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Keywords:

  • miR398 ;
  • heat stress;
  • CSDs;
  • CCS ;
  • HSF ;
  • Arabidopsis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

microRNAs (miRNAs) play important roles in plant growth and development. Previous studies have shown that down-regulation of miR398 in response to oxidative stress permits up-regulation of one of its target genes, CSD2 (copper/zinc superoxide dismutase), and thereby helps plants to cope with oxidative stress. We report here that heat stress rapidly induces miR398 and reduces transcripts of its target genes CSD1, CSD2 and CCS (a gene encoding a copper chaperone for both CSD1 and CSD2). Transgenic plants expressing miR398-resistant forms of CSD1, CSD2 and CCS under the control of their native promoters are more sensitive to heat stress (as indicated by increased damage at the whole-plant level and to flowers) than transgenic plants expressing normal coding sequences of CSD1, CSD2 or CCS under the control of their native promoters. In contrast, csd1, csd2 and ccs mutant plants are more heat-tolerant (as indicated by less damage to flowers) than the wild-type. Expression of genes encoding heat stress transcription factors (HSF genes) and heat shock proteins (HSP genes) is reduced in heat-sensitive transgenic plants expressing miR398-resistant forms of CSD1, CSD2 or CCS but is enhanced in the heat-tolerant csd1, csd2 and ccs plants. Chromatin immunoprecipitation assays revealed that HSFA1b and HSFA7b are the two HSFs responsible for heat induction of miR398. Together, our results suggest that plants use a previously unrecognized strategy to achieve thermotolerance, especially for the protection of reproductive tissues. This strategy involves the down-regulation of CSD genes and their copper chaperone CCS through heat-inducible miR398.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

microRNAs (miRNAs) are non-coding RNAs approximately 20–24 nucleotides (nt) long that were first identified in Caenorhabditis elegans and then found in other animals and plants (Lee et al., 1993; Llave et al., 2002; Reinhart et al., 2002; Rhoades et al., 2002; Lim et al., 2003; Palatnik et al., 2003). miRNAs are processed from their precursors, which have imperfect stem-loop or hairpin structures, by RNase III-like Dicer enzymes in animals or Dicer-like (DCL) proteins in plants. miRNAs play important regulatory roles in plants by targeting mRNAs for cleavage or translational repression (Bartel, 2004; Beauclair et al., 2010; Yu and Wang, 2010; Yang et al., 2012). Many miRNAs are important for plant growth and development (Jones-Rhoades and Bartel, 2004; Baker et al., 2005; Lauter et al., 2005; Jones-Rhoades et al., 2006; Reyes and Chua, 2007; Zhou et al., 2007). An increasing number of reports demonstrate that miRNAs are also key regulators in plant responses to nutrient homeostasis and to biotic and abiotic stresses (Sunkar and Zhu, 2004; Jones-Rhoades and Bartel, 2004; Fujii et al., 2005; Khraiwesh et al., 2012).

Superoxide dismutase (SOD) is an important reactive oxygen species (ROS)-scavenging enzyme that catalyzes conversion of superoxide radicals to H2O2 and O2, a reaction that constitutes the first cellular defense against oxidative stress (Fridovich, 1995). Plants have evolved three types of SODs with different metal ligands: iron SOD (Fe–SOD), manganese SOD (Mn–SOD) and copper/zinc SOD (Cu/Zn–SOD, also known as CSD; Bowler et al., 1992). Cu/Zn–SOD is a major copper enzyme in plants. The Arabidopsis genome encodes three CSD isozymes: CSD1 in the cytoplasm, CSD2 in chloroplasts, and CSD3 in peroxisomes (Kliebenstein et al., 1998). In Arabidopsis, miR398 has four target genes: CSD1, CSD2, CCS (encoded by At1g12520, is a copper chaperone of CSD1 and CSD2, and delivers copper to these two SODs) and COX5b–1 (a zinc-binding subunit of cytochrome c oxidase encoded by At3g15640; Jones-Rhoades and Bartel, 2004; Sunkar and Zhu, 2004; Cohu et al., 2009; Beauclair et al., 2010). When plants are exposed to high light, heavy metals (Cu2+ and Fe3+) or methyl viologen (MV), expression of miR398 is down-regulated, which permits up-regulation of one of its target genes, CSD2 (Sunkar et al., 2006). Over-expression of a miR398-resistant version of CSD2 leads to improved plant performance under oxidative stress caused by high light, heavy metals and MV (Sunkar et al., 2006).

Heat stress adversely affects the distribution and productivity of horticulturally and agriculturally important plants worldwide. When the temperature rises ≥5°C above the optimum, it is experienced as heat stress by all living organisms including plants. Heat stress disrupts normal functions of cellular processes, may severely delay plant growth and development, and may even result in death. A central component of responses to heat stress in all living organisms is the induction of heat shock proteins (HSPs) through the action of heat stress transcription factors (HSFs). HSPs are grouped into five classes based on their approximate molecular weights in kDa: HSP100, HSP90, HSP70, HSP60 and small heat shock proteins (sHSPs, 15–30 kDa; Iba, 2002). HSPs function as molecular chaperones that are essential for maintenance and/or restoration of protein homeostasis. HSFs recognize heat stress elements (HSE: 5′-GAAnnTTC-3′) that are conserved in promoters of heat stress-responsive genes including HSP genes (Busch et al., 2005). Plant genomes encode a family of HSFs with more than 20 members, and plant HSFs are grouped into three classes (A, B and C) according to their oligomerization domains (Nover et al., 2001; Baniwal et al., 2004). The interplay and complexity among HSFs in plants are being actively studied, and research has established that HSFA1 members act as master regulators of heat stress responses and the remaining HSFs in tomato (Solanum lycopersicum) and Arabidopsis (Mishra et al., 2002; Liu et al., 2011; Nishizawa-Yokoi et al., 2011; Yoshida et al., 2011). However, the more upstream regulators of HSFs remain to be identified.

We are interested in determining whether any miRNAs play a role in heat stress responses in plants. We found that one miRNA, miR398, is rapidly induced in Arabidopsis subjected to heat stress, and three of its target genes (CSD1, CSD2 and CCS) are down-regulated. Transgenic plants expressing miR398-resistant versions of CSD1, CSD2 or CCS are hypersensitive to heat stress, and expression of many heat stress-responsive genes (HSF and HSP genes) under high-temperature conditions is reduced in these plants. In contrast, expression levels of HSF and HSP genes in csd1, csd2 and ccs mutant plants are increased under heat stress, and csd1, csd2 and ccs plants are more tolerant to heat stress than wild-type plants. We also identified two HSFs (HSFA1b and HSFA7b) that are responsible for heat induction of miR398. Together, our results reveal an essential regulatory loop for plant thermotolerance that involves HSF genes, miR398 and its target genes CSD1, CSD2 and CCS.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

miR398 is induced by heat stress

To study the physiological and molecular function of miRNAs in the plant heat stress response, we examined the expression profiles of known miRNAs under heat stress in Arabidopsis seedlings grown on MS medium by small RNA Northern hybridization analysis. Expression of miR393 is moderately down-regulated by heat stress, while that of miR169 is slightly induced by heat stress (Figure S1a). We also observed that expression of miR398 is quickly induced by heat stress at 37°C and reaches its peak level 2 h after heat stress is initiated (Figure 1a). In Arabidopsis, the miR398 family is encoded by three loci (miR398a, miR398b and miR398c; Bonnet et al., 2004; Jones-Rhoades and Bartel, 2004; Sunkar and Zhu, 2004). Mature miR398 sequences produced by the three miR398 genes are nearly identical (Figure 1b). Real-time quantitative RT–PCR analysis revealed that expression of miR398 precursors is also elevated under heat stress, and that heat stress induces expression of miR398b to a much higher level than that of the other two miR398 precursors (Figure 1c). We produced transgenic Arabidopsis plants expressing a GUS reporter gene under the control of the miR398b promoter (miR398b:GUS) or the miR398c promoter (miR398c:GUS). Expression of miR398b:GUS is heat-inducible (Figure 1d). These results suggest that potential cis-elements present in the promoter regions of miR398b may be recognized by certain HSFs under heat stress. Consistent with the subtle level of heat induction of miR398c, miR398c:GUS is constitutively expressed (Figure 1d). Yamasaki et al. (2009) reported that SPL7 (SQUAMOSA promoter binding protein-like7) activates miR398 transcription under low-copper conditions by direct binding of the miR398c promoter. The low-copper conditions ([Cu] =0.1 μm in growth medium) described by Yamasaki et al. (2009) are the same as the regular MS medium used in this study (see Experimental procedures). We examined SPL7 expression under heat stress using wild-type seedlings grown on MS medium to determine whether SPL7 is responsive to heat stress. Real-time quantitative RT–PCR analysis showed that expression of SPL7 is down-regulated under heat stress (Figure S1b). These results are consistent with SPL7 expression patterns in aerial tissues of Arabidopsis wild-type seedlings under heat stress summarized from publically available gene expression data (Figure S1c; Kilian et al., 2007). We also investigated miR398b expression by real-time quantitative RT–PCR and miR398 expression by small RNA Northern hybridization analysis using wild-type plants grown on MS medium supplemented with 5 μm CuSO4 under heat stress. The 5 μm CuSO4 present in the growth medium is sufficient to dramatically repress miR398 expression (Yamasaki et al., 2007, 2009). We found that expression of miR398b and miR398 are up-regulated by heat stress in plants grown on MS medium supplemented with 5 μm CuSO4 (Figure S1d,e). These results suggest that the increased expression of mature miR398 under heat stress in the present study is not due to increased expression of miR398c and its activator SPL7. These results further suggest that the increased expression of miR398 under heat stress in the current study is a response to heat stress and not to the low copper level in the growth medium.

image

Figure 1. Expression of miR398 under heat stress.

(a) miR398 expression in response to heat stress as determined by small RNA Northern hybridizations. Small RNA U6 was used as the loading control.

(b) Alignment of three mature miR398 isoforms in Arabidopsis.

(c) Expression of precursors of miR398 in response to heat stress.

(d) Expression patterns of miR398b:GUS or miR398c:GUS in two independent lines of transgenic Arabidopsis plants subjected to 0 or 2 h of heat stress at 37°C.

(e) Expression levels of four miR398 target genes in wild-type plants subjected to 0 (control) or 2 h heat stress at 37°C.

(f) Expression of miR398 in soil-grown wild-type plants of Arabidopsis (1 month old) and corn (15 days old) at 37°C for 0 or 2 h.

(g) Expression of miR398 target genes in 1-month-old soil-grown wild-type plants subjected to 0 (control) or 2 h heat stress at 37°C.

Error bars in (c), (e) and (g) represent the standard deviation (= 6). These experiments were repeated at least four times with similar results, and data from one representative experiment are shown.

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Another potential concern is that the 2% sucrose used in our growth medium (see Experimental procedures) may increase miR398 expression; miR398 expression increases when sucrose levels are increased from 0 to 3% in plant growth medium (Dugas and Bartel, 2008). Because heat induces expression of the miR398 precursor miR398b and mature miR398 in plants grown on a medium containing only 1% sucrose (Figure S1f,g), we conclude that the increased expression of miR398 under heat stress in the current study is a response to heat stress and not to high sucrose levels.

Real-time quantitative RT–PCR analysis indicated that expression of three of the four miR398 target genes (CSD1, CSD2 and CCS) is down-regulated by heat stress (Figure 1e). Dugas and Bartel (2008) observed that transcript levels of COX5b–1 did not decrease in transgenic plants over-expressing miR398c, suggesting that COX5b–1 is a poor target of miR398. In this study, we found that expression of COX5b–1 is not responsive to heat stress, suggesting that it may not be targeted by miR398 under heat stress (Figure 1e). These results are further confirmed by the publically available expression data on these genes using aerial tissues of wild-type Arabidopsis seedlings under heat stress (Figure S2; Kilian et al., 2007).

We also examined expression of miR398 and its four target genes in 1-month-old soil-grown Arabidopsis plants under heat stress. Mature miR398 expression is strikingly induced by heat stress (Figure 1f), and expression of three of four miR398 target genes (CSD1, CSD2 and CCS) is dramatically decreased under heat stress (Figure 1g). The expression of COX5b–1 in soil-grown Arabidopsis plants is not responsive to heat stress (Figure 1g). These results further confirmed that expression of miR398 is induced by heat stress. Furthermore, we examined whether miR398 is up-regulated under heat stress in 15-day-old soil-grown corn (Zea mays) plants. As shown in Figure 1(e), expression of miR398 is moderately induced by heat stress in corn plants.

Transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS are defective in heat-responsive gene regulation and are more sensitive to heat stress

To study the significance of heat induction of miR398, we generated transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS under the control of their native promoters (Figure S3a–c). Transgenic plants expressing the normal forms of CSD1, CSD2 or CCS under the control of their native promoters were used as controls (Figure S3a–c). We first determined the thermotolerance of these transgenic plants. All transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS were more sensitive to heat stress at 37°C (as indicated by increased damage at the whole-plant level and to flowers) than wild-type or control plants expressing the normal forms of CSD1, CSD2 or CCS (Figure 2). These results suggest that heat tolerance requires down-regulation of CSD1, CSD2 and their copper chaperone CCS. We then examined the expression of heat stress-responsive genes in these transgenic plants. Compared to their expression in wild-type or control plants, expression of four HSF genes (HSFA1e, HSFA2, HSFA3 and HSFA7b) was substantially reduced in all transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS (Figure 3a–d). Expression levels of three HSP genes (HSP17.6, HSP70B and HSP90.1) were strongly decreased (relative to their expression in wild-type or control plants) in all transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS (Figure 3e–g).

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Figure 2. Thermotolerance of CSD1, CSD2 and CCS transgenic plants.

(a) Thermotolerance of wild-type (WT) and transgenic plants expressing the normal or miR398-resistant form of CSD1 (mCSD1) under the control of its native promoter (referred to as CSD1 transgenic plants). Three-week-old soil-grown plants were subjected to 0 (control) or 7 days heat stress at 37°C, and damage was recorded 4 days later.

(b) Survival rates of CSD1 transgenic plants as shown in (a), and flowers of separate batches of 1-month-old CSD1 transgenic plants under heat stress (37°C for 4 days). Flowers that fail to produce viable siliques are considered dead.

(c) Thermotolerance of wild-type (WT), and transgenic plants expressing the normal or miR398-resistant form of CSD2 (mCSD2) under the control of its native promoter (referred to as CSD2 transgenic plants) under the conditions described in (a).

(d) Survival rates of CSD2 transgenic plants as shown in (c), and flowers of separate batches of 1-month-old CSD2 transgenic plants under heat stress (37°C for 4 days).

(e) Thermotolerance of wild-type (WT) and transgenic plants expressing the normal or miR398-resistant form of CCS (mCCS) under the control of its native promoter (referred to as CCS transgenic plants) under the conditions described in (a).

(f) Survival rates of CCS transgenic plants as shown in (e), and flowers of separate batches of 1-month-old of CCS transgenic plants under heat stress (37°C for 4 days).

Error bars represent the standard deviation (= 50–100). Data are from one representative individual transgenic plant for each transgene, and there are two to three independent transgenic plants per transgene (Figure S3a–c). These experiments were repeated at least four times with similar results, and data from one representative experiment are shown.

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image

Figure 3. Expression patterns of heat stress-responsive genes in CSD1, CSD2 and CCS transgenic plants.

(a–g) Expression of HSF and HSP genes in CSD1, CSD2 and CCS transgenic plants subjected to 0 or 2 h at 37°C. Error bars represent the standard deviation (= 5). The data are from one representative individual transgenic plant for each transgene, and there are two to three independent transgenic plants per transgene (Figure S3a–c). These experiments were repeated at least four times with similar results, and data from one representative experiment are shown.

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Loss-of-function mutants csd1, csd2 and ccs show enhanced heat-responsive gene expression and are more heat-tolerant

To better understand the role of miR398, CSD1, CSD2 and CCS under heat stress, we isolated loss-of-function mutants of CSD1 and CCS genes (Figure S3d–f) and obtained a knockdown mutant of CSD2 (Figure S3d; Rizhsky et al., 2003). Compared to wild-type plants, the csd1, csd2 and ccs mutant plants are more tolerant of heat stress at 37°C, as indicated by reduced damage to flowers (Figure 4a). The improved thermotolerance in csd1, csd2 and ccs mutant plants is correlated with enhanced expression of HSF and HSP genes (Figure 4b–h). These results further confirm that CSD1, CSD2 and CCS are negative regulators of heat stress-responsive genes and thermotolerance.

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Figure 4. Survival rate of flowers, and expression levels of HSF and HSP genes in csd1, csd2 and ccs mutant plants.

(a) Survival rate of flowers of wild-type (WT), csd1, csd2 and ccs plants under heat stress. One-month-old soil-grown plants were subjected to 10 days heat stress at 37°C, and damage to flowers was recorded 4 days later. Error bars represent the standard deviation (= 50–100).

(b–h) Expression of HSF and HSP genes in WT, csd1, csd2 and ccs seedlings subjected to 0 or 2 h heat stress at 37°C. Error bars represent the standard deviation (= 5).

These experiments in Figure 4 were repeated at least four times with similar results, and data from one representative experiment are shown.

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ROS accumulation in CSD1, CSD2 and CCS transgenic plants and loss-of-function csd1, csd2 and ccs mutant plants

To investigate whether altered heat stress-responsive gene expression and thermotolerance are due to altered redox status under heat stress, we measured ROS levels in transgenic plants expressing the normal or miR398-resistant forms of CSD1, CSD2 or CCS, and in loss-of-function csd1, csd2 and ccs mutant plants.

We first measured the ROS levels in CSD1, CSD2 and CCS transgenic plants. Without heat stress, all transgenic plants expressing the miR398-resistant or normal forms of CSD1, CSD2 or CCS accumulate slightly lower levels of superoxide radicals (as indicated by nitroblue tetrazolium staining) than wild-type plants (Figure 5a). Under heat stress, transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS accumulate much lower levels of superoxide radicals than wild-type and control plants that express normal forms of CSD1, CSD2 or CCS (Figure 5a). With regard to hydrogen peroxide (H2O2) accumulation (as indicated by 3,3′–diaminobenzidine staining), all transgenic plants expressing the miR398-resistant or normal forms of CSD1, CSD2 or CCS accumulate similar amounts of H2O2 as wild-type plants under control conditions (Figure 5a). Under heat stress, transgenic plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS accumulate slightly lower levels of H2O2 than wild-type plants (Figure 5a).

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Figure 5. ROS accumulation in CSD1, CSD2 and CCS transgenic plants and csd and ccs mutants under heat stress, and heat induction of HSF genes under oxidative stress.

(a) Nitroblue tetrazolium staining for superoxide free radicals (top panels) and 3,3′–diaminobenzidine staining for H2O2 (bottom panels) in CSD1, CSD2 and CCS transgenic plants.

(b) Nitroblue tetrazolium staining for superoxide free radicals (left panels) and 3,3′–diaminobenzidine staining for H2O2 (right panels) in csd and ccs mutants.

(c) Expression of HSF genes in plants treated with H2O2 and methyl viologen (MV). Error bars represent the standard deviation (= 5).

These experiments in Figure 5 were repeated at least four times with similar results, and data from one representative experiment are shown.

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We also determined ROS levels in csd1, csd2 and ccs mutant plants under heat stress. Without heat stress, csd1, csd2 and ccs mutant plants accumulate slightly higher levels of superoxide radicals (as indicated by nitroblue tetrazolium staining) than wild-type plants (Figure 5b). With heat stress, the csd1, csd2 and ccs mutant plants accumulate much higher levels of superoxide radicals than wild-type plants (Figure 5b). The pattern is similar for H2O2. Without heat stress, H2O2 accumulation is similar in csd1, csd2 and ccs and their wild-type controls (Figure 5b). With heat stress, however, H2O2 levels are much higher in csd1, csd2 and ccs mutant plants than in wild-type plants (Figure 5b). These results indicate that altered expression of CSD1, CSD2 and CCS genes changes the redox status of cells.

HSF genes are responsive to oxidative stress

It is known that altered redox status affects expression of heat-responsive genes, including HSF and HSP genes (Desikan et al., 2001; Vandenabeele et al., 2003; Volkov et al., 2006). The altered ROS accumulation results suggested that the effects of CSD1, CSD2 or CCS on heat-responsive gene expression may be due to their effect on ROS. We performed real-time quantitative RT–PCR analysis to determine whether oxidative stress affects the expression of the HSFA1e, HSFA2, HSFA3 and HSFA7b genes that showed altered expression patterns in CSD1, CSD2 or CCS transgenic plants and loss-of-function mutant plants. H2O2 induces the expression of HSFA2, HSFA3 and HSFA7b, but not of HSFA1e (Figure 5c). Methyl viologen (MV) also induces the expression of HSFA2 and HSFA3 but not of HSFA1e or HSFA7b (Figure 5c). These results support the hypothesis that expression of HSF genes is responsive to redox status, which is in turn regulated by CSD1, CSD2 and CCS.

HSFA1b and HSFA7b bind directly to the promoter regions of miR398b

Heat stress elements (HSE: 5′-GAAnnTTC-3′) were found in the promoter regions of all three miR398 genes (Figure S4). Because only miR398b is strongly responsive to heat stress (Figure 1c,d), we generated transgenic plants expressing FLAG-tagged HSFs under the control of their native promoters in Arabidopsis to search for HSFs that bind to the promoter of miR398b under heat stress (Figure S5a–e). We then performed chromatin immunoprecipitation (ChIP) assays followed by real-time quantitative PCR (qPCR) analysis (ChIP-qPCR). We found that HSFA1b and HSFA7b, but not HSFA1a, HSFA7a or HSFB2a, are able to bind directly to the promoter regions of miR398b where core HSEs are located (Figure 6a,b and Figure S5f–h). Thus, we have identified two HSFs that are responsible for the heat induction of miR398.

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Figure 6. Binding of HSFA1b and HSFA7b to miR398b promoter regions under heat stress and a working model for miR398 function under heat stress.

(a) Binding capacity of HSFA1b to three areas of the miR398b gene under heat stress as determined by ChIP-qPCR analysis.

(b) Binding capacity of HSFA7b to three areas of the miR398b gene under heat stress as determined by ChIP-qPCR analysis.

Regions of amplification: A (containing one HSE, GAAGGTTC) = −2189 to −2073; B (containing one HSE, GAATTTTC) = −679 to −458; C (containing one HSE, GAAAGTTC) = −343 to −190; D (containing no HSE; negative control) = +337 to +479 base pairs relative to the stem loop start site.

(c) Working model for miR398 function under heat stress. Heat stress induces expression of HSF genes, which up-regulate expression of miR398. Heat-induced miR398 negatively targets three genes (CSD1, CSD2 and CCS) that control ROS accumulation under heat stress. The altered redox status in the cells contributes to the accumulation of HSFs and expression of other heat stress-responsive genes that are critical for thermotolerance in plants.

Values in (a) and (b) are results collected from pooled samples of five independent transgenic plants (Figure S5a,b). Error bars in (a) and (b) represent the standard deviation (= 6). These experiments were repeated at least four times with similar results, and data from one representative experiment are shown.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

We have shown that heat-inducible miR398 is required for thermotolerance through down-regulation of its target genes (CSD1, CSD2 and CCS). miR398 is up-regulated by heat stress, and this up-regulation is not due to high sucrose in the growth medium (Figure 1a,c,d,f and Figure S1f,g). A previous report showed that endogenous sucrose levels increased at the end of the light period under warmer temperatures (31°C light/29°C dark) in mature leaves of potato (Solanum tuberosum) plants, but sucrose levels remained essentially the same at the end of the dark period (Lafta and Lorenzen, 1995). Net photosynthesis rates decrease at temperatures above 30°C in most plant species, including corn and wheat (Triticum aestivum), as a result of inactivation of Rubisco (ribulose-1,5–bisphosphate carboxylase oxygenase; Law and Crafts-Brandner, 1999; Crafts-Brandner and Salvucci, 2002). In this study, we start heat stress treatments at 37°C in the early morning (approximately 9 am, when plants have been exposed to light for 3 h). Endogenous sucrose levels may not increase significantly under our experimental conditions due to shorter exposure time to light (approximately 5 h from the beginning of light period) and inactivation of Rubisco at 37°C. Therefore, heat-induction of miR398 is probably not caused by an increase in plant endogenous sucrose levels.

Except for one gene encoding a subunit of cytochrome c oxidase (COX5b–1), all the targets of miR398, including CSD1, CSD2 and CCS, are down-regulated by heat stress (Figure 1e,g). These results are further confirmed by the publically available expression data on these genes using aerial tissues of wild-type Arabidopsis seedlings under heat stress (Figure S2; Kilian et al., 2007). Two lines of evidence support our conclusion that down-regulation of CSD1, CSD2 and CCS by heat-induced miR398 is required for thermotolerance. First, plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS are more sensitive to heat stress and display reduced expression levels of heat-induced HSF and HSP genes (Figures 2 and 3). Second, csd1, csd2 and ccs mutant plants are more heat-tolerant, and accumulate increased levels of transcripts of heat-inducible HSF and HSP genes (Figure 4). Therefore, thermotolerance may be achieved by down-regulation of CSD1, CSD2 and CCS through heat-inducible miR398.

Two HSFs are responsible for the heat induction of miR398. Sequence searches detected putative HSF binding sites (HSEs) in the promoter regions of miR398b, and a subsequent miR398b:GUS experiment demonstrated that miR398b is heat-inducible (Figure 1d). We identified the two HSFs that bind directly to the promoter regions of miR398b by chromatin immunoprecipitation (ChIP) assays followed by real-time quantitative PCR analysis. ChIP-qPCR analysis revealed that HSFA1b and HSFA7b bind to the miR398b promoter under heat stress (Figure 6a,b). Previous research demonstrated that members of the HSFA1 sub-family are master regulators of heat stress responses (Mishra et al., 2002; Liu et al., 2011; Nishizawa-Yokoi et al., 2011; Yoshida et al., 2011). In Arabidopsis, there are four members of the HSFA1 sub-family: HSFA1a, HSFA1b, HSFA1d and HSFA1e. Thus, miR398 is regulated by at least one of the master regulators for heat stress responses. A feedback loop for HSFA7b was also observed. Heat induction of miR398 requires HSFA7b, and miR398 in turn positively affects the heat-induced accumulation of HSFA7b and other HSF genes (Figure 6c).

The altered expression of heat stress-responsive genes observed in plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS as well as csd and ccs mutant plants may be explained by altered activity of HSFA1e. We have two lines of evidence to support this notion. First, decreased heat induction of other HSF and HSP genes is correlated with a reduced HSFA1e transcript level in plants expressing the miR398-resistant forms of CSD1, CSD2 or CCS, (Figure 3). Second, the increased transcript level of HSFA1e is correlated with greater expression of HSF and HSP genes in csd1, csd2 and ccs mutant plants (Figure 4). Furthermore, we suspect that regulation of HSF and HSP genes and heat stress responses by miR398 involves oxidative stress, partly because the signal transduction pathways of heat stress and oxidative stress are inter-connected (Kotak et al., 2007). Production of H2O2 transiently increases after very short periods of exposure to high temperature as a result of NADPH oxidase (Vacca et al., 2004). The heat stress-induced H2O2 induces heat stress-responsive genes in Arabidopsis (Volkov et al., 2006), and this process may be controlled by sensing of H2O2 by HSFs (Miller and Mittler, 2006). In addition, plants become heat-tolerant when pre-treated with H2O2, while mutants defective in NADPH oxidases display compromised thermotolerance (Larkindale and Huang, 2004; Larkindale et al., 2005). As shown here, the redox status is altered in transgenic plants expressing miR398-resistant forms of CSD1, CSD2 or CCS, and csd1, csd2 and ccs mutant plants (Figure 5a,b). As expected, when expression of CSD1, CSD2 or CCS is at a higher level, as in the transgenic plants expressing miR398-resistant forms of CSD1, CSD2 or CCS, superoxide radicals accumulate to a much lower level. In contrast, when CSD1, CSD2 or CCS is not functional, superoxide radicals are over-accumulated in the corresponding mutant plants. Because of an unknown mechanism presumably involving altered NADPH oxidase activity, the H2O2 levels are higher in the csd1, csd2 and ccs mutant backgrounds, but are lower in the transgenic plants expressing miR398-resistant forms of CSD1, CSD2 or CCS under heat stress than in wild-type plants. We have also shown that oxidative stress may induce expression of HSF genes (Figure 5c). Our results suggest that down-regulation of CSD1, CSD2 and CCS by heat-induced miR398 alters the cellular redox status and creates a situation similar to the redox status in csd1, csd2 and ccs mutant plants. The altered redox status may be sensed directly or indirectly by certain plant HSFs to regulate the expression of HSF and HSP genes.

Although multiple abiotic stresses (including low availability of copper, high levels of sucrose, ABA and salt, or drought stress) may lead to up-regulation of miR398 in Arabidopsis, Populus tremula and Medicago truncatula plants (Yamasaki et al., 2007, 2009; Dugas and Bartel, 2008; Jia et al., 2009; Trindade et al., 2010), except for the low copper response mediated by SPL7, no biological function of such up-regulation had been determined before the present study. Under field conditions, multiple stresses may occur simultaneously, for example, a combination of drought and heat stress. The combination of drought and heat stress led to greater damage to plants compared with the effect caused by each individual stress (Craufurd and Peacock, 1993; Savin and Nicolas, 1996; Jiang and Huang, 2001; Wang and Huang, 2004). As mentioned above, miR398 is up-regulated by drought stress in Medicago truncatula (Trindade et al., 2010). It is very possible that miR398 is up-regulated under a combination of drought and heat stress in order to degrade transcripts of its target genes. Yu et al. (2012) reported that the miR398 level is reduced in Chinese cabbage (Brassica rapa) under an extremely high temperature (46°C) for 1 h, although the study did not address the functional significance of such a regulation. In this study, we provide multiple lines of evidence that miR398 is up-regulated by heat stress (37°C) in Arabidopsis. The up-regulation of miR398 by heat stress revealed in this study is probably independent of copper and sucrose levels in the plant growth medium. We have shown that heat induction of miR398 in Arabidopsis results in down-regulation of expression of CSD genes and the CSS gene encoding their copper chaperone, and consequent accumulation of HSFs and HSPs required for thermotolerance (Figure 6c). Because miR398 family members and their target genes are greatly conserved across plant species (Figure S5i; Sunkar and Zhu, 2004; Dugas and Bartel, 2008; Beauclair et al., 2010), down-regulation of expression of CSD genes and the CSS gene by heat-inducible miR398 may be a common mechanism by which plants cope with the deleterious effects of heat stress. We determined miR398 levels in soil-grown corn plants subjected to heat stress at 37°C for 0 or 2 h, and found that miR398 is induced by heat stress in corn plants (Figure 1f). Heatwaves experienced in the summer in the corn belt of the USA and other regions of the world greatly reduce corn yield by damaging the reproductive tissues of corn, so manipulation of miR398 and/or its target genes may be viable strategies for improving the thermotolerance and yield stability of corn.

Experimental Procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

Plant materials and growth conditions

Arabidopsis thaliana (ecotype Columbia) was used as the wild-type in this study. Seeds of the csd2 knockdown (Rizhsky et al., 2003) were kindly provided by Ron Mittler (Department of Biological Sciences, University of North Texas). Seeds of csd1 (SALK_024857) and ccs (SALK_025986) were obtained from the Arabidopsis Biological Resource Center (Columbus, OH). Homozygous plants were identified by diagnostic PCR analysis using the primers listed in Table S1. Arabidopsis seedlings on Murashige and Skoog (MS) medium agar plates (1 x MS salts, 2% sucrose, 0.6% agar, pH 5.7) were routinely grown under cool white light (approximately 120 μmol m−2 sec−1) at 21°C with a 16 h light/8 h dark photoperiod (light is on at 6 am). The MS salts used (Murashige and Skoog, 1962) were from Phyto Technology Laboratories (product ID M519, http://www.phytotechlab.com), where the sole source of copper in MS is from CuSO4 at a concentration of 0.1 μm. Soil-grown Arabidopsis plants were kept under cool white light (approximately 100 μmol m−2 sec−1) with a 16 h light/8 h dark photoperiod at 21°C and a 1:1 ratio of potting soil Metro Mix 360 and LC1 (Sun Gro Horticulture, http://www.sungro.com). Corn (Zea mays L., inbred line B73) seedlings were grown in a 1:1 ratio of potting soil Metro Mix 360 and LC1 with a 14 h light/10 h dark photoperiod at 26°C under cool white light (approximately 450 μmol m−2 sec−1). The corn plants were supplied with Hoagland solution twice weekly.

Generation of miR398b:GUS and miR398c:GUS constructs

The DNA fragments containing the miR398b and miR398c promoters were amplified by PCR using the primers listed in Table S1. The PCR products were cloned into the binary vector pMDC164 by Gateway technology (Invitrogen, http://www.invitrogen.com/site/us/en/home.html). The resulting constructs (miR398b:GUS and miR398c:GUS) were transformed into Arabidopsis wild-type plants (ecotype Columbia) via floral-dip transformation mediated by Agrobacterium tumefaciens (strain GV3101; Clough and Bent, 1998). Seedlings or tissues from the T2 populations were first immersed in a 5–bromo-4–chloro-3–indoyl glucuronide (X–Gluc) solution (2 mm X–Gluc, 100 mm sodium phosphate buffer pH 7.5, 0.5% Triton X–100, 2 mm K3[Fe(CN)6], 2 mm K4[Fe(CN)6], 0.02% NaN3), vacuum-infiltrated for 10 min and then incubated at 37°C for at least 12 h in the dark, followed by incubation in 70% ethanol to remove chlorophyll (Jefferson et al., 1987).

Generation of CSD1:CSD1, CSD1:mCSD1, CSD2:CSD2, CSD2:mCSD2, CCS:CCS and CCS:mCCS constructs

CSD1:CSD1, CSD2:CSD2 and CCS:CCS plasmids in pDONR–zeo (Invitrogen) were constructed using genomic DNA fragments (including native promoters, coding sequences and 3′ UTRs) that were amplified by PCR reactions with BAC clones F22O13, F24D13 and F5O11 as templates using the primers listed in Table S1. miR398-resistant versions of these constructs (CSD1:mCSD1, CSD2:mCSD2 and CCS:mCCS), as described previously (Sunkar et al., 2006; Dugas and Bartel, 2008; Beauclair et al., 2010), were generated by site-directed mutagenesis using the primers in Table S1 and CSD1:CSD1, CSD2:CSD2 and CCS:CCS plasmids in pDONR-zeo as templates. CSD1:CSD1, CSD1:mCSD1, CSD2:CSD2, CSD2:mCSD2, CCS:CCS and CCS:mCCS in pDONR–zeo were transferred to the binary vector pMDC99 (Invitrogen). The resulting constructs were transformed into wild-type (Columbia) plants via floral-dip transformation mediated by Agrobacterium tumefaciens (strain GV3101).

Real-time quantitative RT–PCR analysis

Fifteen-day-old seedlings grown on MS medium were used for total RNA extraction with Trizol reagent (Invitrogen). Total RNA was treated with DNase I (New England Biolabs, https://www.neb.com) for potential genomic DNA contamination. For real-time RT–PCR (real-time quantitative RT–PCR) analysis, 5 μg of total RNA was used for synthesis of first-strand cDNA with the Maxima first-strand cDNA synthesis kit (Fermentas, www.thermoscientificbio.com/Fermentas) in a 20 μl reaction volume according to the manufacturer's instructions. The cDNA reaction mixture was diluted twice, and 5 μl was used as a template in a 20 μl PCR reaction. PCR reactions included a pre-incubation at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 15 sec, annealing at 56°C for 40 sec, and extension at 72°C for 45 sec. All the reactions were performed in a CFX96 real-time PCR detection system (Bio–Rad, www.bio-rad.com/) using iQ SYBR Green Supermix (Bio–Rad). Each experiment had five to six biological replicates (three technical replicates for each biological replicate). Each experiment was repeated at least four times. The comparative Ct method was used. Beta-Tubulin8 (TUB8) was used as the reference gene. The primers used in this study are listed in Table S1.

Determination of ROS levels

Superoxide free radicals were detected as described previously (Lee et al., 2002) with minor modifications. Fifteen-day-old seedlings grown on MS medium were treated at 21 or 37°C for 2 h. These seedlings were vacuum-infiltrated with 0.1 mg ml−1 nitroblue tetrazolium (Sigma, www.sigmaaldrich.com) in 25 mm HEPES buffer (pH 7.6). In a control treatment, 10 mm MnCl2 and 10 units ml−1 of superoxide dismutase were added to the 0.1 mg ml−1 nitroblue tetrazolium solution. Samples were subsequently incubated at room temperature in darkness for 2 h. Chlorophyll was removed using 70% ethanol.

Hydrogen peroxide (H2O2) was detected by 3,3′–diaminobenzidine staining as described previously (Lee et al., 2002) with minor modifications. To detect H2O2, seedlings of the same batch of plants used for superoxide detection were vacuum-infiltrated with 0.1 mg ml−1 3,3′–diaminobenzidine (Sigma) in 50 mm Tris-acetate buffer (pH 5.0). As a control, ascorbic acid at a final concentration of 10 mm was added to the staining solution. Samples were incubated for 24 h at room temperature in darkness. Chlorophyll was removed using 70% ethanol.

Chromatin immunoprecipitation (ChIP) assays

Genomic DNA fragments of HSFA1a, HSFA1b, HSFB2a, HSFA7a and HSFA7b (including their native promoters) genes were amplified by PCR and cloned into pEarlyGate302 (Earley et al., 2006). The resulting constructs were then transformed into Arabidopsis wild-type plants via floral-dip transformation mediated by Agrobacterium tumefaciens (strain GV3101). ChIP assays were performed using pooled 15-day-old seedlings of five independent transgenic plants in the T3 generation grown on MS medium as described previously (Gendrel et al., 2002). Briefly, seedlings that had been heat-treated at 37°C for 2 h were cross-linked with 1% formaldehyde, and chromatin was isolated, sonicated using a Biodismembrator model 120 (Fisher), and pre-cleared using salmon sperm DNA/protein G agarose beads for 1 h. Samples were then immunoprecipitated using anti-FLAG antibody (Sigma, F1804) at 4°C overnight. The chromatin/antibody complex was precipitated using salmon sperm DNA/protein G agarose beads, washed for 5 min with each of four buffers (low salt buffer, high salt buffer, LiCl buffer, and TE buffer; Gendrel et al., 2002) at 4°C, and reverse-cross-linked in elution buffer (1% SDS, 0.1 m NaHCO3) for 6 h at 65°C. Proteins in the complex were removed by digestion with proteinase K at 45°C for 1 h. DNA was precipitated in the presence of two volumes of ethanol, a one-tenth volume of 3 m sodium acetate (pH 5.2), and 2 μg glycogen. Real-time quantitative PCR analysis was performed on immunoprecipitated DNA using a Bio–Rad CFX96 real-time PCR detection system.

Small RNA Northern hybridization analysis

Total RNA was extracted from 15-day-old seedlings using Trizol reagent (Invitrogen). High-molecular-weight RNAs and low-molecular-weight RNAs were separated by adding an equal volume of 20% poly(ethylene glycol; mean molecular weight 8000) in 1 m NaCl on ice for 2–4 h. The supernatant containing low-molecular-weight RNAs was further purified by phenol/chloroform extraction, and precipitated using isopropyl alcohol.

For small RNA Northern hybridization analysis, 30 μg of small RNAs were resolved on a 17% polyacrylamide/7 m urea gel and transferred to Hybond N+ membranes (Amersham Biosciences, www.gelifesciences.com). The membranes were UV-cross-linked, and baked in an oven at 80°C for 1 h. DNA oligos complementary to miRNAs and U6 were labeled with γ–ATP−32P using T4 polynucleotide kinase. Hybridization was performed overnight at 38°C using Perfect Hyb Plus buffer (Sigma-Aldrich). Blots were washed twice for 15 min each time in 2 x SSC/0.1% SDS at 38°C.

Accession Numbers

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

The Arabidopsis Genome Initiative database accession numbers for the sequences referred to in this paper are given in parentheses: miR398a (At2g03445), miR398b (At5g14545), miR398c (At5g14565), CSD1 (At1g08830), CSD2 (At2g28190), CCS (At1g12520), COX5b–1 (At3g15640), HSFA1b (At5g16820), HSFA1e (At3g02990), HSFA2 (At2g26150), HSFA3 (At5g03720), HSFA7b (At3g63350), HSP17.6 (At1g59860), HSP70B (At1g16030) and HSP90.1 (At5g52640).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported by US National Science Foundation grants IOS0919745 and MCB0950242 to J.Z, and by US National Science Foundation grant DBI0922650.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Accession Numbers
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
tpj12169-sup-0001-FigS1-S5.pdfapplication/PDF386K

Figure S1. Expression of miR169, miR393 and SPL7 under heat stress, and heat induction of miR398 under low-sucrose and high-copper conditions.

Figure S2. Expression levels of CSD1, CSD2, CCS and COX5b-1 in aerial tissues of wild-type Arabidopsis seedlings under heat stress.

Figure S3. Expression levels of CSD1, CSD2 and CCS in corresponding transgenic plants, and expression of CSD1 and CCS in csd1 and ccs mutant plants.

Figure S4. Heat stress elements in putative promoter regions of three miR398 genes.

Figure S5. Expression levels of HSFA1b, HSFA7b, HSFA1a, HSFA7a and HSFB2a in corresponding transgenic plants, and binding capacity of HSFA1a, HSFA7a and HSFB2a to the promoter regions of miR398b.

tpj12169-sup-0002-TableS1.pdfapplication/PDF528KTable S1. Primers used in this study.
tpj12169-sup-0003-Legends.docxWord document21K 

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