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First published online February 11, 2009; 10.1104/pp.108.133744 Plant Physiology 149:1773-1784 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Molecular and Genetic Evidence for the Key Role of AtCaM3 in Heat-Shock Signal Transduction in Arabidopsis1,[W],[OA]Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang 050016, China (W.Z., Y.-J.G., S.-Z.Z., P.X., S.-Q.Z., D.-Y.S.); Institute of Genetics and Physiology, Hebei Academy of Agricultural Sciences, Shijiazhuang 050051, China (R.-G.Z.); and Center for Agricultural Resources, Research Institute of Genetics and Development, Shijiazhuang 050021, China (W.Z.)
Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45°C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were down-regulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway.
In most organisms, temperatures above those needed for normal growth act as a form of stress. In plants, heat shock (HS) affects growth and crop production. Most organisms, including higher plants, synthesize heat shock proteins (HSPs) in response to high-temperature stress (Vierling, 1991  ; Queitsch et al., 2000; Nieto-Sotelo et al., 2002; Charng et al., 2006; Yang et al., 2006). In eukaryotes, expression of HSPs is regulated by heat shock transcription factors (HSFs). Upon HS, HSFs become activated and initiate the transcription of HSPs through binding to heat shock promoter elements (HSEs) in the promoter regions of genes encoding HSPs (Nover et al., 2001; Mishra et al., 2002; Baniwal et al., 2004; Charng et al., 2007). Many HSFs and HSPs have been shown to be involved in improving plant thermotolerance (Queitsch et al., 2000; Mishra et al., 2002; Wunderlich et al., 2003; Sanmiya et al., 2004; Charng et al., 2006, 2007; Nishizawa et al., 2006). Although the downstream components (HSFs, HSEs, and HSPs) of heat stress response have been studied in detail, the signal transduction pathway through which HS influences the binding activity of HSFs to HSEs to regulate HSP expression is not well understood. The aim of this study was to identify possible upstream components of HS signal transduction.
Several hypotheses regarding the HS signal transduction pathway have been proposed. For example, Ananthan et al. (1986)
In both animals and plants, data indicate that HS can elicit changes in the levels of intracellular Ca2+, one of the most ubiquitous cellular second messengers. Gong et al. (1998b)
Calmodulin (CaM) is a well-studied intracellular calcium sensor that mediates Ca2+ signal transduction (Snedden and Fromm, 1998
Arabidopsis contains at least nine different CaM genes. AtCaM1 through AtCaM7 share a high level of sequence identity (>95%), differing from each other only by a few nucleic acids; AtCaM8 and AtCaM9 are more divergent from the other forms (Reddy et al., 2000
In this paper, we generated T-DNA knockout mutants and transgenic plants to provide direct molecular genetic evidence that endogenous AtCaM3 is a key component in HS signal transduction. We also confirmed the existence of a previously proposed, new Ca2+-CaM HS signal transduction pathway (Liu et al., 2003
AtCaM3 T-DNA Insertion Mutant Decreases the Thermotolerance of Arabidopsis Seedlings To determine whether AtCaM3 is involved in the HS signal transduction pathway, we first utilized a reverse genetic approach by screening Arabidopsis T-DNA insertion mutants effecting AtCaM family members. T-DNA insertion mutants for AtCaM genes were obtained from the Arabidopsis Biological Resource Center. Of all the mutants obtained, only the homozygous T-DNA insertion mutants cam2, cam3, and cam4, which separately affect AtCaM2, AtCaM3, and AtCaM4, respectively, were detected to be deficient in transcripts by reverse transcription (RT)-PCR analysis in this experiment (Fig. 1B ). The positions of the individual T-DNA insertions are shown in Figure 1A.
AtCaM2 and AtCaM4 were used as controls and compared with AtCaM3 in a thermotolerance assay. The cam2, cam3, and cam4 mutants exhibited no phenotypic differences compared with wild-type plants under normal growth conditions (Fig. 2A ). However, the thermotolerance assay in which 6-d-old seedlings were heat shocked at 45°C for 50 min and then allowed to recover for 6 d at 22°C showed that thermotolerance of the cam2 and cam4 mutant seedlings was similar to that of the wild-type seedlings and that only the cam3 mutant exhibited a significant impairment in thermotolerance (Fig. 2A). The survival rate of the cam3 mutants was about 43% lower than that of the wild-type plants (Fig. 2B).
With the exception of gene expression analyses by RT-PCR (Fig. 1B), to further determine whether the reduction in AtCaM3 was responsible for the increased heat sensitivity, we examined the endogenous total AtCaM protein (translated by all AtCaM genes) levels in wild-type plants and in cam2, cam3, and cam4 mutants after HS at 37°C by western blot. As shown in Figure 2C, AtCaM protein was lower in the cam3 mutant than in wild-type plants or the cam2 and cam4 mutants.
The expression of AtCaM3 after HS was studied in Arabidopsis in order to gain insight into the function of its protein product. The AtCaM3 promoter was fused to the reporter gene GUS and introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation. AtCaM3::GUS transgenic plants were used to analyze the expression pattern of AtCaM3 under HS conditions. Four different transgenic lines were tested. Plants were grown at 22°C for 10 d and then treated at 37°C before they were harvested for GUS activity assays. Assay results showed that, compared with the activity at 0 h, HS treatment caused GUS activity to increase rapidly by 2-fold in the first 2 h. When heat treatment was extended to 4 h, GUS activity was slightly lower than was observed for the 2-h treatment but was still 1.8-fold higher than at 0 h (Fig. 3A
). These GUS activity assays confirmed the results of previous real-time RT-PCR experiments, in which the expression of the AtCaM3 gene was significantly up-regulated after HS (Liu et al., 2005
We also examined the tissue- and organ-specific expression of AtCaM3 in Arabidopsis. As shown in Figure 3B, AtCaM3::GUS was expressed ubiquitously in young seedlings as well as in rosette and cauline leaves (Fig. 3B, a–c). When plants entered the flowering stage, AtCaM3::GUS expression became concentrated in the sepals, stigma, and filaments but not in the anthers or petals (Fig. 3B, e–h). AtCaM3::GUS was also strongly expressed in the separation layer of the abscission zone of flower organs in which the petal, sepal, and stamen had fallen off after anthesis (Fig. 3B, d and i). These results are consistent with the results of previous microarray analyses (Zimmermann et al., 2004 ). GUS assays revealed that AtCaM3 was expressed in most organs and tissues of the plant, especially in the young seedling.
To confirm whether the suppression of AtCaM3 gene expression in cam3 was responsible for the phenotype of impaired thermotolerance, we also performed complementation analyses. A binary vector containing the coding region of AtCaM3 under the control of the 35S promoter was constructed and used to transform the cam3 mutant by the floral-dip method. Due to the more than 95% nucleic acid sequence identity of the seven AtCaM members, to ensure the specificity of the primers, the NOS terminator region of the binary vector was used to design the reverse primer used to identify the transgenic plants. RT-PCR showed that ectogenous AtCaM3 expression was only found in the complemented transgenic lines. By contrast, ectogenous AtCaM3 cDNA was not amplified in the wild-type or cam3 plants (Fig. 4A ). These data confirmed that the cam3 mutant was successfully transformed with the AtCaM3 cDNA. Six homozygous lines of complemented transgenic seedlings (cam3/AtCaM3) were obtained and identified. Three of these lines (1-2, 7-4, and 43-1) were chosen for thermotolerance studies. In these experiments, none of the cam3/AtCaM3 transgenic plants showed variant phenotypes under normal growth conditions as compared with the wild-type and cam3 plants (Fig. 4B). However, the cam3/AtCaM3 transgenic lines completely rescued the heat-hypersensitive phenotype of the cam3 mutants (Fig. 4C) and exhibited survival rates that were similar to those of the wild-type plants (Fig. 4D).
It has been reported that heat induces oxidative damage, and considerable interlinking between heat and oxidative stress responses have been suggested (Gong et al., 1997a , 1998a; Dat et al., 1998). As an additional test of heat sensitivity, the cam3 mutants and complemented transgenic lines were assayed for the accumulation of thiobarbituric acid-reactive substances (TBARS) following heat stress. Plant tissues used in TBARS assays were harvested 2 d after HS at 45°C for 1 h (Larkindale and Knight, 2002; Larkindale et al., 2005). Damage to seedlings, including bleached leaves and plant bleaching, suggested that the damage caused by heating may have been due to oxidative stress occurring during the recovery phase. TBARS levels in three cam3/AtCaM3 transgenic lines were similar to those detected in wild-type plants (Fig. 4E). However, TBARS levels in cam3 mutant plants, which showed decreased thermotolerance, were 250% higher than those detected in wild-type plants (Fig. 4E).
To further confirm the function of AtCaM3 in thermotolerance, transgenic plants were generated by transformation of the AtCaM3 gene fused to GUS under the control of the 35S promoter (35S::AtCaM3-GUS) in the ecotype Columbia background. Because of the high level of nucleic acid sequence identity among AtCaM multigene family members, no specific primers could be used to determine the expression level of AtCaM3 by real-time RT-PCR. Thus, GUS was used as a label to show AtCaM3 expression levels in the different transgenic lines. After testing the level of AtCaM3 expression (as indicated by GUS activity) of seven independent T3 homozygous transgenic lines, three lines (3-7, 3-21, and 3-22) with strong GUS activity compared with wild-type control plants (Fig. 5A ) were used in the following experiments. Transgenic plants carrying 35S::GUS were used as a vector control.
The three 35S::AtCaM3-GUS homozygous transgenic lines, a vector control line, and wild-type plants were plated together on the same Murashige and Skoog plate. There were no significant phenotypic differences between the transgenic and wild-type plants under normal growth conditions (Fig. 5B). The seedlings were grown at 22°C for 6 d followed by exposure to 45°C for 70 min, then they were allowed to recover at 22°C for another 6 d. As shown in Figure 5C, most of the wild-type and vector control plants could not survive under this lethal-level heat stress. The cotyledons of these plants were bleached and dried. However, although they all exhibited a delay in growth, seedlings overexpressing AtCaM3 survived, as demonstrated by their green cotyledons and the presence of young leaves (Fig. 5C). The overall survival rate of the AtCaM3-GUS-overexpressing transgenic lines was 40% to 60% higher than in either the wild-type or vector control plants (Fig. 5D). In the three AtCaM3-GUS-overexpressing transgenic lines, TBARS levels were 24% to 40% lower than those in either the wild-type plants or the 35S::GUS vector control plants (Fig. 5E).
In order to understand the underlying mechanism of AtCaM3-induced thermotolerance in Arabidopsis, the binding activity of HSF to HSE in AtCaM3 knockout mutant, AtCaM3-GUS-overexpressing, and wild-type plants was analyzed by electrophoretic mobility-shift assay. Results indicate that after HS the binding activity of HSF to HSE in the cam3 mutant plants was weaker than that detected in the wild-type plants. In the three AtCaM3-GUS-overexpressing transgenic lines (3-7, 3-21, and 3-22), the binding activity was much stronger than in the wild-type plants (Fig. 6A ). As a control, there was no binding when the wild-type plant was not heated, which suggested that the band shift was induced specifically by heat (Fig. 6A). These data indicate that changes in AtCaM3 expression influence the binding activity of HSF to HSE.
The effect of AtCaM3 on the transcriptional regulation of HSPs was also examined by real-time quantitative RT-PCR. AtHSP18.2, AtHSP25.3, and AtHSP83 were chosen as marker genes. After HS at 37°C for 1 h, levels of AtHsp18.2 mRNA were 66% lower in the cam3 mutants than in the wild-type plants, while levels in the three cam3/AtCaM3 transgenic lines were rescued such that they were 56% to 63% higher than in the wild-type plants (Fig. 6B). Thus, overexpression of AtCaM3 increased the expression of AtHsp18.2. The relative level of AtHsp18.2 expression in the AtCaM3-GUS-overexpressing transgenic lines was 70% to 130% higher than in the wild-type plants (Fig. 6B). Similar results were obtained for the expression of AtHsp25.3 and AtHsp83 in the mutant and different transgenic lines. After HS at 37°C for 1 h, AtHsp25.3 and AtHsp83 mRNA levels in the cam3 mutants were reduced to 47% and 64% of levels detected in the wild-type plants (Fig. 6, C and D), while complemented transgenic lines showed fully restored AtHsp25.3 and AtHsp83 expression (Fig. 6, C and D). Similarly, the expression of AtHsp25.3 and AtHsp83 in the AtCaM3-GUS-overexpressing transgenic lines was 40% to 160% higher than in the wild-type plants (Fig. 6, C and D). The effect of AtCaM3 on the accumulation of AtHSP18.2 was further documented through examination of protein changes by western blot. Ten-day-old seedlings were heat treated at 37°C for 2 h or maintained at 22°C and cellular protein was extracted. AtHSP18.2, a heat-induced protein, was not detected in plants incubated at the normal growth temperature of 22°C. HS at 37°C for 2 h rapidly induced the expression of AtHSP18.2 protein (Fig. 7A ). Similar to changes seen in RNA transcriptional regulation, AtHSP18.2 accumulation in the cam3 mutants was lower than in the wild-type plants (Fig. 7A). In the three cam3/AtCaM3 transgenic lines (1-2, 7-4, and 43-1), AtHSP18.2 accumulation was rescued as compared with the cam3 mutant, with AtHSP18.2 levels that were similar to those in the wild-type plants (Fig. 7B). AtHSP18.2 levels in the three AtCaM3-GUS-overexpressing lines (3-7, 3-21, and 3-22) were higher than in the wild-type plants (Fig. 7C). In these experiments, tubulin protein expression was used to ensure equal sample loading. These results suggest a link between changes in the expression and accumulation of HSPs in both cam3 mutant and transgenic plants and changes in plant thermotolerance.
The Effect of AtCaM3 on Thermotolerance in Arabidopsis
As one of the most conserved cellular proteins, CaM has been well studied in its role as an intracellular Ca2+ sensor. CaM participates in numerous signaling pathways, and expression of CaM genes in plants is regulated by many types of environmental stresses, including wind, touch, wounding, osmotic stress, pathogens, and cold stress (Braam et al., 1997 To obtain direct molecular genetic evidence for the function of AtCaM3 in HS signaling pathways, AtCaM3 T-DNA knockout mutants, complemented transgenic plants, and AtCaM3-GUS-overexpressing plants were generated. Thermotolerance testing indicated that knockout of AtCaM3 by T-DNA insertion clearly decreased the thermotolerance of Arabidopsis, whereas complementary transformants were able to completely rescue the decreased thermotolerance of the cam3 mutant (Fig. 4). Moreover, overexpression of AtCaM3 resulted in transgenic plants with a thermotolerance much higher than that observed in wild-type plants (Fig. 5). As shown in Figure 2, single AtCaM gene knockout (including the cam3 mutant) did not exhibit any phenotypic differences as compared with the wild-type plants under normal growth conditions. These data suggested the existence of functional redundancy between different AtCaM members for plant growth. But the absence of a single AtCaM3 gene or overexpression could change thermotolerance under HS conditions. As shown in AtCaM3::GUS activity tests (Fig. 3A) and western-blot analyses (Fig. 2C), because AtCaM3 was heat induced, AtCaM3 protein in the AtCaM3 T-DNA insertion mutant was not up-regulated after HS. Using a polyclonal antibody, the cam3 mutant showed lower total AtCaM protein levels than the wild type and other mutant (cam2 and cam4) plants. These results suggested that higher AtCaM3 protein levels were linked to high thermotolerance.
Even though AtCaM2 and AtCaM4 have a high amino acid sequence identity with AtCaM3 (Supplemental Fig. S3; Supplemental Table S1), the cam2 and cam4 mutants did not show obvious differences in thermotolerance compared with wild-type plants after HS at 45°C, unlike the cam3 mutant (Fig. 2A). The reason for this phenotypic difference may be that different CaM genes are differentially regulated by distinct cis-regulatory elements under differing stress conditions. Moreover, even CaM genes encoding the same protein can be differentially regulated in response to different external stimuli, such as disease resistance and cold (Takezawa et al., 1995
In eukaryotes, HSFs are the downstream components of the HS signal transduction chain, which regulates the expression of genes encoding HSPs. HSPs, in turn, are known to contribute to thermotolerance (Kotak et al., 2007
HSF binding to HSE activates the transcription of HSP genes (Baniwal et al., 2004
Several HS signal transduction pathways have been proposed. Ananthan et al. (1986)
We have proposed a Ca2+-CaM pathway involved in HS signal transduction (Liu et al., 2003 According to this model, the HS signal is perceived by an as yet unidentified receptor, leading to an increase in the cytosolic concentration of Ca2+, perhaps through the regulation of inositol 1,4,5-triphosphate/phospholipase C. This elevated [Ca2+]i directly activates AtCaM3 and, in turn, stimulates AtCBK3, which ultimately regulates the phosphorylation and DNA-binding activity of HSFs. By binding to HSEs, HSFs may initiate the transcription of HSP genes as part of the plant's adaptation to environmental heat stress.
Plant Materials and Growth Conditions Seeds of Arabidopsis (Arabidopsis thaliana ecotype Columbia) were surface sterilized and plated on Murashige and Skoog medium containing 1.0% (w/v) Suc and 0.3% (w/v) Phytagel (Sigma). The seeds were vernalized at 4°C for 3 d and then grown under long-day conditions (16 h of light/8 h of dark) at 22°C. The T-DNA insertion lines for AtCaM3 (SALK_001357) and AtCaM2 (SALK_114166) were obtained from the Arabidopsis Biological Resource Center. The T-DNA insertion line for AtCaM4 (GABI_309E09) was ordered from GABI-Kat. T-DNA insertion lines were screened and isolated as described by the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/tdna_FAQs.html). Homozygous mutants were used in further analyses.
The 35S cauliflower mosaic virus promoter (restriction enzymes PstI and XbaI) and the reporter gene GUS (restriction enzymes BamHI and SacI) were cloned into the vector pCAMBIA1300, yielding pCAMBIA1300M. To generate AtCaM3::GUS transgenic plants, a fragment of the AtCaM3 promoter containing 1.2 kb of the upstream region of the gene was amplified by PCR using the forward primer 5'-AACTGCAGAGGAGGTAAAGGCCAACAC-3' and the reverse primer 5'-GCTCTAGACCTTGTCGAATAAGCTGAAAG-3'. The amplified fragment was cloned into pCAMBIA1300M using the PstI and XbaI restriction sites, thus replacing the 35S promoter and generating the AtCaM3::GUS construct. To generate 35S::AtCaM3 for complementation experiments, the AtCaM3 coding region was amplified by PCR from the AtCaM3 cDNA using the primers CaM3F1 (5'-CTCTAGAATGGCGGATCAGCTCACCGA-3') and CaM3R1 (5'-CGAGCTCTCACTTAGCCATCATGACCTTAAC-3'). The amplified products (450 bp) were cloned into the binary vector pCAMBIA1300M using the XbaI and SacI restriction sites, thus replacing GUS. To generate 35S::AtCaM3-GUS for plants overexpressing AtCaM3 in the wild-type background, the AtCaM3 cDNA was amplified by RT-PCR using the primers CaM3F1 (5'-CTCTAGAATGGCGGATCAGCTCACCGA-3') and CaM3R2 (5'-CGGATCCCCTTAGCCATCATGACCTTAAC-3'). The 35S::AtCaM3-GUS fusion construct was generated by cloning the 450-bp PCR product into pCAMBIA1300M using the XbaI and BamHI restriction sites (underlined).
Transformation of the different constructs into Arabidopsis (ecotype Columbia) was carried out by the floral dipping method (Clough and Bent, 1998
Histochemical staining for GUS expression was performed according to the method of Jefferson (1987)
GUS activity was quantified according to the method of Jefferson (1987)
Total RNA was isolated from 10-d-old seedlings using the Trizol reagent (Invitrogen). Transcript abundance in wild-type and mutant plants was determined by RT-PCR using a one-step RT-PCR Kit (TaKaRa). The AtCaM3 (At3g56800) transcript was amplified using the forward primer 5'-CGTACCCGATAAATACGGTTG-3' and the reverse primer 5'-GACCTAATTTGCATTTCACAAAACC-3'. The AtCaM2 (At2g41110) transcript was amplified using the forward primer 5'-TTCGTTTTTCCCTTTTTCTCC-3' and the reverse primer 5'-CAAGAATCAGGTTCTGAACCTG-3'. The AtCaM4 (At1g66410) transcript was amplified using the forward primer 5'-CCAAAGAAACGAGAAGAAGAAGC-3' and the reverse primer 5'-GGCTCAAATCAAACCCAAGAC-3'. The actin (At2g37620) transcript served as a control and was amplified using the forward primer 5'-AGGCACCTCTTAACCCTAAAGC-3' and the reverse primer 5'-GGACAACGGAATCTCTCAGC-3'. PCR products were analyzed by agarose gel electrophoresis and stained with ethidium bromide.
All plants were grown under the same conditions, and seeds were harvested at the same time for use in thermotolerance assays. For thermotolerance testing, 6-d-old seedlings were exposed to 45°C for 50 min (to kill CaM mutants) or 70 min (to kill wild-type plants) and then allowed to recover at 22°C for an additional 6 d (Larkindale et al., 2005
TBARS was assayed according to the method of Heath and Packer (1968)
The HSE (Scharf et al., 1990
Total RNA (500 ng) isolated using the Trizol reagent (Invitrogen) from 10-d-old seedlings was used together with the PrimeScript RT Reagent Kit (TaKaRa) for first-stand cDNA synthesis according to the manufacturer's instructions. For RT-PCR, SYBR Premix Ex Taq (TaKaRa) was used. The PCR program was as follows: initial polymerase activation for 10 s at 95°C, and 40 cycles of 95°C for 5 s followed by 60°C for 31 s. The reactions were carried out using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Primer pairs were designed using Primer Express (Applied Biosystems). The primers used for AtHsp18.2 (At5g59720) were 5'-TCGTGATGTGGCAGCGTTTA-3' (forward) and 5'-AAGTCCGCTTTGAACACATGTG-3' (reverse); those for AtHsp25.3 (At4g27670) were 5'-GACGTCTCTCCTTTCGGATTGT-3' (forward) and 5'-CTCCACTTCCTCCTCTGTTTCTTC –3' (reverse); and those for AtHsp83 (At5g52640) were 5'-GCTGCTAGGATTCACAGGATG-3' (forward) and 5'-TCCTCCATCTTGCTC TCTTCA-3' (reverse). The primers used for the internal control actin (At2g37620) were 5'-TGTGCTCAGTGGTGGAACCA-3' (forward) and 5'-GGAGCCAAAGCAGTGATCTCTT-3' (reverse).
Ten-day-old seedlings were kept at 37°C for 2 h and then ground in liquid nitrogen. Total protein was extracted using extraction buffer (10 mM HEPES, pH 7.9, 0.4 M NaCl, 0.5 mM dithiothreitol, 0.1 mM EDTA, 5% glycerol, and 0.5 mM phenylmethylsulfonyl fluoride), and the extracts were clarified by centrifugation at 14,000g for 20 min at 4°C. Supernatants were transferred to fresh tubes, and the protein content was determined (Bradford, 1976
The following materials are available in the online version of this article.
We thank Dr. Wen-Qiang Tang (Department of Plant Biology, Carnegie Institution of Washington) for critical reading of the manuscript and comments and Drs. Su-Juan Cui, Ying Sun, Yi Guo, and Jun-Feng Zhao (Institute of Molecular Cell Biology, Hebei Normal University) for technical assistance. Received December 7, 2008; accepted February 10, 2009; published February 11, 2009.
1 This work was supported by the National Basic Research Program of China (grant nos. 2006CB100101 and 2006CB910600) and the Natural Science Foundation of China (grant no. 90208004).  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.plantphysiol.org) is: Da-Ye Sun (dayesun{at}gmail.com).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.133744 * Corresponding author; e-mail dayesun{at}gmail.com.
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-32P]ATP using T4 polynucleotide kinase (TaKaRa). Ten-day-old seedlings were ground in liquid nitrogen and extracted with buffer (10 mM Tris, pH 8.0, 1 mM EDTA, 10 mM boric acid, and 0.1 mM phenylmethylsulfonyl fluoride). After centrifugation, the supernatants were used as whole cell extracts. These extracts were heat shocked at 37°C for 1 h. Electrophoretic mobility-shift assays were carried out according to the method of Li et al. (2004)
