able to dephosphorylate synthetic phosphopeptide substrates resembling the phosphorylated CTD domain of the largest subunit of RNA pol II. To date, no catalytic activity of CPL1 toward native RNA pol II has been demonstrated. HYL1 is also probably dephosphorylated by CPL1 (Manavella et al., 2012). Based on the above evidence, we determined the phosphorylation status of NAC019 in rcf2-1 plants and found that the proportion of the unphosphorylated form of NAC019 is reduced in rcf2-1 plants under heat stress, suggesting that NAC019 might be dephosphorylated by RCF2 (Figure 2E). In addition, we found that the expression of NAC019 is reduced in heat-stressed or unstressed rcf2-1 mutant plants (Supplemental Figure 7C). These results indicate that RCF2 is not only important for posttranslational modification of NAC019 but is also required for NAC019 expression at the transcriptional or posttranscriptional level. We subsequently investigated the role of NAC019 in the heat stress responses. The nac019 mutant plants show increased sensitivity to heat stress, especially in the reproductive stage, when NAC019 reaches its peak expression level (Figures 3A to 3C; Supplemental Figure 7A). The reduced thermotolerance of nac019 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 nac019 plants (Figures 3D to 3I).
The NAC transcription factor complete recognition sequence was determined with a yeast one-hybrid system (Tran et al., 2004), and the recognition sequence contains CATGT and harbors CACG as a core binding site. Bu et al. (2008) reported that NAC019 shows high affinity with site CATGTCCACG (CATGT and CACG are spaced by one nucleotide), and NAC019 can bind to either site CATGT or site CACG. Because consensus NAC019 binding sites were found in the promoter regions of the five HSFs that were differentially expressed in nac019 mutant plants, we then performed EMSA, and a high-affinity DNA–protein complex was detected and the DNA binding activity was reduced with the addition of competitor (Supplemental Figure 10A), indicating that NAC019 binds to the HSFA6b promoter in vitro. The cis-promoter element (a 28-bp segment) in HSFA6b contains a NAC recognition site, CATGT, and a core binding site, CACG, which is followed by a 19-bp spacer DNA sequence; cis-promoter elements in promoter regions of three other HSFs (HSFA1b, HSFA7a, and HSFC1) contain CATGT and CACG spaced by 18-bp (HSFA1b and HSFC1) and 51-bp (HSFA7a) DNA sequences (Supplemental Figures 8 and 9). ChIP-qPCR analyses further confirmed that NAC019 binds to cis-elements in the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1 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 enriched in promoter regions of HSFA1b, HSFA6b, HSFA7a, and HSFC1 where NAC019 is bound. Because RCF2 interacts with NAC019 in vivo, our data suggest that RCF2 interacts with NAC019 on the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1 (Supplemental Figure 11). Our data also suggest that HSFB1 is an indirect target of RCF2-NAC019. Furthermore, we showed through dual luciferase reporter assays that RCF2 is able to enhance the transactivation of NAC019 on the HSFA6b promoter and that RCF2(D161A), the phosphatase-inactive form of RCF2 (Hausmann et al., 2005), eliminates the enhanced transactivation of RCF2 and NAC019 on the HSFA6b promoter (Figure 4F). Finally, we showed that RCF2(D161A) driven by the RCF2 native promoter is not functional in vivo to control heat stress–responsive gene expression (Supplemental Figures 12A and 12B). Our data suggest that the phosphatase activity of RCF2 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 the largest subunit of RNA pol II under heat stress. Since we lack a mutant form of RCF2 that fails to dephosphorylate NAC019 but is still able to dephosphorylate the CTD of the largest subunit of RNA pol II, we do not know whether RCF2 phosphatase activity on the CTD of the largest subunit of RNA pol II is critical for heat stress responses. The importance of RCF2 and NAC019 in thermotolerance is further supported by gain-of-function studies. Transgenic Arabidopsis plants constitutively expressing RCF2 or NAC019 are heat tolerant (Figures 5 and 6). The increased thermotolerance in RCF2 overexpression or NAC019 overexpression plants is correlated with the elevated accumulation of heat-responsive genes (Figures 5 and 6). These results further support the inference that both RCF2 and NAC019 are positive regulators for heat-responsive gene expression and thermotolerance in plants (Figure 7). It is clear that, although every plant tissue is vulnerable to heat stress, the reproductive tissues are particularly susceptible (Zinn et al., 2010). Because the peak expression levels of RCF2 and NAC019 occur at the reproductive stage and RCF2 and NAC019 protect plant reproductive tissues (flowers) from heat stress (Figures 1B and 3B; Supplemental Figures 5A and 7A), and because RCF2 and NAC019 are highly conserved across plant species (Supplemental Figure 14), the manipulation of RCF2 and NAC019 levels in heat-sensitive crops such as maize and soybean may increase their thermotolerance and especially the thermotolerance of their reproductive tissues.
Working Model for RCF2 and NAC019 Function in Heat Stress–Responsive Gene Expression and Thermotolerance.
Heat stress induces the expression of HSFs, which are responsible for the accumulation of HSPs. Heat stress also regulates RCF2 expression. RCF2 interacts with and dephosphorylates NAC019, and both proteins are required for the heat induction of HSFs and thermotolerance.
METHODS
Plant Materials and Growth Conditions
A firefly luciferase reporter gene driven by the cold stress–responsive CBF2 promoter (−1500 to −1 bp upstream of the transcription start site) was introduced into Arabidopsis thaliana plants in the Columbia glabrous1 background. Seeds from one homozygous line expressing a single functional copy of the CBF2:LUC gene (referred to as the wild type) were mutagenized with ethyl methanesulfonate. The rcf2-1 mutant with altered CBF2:LUC gene expression was isolated from M2 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 the ABRC: SALK_096295 (nac019) and CS24934 (fry2-1). Arabidopsis seedlings on Murashige and Skoog (MS) medium agar plates (1× MS salts, 2% Suc, and 0.6% agar, pH 5.7) were routinely grown under cool-white light (∼120 μmol m−2 s−1) at 21 ± 1°C with a 16-h-light/8-h-dark photoperiod. Soil-grown plants were kept under cool-white light (∼100 μmol m−2 s−1) with a 16-h-light/8-h-dark photoperiod at 21 ± 1°C and with a 1:1 ratio of potting 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, and 1632 mutant plants were chosen from the F2 generation based on altered CBF2:LUC phenotype. Simple sequence length polymorphism markers were designed according to the information in the Cereon Arabidopsis Polymorphism Collection and were used to analyze recombination events (Jander et al., 2002). Initial mapping revealed that the rcf2-1 mutation is located on the upper arm of chromosome 4 between F9F13 and F7K2. Fine mapping within this chromosomal interval narrowed the RCF2 locus to the BAC clone F17L22. All candidate genes in this BAC were sequenced from the rcf2-1 mutant and compared with those in GenBank to find the rcf2-1 mutation.
For complementation of the rcf2-1 mutant, a 7104-bp genomic fragment of At4g21670 that included 1695 bp upstream of the translation initiation codon was amplified with F17L22 as a template (for primer sequences, see Supplemental Table 2). The amplified fragment was first cloned through Gateway technology (Invitrogen) into the binary vector pEarleyGate302. The resulting construct (RCF2:RCF2-FLAG) was transferred into Agrobacterium tumefaciens (strain GV3101), and rcf2-1 plants were transformed by the floral dip method (Clough and Bent, 1998).
Gene Complementation of nac019
For complementation of the nac019 mutant, a genomic fragment of the NAC019 gene including its promoter, introns, and exons (3′-untranslated region excluded) was amplified with F14G24 as a template (for primer sequences, see Supplemental Table 2). The amplified fragment was first cloned through Gateway technology (Invitrogen) into the binary vector pEarleyGate301. The resulting construct (NAC019:NAC019-HA) was transferred into Agrobacterium (strain GV3101), and nac019 plants were transformed by the floral dip method.
Overexpression of RCF2 and NAC019
Coding regions of RCF2 and NAC019 were amplified by PCR and cloned into pEarleyGate202 and pEarleyGate201, 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 dip method. The construct (35S:FLAG-RCF2 or 35S:HA-NAC019) was also transferred 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-Time RT-PCR Analysis
Fourteen-day-old seedlings grown on MS medium (1× MS salts, 2% Suc, and 0.6% agar, pH 5.7) were used for RNA isolation. Total RNA was extracted from wild-type, mutant, and/or transgenic plants with Trizol reagent (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 and rcf2-1, 5 μg of total RNA was used for the synthesis of the first-strand cDNA with the Maxima First-Strand cDNA Synthesis kit in a total volume of 20 μL (Fermentas). RCF2 fragments were amplified with a pair of primers (RCF2 RT F and RCF2 RT R) with cDNA templates using Taq DNA polymerase (Thermo Scientific Fermentas). The RT-PCR products were subcloned 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 μg of total RNA was used for synthesis of the first-strand cDNA with the Maxima First-Strand cDNA Synthesis kit in a total volume of 20 μL (Fermentas). The cDNA reaction mixture was diluted two times, and 5 μL was used as a template in a 20-μL PCR. PCRs included a preincubation at 95°C for 2 min followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 40 s, and extension at 72°C for 45 s. All reactions were performed in the CFX96 Real-Time PCR Detection system (Bio-Rad) using Maxima SYBR Green/Fluorescein qPCR Master Mix (Fermentas). Each experiment had six biological replicates (three technical replicates for each biological replicate). The comparative threshold cycle method was applied. TUB8 was used as a reference gene. The primers used in this study are listed in Supplemental 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 cloned into pSPYNE-35S and pSPYCE-35S, respectively. The resulting constructs (RCF2-nYFP and NAC019-cYFP) were transformed into Agrobacterium strain C58C1 and coinfiltrated with 35S:p19 (p19 is an RNA-silencing repressor protein from Tomato bushy stunt virus [Voinnet et al., 2003]) in Agrobacterium strain C58C1 into the 3-week-old leaves of tobacco (Nicotiana tabacum) plants. The infiltrated tobacco plants were grown for an additional 3 d in a growth chamber under a 16-h-light/8-h-dark photoperiod at 21°C. Yellow fluorescent protein signals in transformed tobacco leaves were then detected with a Leica SP5X confocal microscope (Leica Microsystems).
For Split-LUC assay, coding regions of RCF2 and NAC019 were amplified by PCR and cloned into the Gateway-compatible firefly luciferase complementation imaging vectors (modified from original plasmids described by Chen et al. [2008]). The resulting constructs (RCF2-nLUC and NAC019-cLUC) were transformed into Agrobacterium strain C58C1 and coinfiltrated with 35S:p19 in Agrobacterium strain C58C1 into the 3-week-old leaves of tobacco plants. The infiltrated tobacco plants were grown for an additional 3 d in a growth chamber under a 16-h-light/8-h-dark photoperiod 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 (Leister et al., 2005; Choi et al., 2012) with minor modifications. Briefly, coding regions of RCF2 and NAC019 were amplified by PCR and cloned into pEarleyGate 202 and pEarleyGate 201, respectively. The resulting constructs (FLAG-RCF2 and HA-NAC019) were transformed into Agrobacterium strain C58C1 and coinfiltrated with 35S:p19 in Agrobacterium strain C58C1 into the 3-week-old leaves of tobacco plants. The infiltrated tobacco plants were grown for an additional 3 d in a growth chamber under a 16-h-light/8-h-dark photoperiod at 21°C. Proteins were extracted from leaf samples with extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× Halt protease inhibitor cocktail [Fisher Scientific]). The protein extracts were incubated overnight with anti-hemagglutinin (HA) antibody (Sigma-Aldrich). The immunocomplexes were collected by adding protein A agarose beads and were washed with immunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 10% glycerol, 0.15% Triton X-100, 1 mM PMSF, and 1× Halt protease inhibitor cocktail [Fisher Scientific]). The pellet (immunocomplexes with beads) was resuspended in 2× SDS-PAGE loading buffer. Eluted proteins were analyzed by immunoblotting using anti-FLAG antibody (Sigma-Aldrich) or anti-HA antibody (Sigma-Aldrich). Chemiluminescence signals were detected by autoradiography.
Mobility Shift Assay to Detect Phosphorylated NAC019 in Vivo
The 35S:HA-NAC019 construct generated for the overexpression of NAC019 was also transferred into Agrobacterium (strain GV3101), and rcf2-1 mutant plants were transformed by the floral dip method. Proteins were extracted from 3-week-old wild-type and rcf2-1 transgenic plants expressing 35S:HA-NAC019 subjected to heat stress at 37°C for 0 or 1 h with extraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 10% glycerol, 1% Triton X-100, 1 mM PMSF, and 1× Halt protease inhibitor cocktail [Fisher Scientific]) and separated on a 12% SDS-PAGE gel containing 50 μM Phos-tag (NARD Institute) and 100 μM MnCl2. Separated proteins were then transferred to a polyvinylidene difluoride membrane and blotted with anti-HA antibody (Sigma-Aldrich). Proteins extracted from 3-week-old wild-type plants expressing 35S:HA-NAC019 were 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 and unphosphorylated NAC019. All of the proteins tested were also separated on a regular SDS-PAGE gel and blotted with anti-HA antibody as a loading control.
ChIP Assays
The construct (RCF2:RCF2-FLAG or NAC019:NAC019-HA) used for gene complementation assays described above was also transformed into wild-type plants through Agrobacterium (strain GV3101)–mediated transformation by the floral dip method. ChIP assays were performed with 15-d-old seedlings expressing RCF2:RCF2-FLAG or NAC019:NAC019-HA as described (Guan et al., 2013b). Briefly, seedlings were cross-linked with 1% formaldehyde, and chromatin was isolated, sonicated (Fisher Biodismembrator; 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 immunoprecipitated with anti-FLAG (Sigma-Aldrich; F1804) or anti-HA (Sigma-Aldrich; H6908) antibody at 4°C overnight. The chromatin antibody complex was precipitated with salmon sperm DNA/protein G or A agarose beads, washed with four different buffers for 5 min per buffer, and reverse cross-linked in elution buffer (1% SDS and 0.1 M NaHCO3) containing 200 mM NaCl for 6 h at 65°C. Proteins in the complex were removed by proteinase K at 45°C for 1 h. DNA was precipitated in the presence of 2 volumes of ethanol, one-tenth volume of 3 M sodium acetate, pH 5.2, and 2 μg of glycogen. Real-time PCR analysis was performed with immunoprecipitated DNA using a Bio-Rad CFX96 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 the translation start site) containing the cis-element CATGT and CACG that are recognized by NAC019 (as determined by ChIP-qPCR analysis) was amplified by PCR with BAC clone MWI23 as a template. The PCR product was cloned into the transient expression vector pGreenII 0800-LUC to serve as a reporter plasmid (HSFA6b:LUC). The coding regions of NAC019 and RCF2 (one copy of FLAG-encoding sequence was included in the forward PCR primer before the translation start site of RCF2) were amplified by PCR and cloned into the transient expression vector pGreenII 62-SK to serve as effect plasmids (pGreenII 62Sk-NAC019 and pGreenII-FLAG-RCF2, abbreviated as NAC019 and RCF2, respectively). The RCF2 coding region (one copy of FLAG-encoding sequence was included in the forward PCR primer before the translation start site of RCF2) with one amino acid substitution in the phosphatase catalytic domain (D161A) was amplified by PCR and cloned into the transient expression vector pGreenII 62-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 with Agrobacterium (strain C58C1) harboring the above plasmids in the following 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 plants were 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. The activities of firefly luciferase under the control of the HSFA6b promoter (HSFA6b:LUC) and Renilla luciferase under the control of the 35S promoter (35S:LUC; both the HSFA6b:LUC and 35S:LUC transgenes are present on the HSFA6b:LUC reporter plasmid, and 35S:LUC served as an internal control) were measured using the reagents contained in the Dual-Luciferase Reporter Assay system (Promega) with the Modulus Microplate Multimode Reader (Turner BioSystem) as described (Hellens et al., 2005). Normalized data (the ratio of luminescent signal intensity from the HSFA6b:LUC reporter and luminescent signal intensity from the internal control reporter, 35S:LUC) from 15 independent biological samples are presented.
EMSAs to Detect the Binding of NAC019 to the HSFA6b Promoter in Vitro
The coding regions of NAC019 and RCF2 were amplified by PCR and cloned into the pMAL-c5E vector (New England Biolabs). The resulting constructs (MBP-NAC019 and MBP-RCF2) were transformed into Escherichia coli strain Rosetta (DE3) pLysS (EMD Millipore). The production and purification of MBP-NAC019 and MBP-RCF2 fusion proteins were performed following the manufacturer’s instructions (New England Biolabs). Complementary pairs of 5′-end biotin-labeled and unlabeled oligonucleotides that contain 3× tandem repeats of NAC019 recognition sites (for sequences, see Supplemental Table 2) were annealed and used as probes for the EMSA studies. The EMSA reactions were performed with a LightShift Chemiluminescent EMSA kit (Pierce). In the reaction, purified MBP-NAC019 or MBP-RCF2 protein was incubated with binding buffer (12 mM Tris-HCl, 60 mM KCl, 2.5% glycerol, 5 mM MgCl2, 50 ng/μL poly[dI.dC], 0.05% Nonidet P-40, 0.1 mM EDTA, and unlabeled probe) for 20 min on ice before labeled probe was added and incubated for an additional 20 min at room temperature. After incubation, the reaction mixture was loaded on a 4% polyacrylamide gel (acrylamide:bisacrylamide, 37.5:1; Bio-Rad) and run in 0.5× 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 according to the manufacturer’s instructions (Pierce).
Determination of Chlorophyll Content
Chlorophyll content was determined as described (Lichtenthaler and Wellburn, 1983) with minor modifications. Chlorophyll was extracted by incubating ground seedlings in 80% acetone overnight at 4°C in darkness and with continuous shaking. The contents of chlorophyll a and b were calculated as 7.49A664.9 + 20.3A648.2.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative 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 Transcripts 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, and Developmental 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 the Thermotolerance and Expression of Heat Stress–Responsive Genes.
Supplemental Figure 7. NAC019 Expression Profiles, and Heat Stress–Responsive Genes in nac019 Complementation Lines.
Supplemental Figure 8. NAC019 Recognition Sites (CATGT) and Core Binding Sites (CACG) in the Promoter Regions of HSFA1b, HSFA6b, and HSFA7a.
Supplemental Figure 9. NAC019 Recognition Sites (CATGT) and Core Binding 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 of HSFA1b, HSFA6b, HSFA7a, and HSFC1.
Supplemental Figure 12. Expression Levels of HSFA7b and HSP101 in 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 with Their Close Homologs.
Supplemental Table 1. Genetic Analysis of the rcf2-1 Mutant (Wild Type [Female] × rcf2-1 [Male] Cross).
Supplemental Table 2. Primers Used in This Study.
Acknowledgments
We thank Hisashi Koiwa for providing RCF2 full-length cDNA and Dong Liu for providing the Gateway-compatible Split-LUC vectors. We thank Jheesoo Ahn, Grace Wang, Xiaohui Hu, and Rongrong Wang for technical assistance. This work was supported by the National Science Foundation (Grant MCB0950242 to J.Z.).
AUTHOR CONTRIBUTIONS
Q.G. and J.Z. designed the research. Q.G., X.Y., H.Z., and J.Z. performed the research (J.Z. isolated the rcf2-1 mutant; Q.G. and X.Y. performed map-based cloning of rcf2-1; H.Z. made the RCF2:RCF2-FLAG and NAC019:NAC019-HA constructs; Q.G. performed the rest of the experiments). Q.G. and J.Z. analyzed the data. Q.G. and J.Z. wrote the article.
Footnotes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jianhua Zhu (jhzhu{at}umd.edu).
↵[W] Online version contains Web-only data.
Glossary
- CTD
- C-terminal domain
- NLS
- nuclear localization signal
- dsRBM
- double-strand RNA binding motif
- ABA
- abscisic acid
- qPCR
- quantitative RT-PCR
- BiFC
- bimolecular fluorescence complementation
- Split-LUC
- split luciferase complementation
- pol II
- polymerase II
- EMSA
- electrophoretic mobility shift assay
- ChIP
- chromatin immunoprecipitation
- MS
- Murashige and Skoog
- PMSF
- phenylmethylsulfonyl fluoride
- HA
- hemagglutinin
- Received September 22, 2013.
- Revised November 18, 2013.
- Accepted December 15, 2013.
- Published January 10, 2014.
References
