OST 1‐mediated BTF 3L phosphorylation positively regulates CBF s during plant cold responses
2018; Springer Nature; Volume: 37; Issue: 8 Linguagem: Inglês
10.15252/embj.201798228
ISSN1460-2075
AutoresYanglin Ding, Yuxin Jia, Yiting Shi, Xiaoyan Zhang, Chun‐Peng Song, Zhizhong Gong, Shuhua Yang,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle5 March 2018free access Source DataTransparent process OST1-mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses Yanglin Ding State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Yuxin Jia State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Yiting Shi State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Xiaoyan Zhang State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Chunpeng Song Institute of Plant Stress Biology, Henan University, Kaifeng, China Collaborative Innovation Center of Crop Stress Biology, Kaifeng, Henan, China Search for more papers by this author Zhizhong Gong State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Shuhua Yang Corresponding Author [email protected] orcid.org/0000-0003-1229-7166 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Yanglin Ding State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Yuxin Jia State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Yiting Shi State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Xiaoyan Zhang State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Chunpeng Song Institute of Plant Stress Biology, Henan University, Kaifeng, China Collaborative Innovation Center of Crop Stress Biology, Kaifeng, Henan, China Search for more papers by this author Zhizhong Gong State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Shuhua Yang Corresponding Author [email protected] orcid.org/0000-0003-1229-7166 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China Search for more papers by this author Author Information Yanglin Ding1, Yuxin Jia1, Yiting Shi1, Xiaoyan Zhang1, Chunpeng Song2,3, Zhizhong Gong1 and Shuhua Yang *,1 1State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, China 2Institute of Plant Stress Biology, Henan University, Kaifeng, China 3Collaborative Innovation Center of Crop Stress Biology, Kaifeng, Henan, China *Corresponding author. Tel: +86-10-62734838; E-mail: [email protected] EMBO J (2018)37:e98228https://doi.org/10.15252/embj.201798228 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3-like), β-subunits of a nascent polypeptide-associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo, and facilitates their interaction with C-repeat-binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1-BTF3-CBF module regulates plant response to cold stress. Synopsis In addition to the regulation of CBF transcription factor expression, the cold-induced kinase OST1 promotes plant freezing tolerance by phosphorylating BTF3 and BTF3L, subunits of the nascent polypeptide-associated complex. BTF3 and BTF3L positively regulate plant freezing tolerance. OST1 interacts with and phosphorylates BTF3 proteins to enhance their binding to CBFs. BTF3L increases CBF protein stability under cold stress. BTF3 and BTF3L indirectly promote CBF gene expression under cold stress. Introduction Cold stress dramatically affects plant growth and development, crop productivity, and quality. Many temperate plants acquire freezing tolerance upon prior exposure to non-freezing temperatures for a period of time, in a process known as cold acclimation (Guy, 1990; Thomashow, 1999). Upon exposure to cold temperatures, a set of COR (Cold-regulated) genes are induced, leading to the production of some protective proteins including detoxification enzymes, key enzymes in osmolyte biosynthesis and fatty acid metabolism, and antifreeze proteins that protect cells from freezing injury (Guy, 1990; Thomashow, 1999). CBFs (C-repeat-binding factors) or DREB1s (dehydration-responsive element-binding proteins) are the major transcription factors involved in cold acclimation. The corresponding transcripts are rapidly induced after cold exposure and directly modulate the expression of downstream COR genes (also known as CBF regulons) (Stockinger et al, 1997; Liu et al, 1998; Thomashow, 1999). In Arabidopsis thaliana, CBF genes are positively regulated by several transcription activators, including ICE1 (inducer of CBF expression 1), CAMTAs (calmodulin binding transcription activators), and BRASSINAZOLE-RESISTANT 1 (Chinnusamy et al, 2003; Doherty et al, 2009; Li et al, 2017b). Conversely, some transcription repressors that inhibit CBF expression and decrease plant freezing tolerance include MYB15, ETHYLENE INSENSITIVE 3, PIF3 (phytochrome-interacting factor 3), and PIF4/7 (Agarwal et al, 2006; Lee & Thomashow, 2012; Shi et al, 2012; Jiang et al, 2017). Furthermore, emerging evidence shows that some factors affect CBF expression indirectly, such as CRLK1 (Ca2+-binding calcium/calmodulin-regulated receptor-like kinase), the rice (Oryza sativa), COLD1 (cold sensor chilling-tolerance divergence 1), and the E3 ligases HOS1 (high expression of osmotically responsive gene 1) and SIZ1 (SAP and Miz) (Dong et al, 2006; Miura et al, 2007; Yang et al, 2010a,b; Ma et al, 2015). In previous study, it was reported that OST1 (open stomata 1) plays a key role in plant freezing tolerance (Ding et al, 2015). Cold-activated OST1 interacts with and phosphorylates ICE1 protein to stabilize ICE1 protein under cold stress (Ding et al, 2015). Moreover, MPK3/6 was shown to negatively regulate ICE1 protein stability, leading to decreased plant freezing tolerance in Arabidopsis (Li et al, 2017a; Zhao et al, 2017). However, OsMPK3 positively regulates rice chilling tolerance by inhibiting OsICE1 degradation under cold stress (Zhang et al, 2017). Most recently, it was discovered that the plasma membrane-localized CRPK1 (cold-responsive protein kinase 1) negatively regulates plant freezing tolerance. Phosphorylated 14-3-3 proteins mediated by CRPK1 are shuttled from the cytosol to the nucleus and interact with CBF proteins, thereby destabilizing CBF proteins under cold stress (Liu et al, 2017). Nascent polypeptide-associated complex (NAC) has been reported to function as initial factors that reversibly interact with nascent polypeptides as they emerge from the ribosome exit tunnel to prevent the peptides from accidentally binding with other cytosolic factors (Rospert et al, 2002). NAC is conserved from Archaea to higher eukaryotes. In Archaea, a homodimeric NAC is formed with two α-subunits, whereas a heterodimer of α-NAC and β-NAC exists in other species (Preissler & Deuerling, 2012). NAC plays diverse roles in mammals and yeasts, including shielding nascent polypeptides in the ribosomal exit tunnel, acting as transcription activators, mediating endoplasmic reticulum (ER) stress, and regulating translocation into the ER and mitochondria (Rospert et al, 2002; Preissler & Deuerling, 2012). Furthermore, NAC has been shown to promote the folding of newly synthesized proteins as a component of ribosome-associated chaperones (Preissler & Deuerling, 2012). Interestingly, loss of NAC can result in developmental defects and early embryonic lethality in mice (Mus sp.), fruit flies (Drosophila melanogaster), and Caenorhabditis elegans, suggesting that NAC is essential for growth and development (Deng & Behringer, 1995; Markesich et al, 2000). However, the loss of NAC does not result in obvious defects in yeast (Reimann et al, 1999). Rather, when both NAC and ribosome-associated HSP70 homologs are absent, substantial growth defects are observed, suggesting that NAC may be connected with the second ribosome-associated chaperone system (Koplin et al, 2010). Although NAC has multiple functions in mammals and yeasts, the knowledge about the exact roles of NAC in plants is very limited. NAC has been implicated in plant development and stress responses (Yang et al, 2007; Huh et al, 2012). However, the molecular function of NAC in these processes remains to be explored. In this study, we found that OST1 interacts with and phosphorylates the β-subunits of NAC, BTF3 (basic transcription factor 3), and BTF3-like protein (BTF3L) to positively regulate cold stress responses in A. thaliana. Biochemical and genetic analyses showed that BTF3 and BTF3L act as substrates for OST1 to enhance plant freezing tolerance by promoting protein stability and the transcription of CBFs. Thus, the OST1-BTF3L module represents a novel mechanism that positively regulates the CBF signaling pathway during plant cold stress responses. Results OST1 interacts with BTF3 and BTF3L To elucidate the molecular mechanism modulated by OST1 in the cold signaling pathway, we performed a liquid chromatography–mass spectrometry (LC-MS) assay using transgenic plants overexpressing OST1-Myc (Ding et al, 2015). The nascent polypeptide-associated complex (NAC) BTF3-like protein subunit (BTF3L) (At1g73230) was identified as a putative OST1-interacting protein (Appendix Table S1). To test the interaction between BTF3L and OST1 in vitro, we conducted yeast two-hybrid assays by co-transforming BTF3L-AD and OST1-BD into yeast strain AH109. We found that BTF3L interacted with OST1 in yeast cells (Fig 1A). Next, we performed in vitro pull-down assays to analyze the physical interaction between BTF3L and OST1. GST-BTF3L or GST was precipitated with GST agarose and then incubated with His-OST1, followed by detection with anti-His antibody. OST1 protein was pulled down by GST-BTF3L but not GST (Fig 1B). Additional evidence that BTF3L interacts with OST1 came from split luciferase complementation assay. Constructs expressing OST1-nLuc and BTF3L-nLuc were co-transformed into Nicotiana benthamiana leaves. We found that BTF3L interacted with OST1 in planta, whereas no interaction was observed when GUS was co-transformed with OST1 or BTF3L, respectively (Fig 1C). Immunoblot analysis confirmed that all of these proteins were expressed (Appendix Fig S1A). To determine the location of the interaction between OST1 and BTF3L, bimolecular fluorescence complementation assay (BiFC) was performed. OST1 interacted with BTF3L in the cytosol and nuclei of leaf pavement cells (Fig 1D). Next, we performed co-immunoprecipitation (co-IP) assays using proteins extracted from N. benthamiana leaves transiently expressing different construct combinations (OST1-Myc/BTF3L-GFP, Myc/OST1-GFP). OST1-Myc, but not Myc, co-immunoprecipitated BTF3L-GFP (Fig 1E). BTF3, a homolog of BTF3L, also interacted with OST1 in yeast two-hybrid, split luciferase complementation, and BiFC assays (Appendix Fig S1B–E). These results demonstrate that BTF3 proteins interact with OST1 in vitro and in vivo. Figure 1. OST1 interacts with BTF3L Yeast two-hybrid assay demonstrating the interaction between OST1 and BTF3L. Yeast cells were grown on SC/−Leu/−Trp (−LW) for 2 days or SC/−Leu/−Trp/−Ade/−His (−LWAH) medium for 5 days. Pull-down assay showing the interaction of OST1 and BTF3L. Purified GST-BTF3L or GST proteins were immunoprecipitated with GST beads. Immunoprecipitated proteins were incubated with His-OST1, and anti-His antibody was used to detect His-OST1. Split luciferase complementation assay showing the interaction of OST and BTF3L in Nicotiana benthamiana leaves. OST1-nLuc/BTF3L-cLuc, OST1-nLuc/GUS-cLuc, and GUS-nLuc/BTF3L-cLuc were co-transformed into N. benthamiana leaves and examined after 48 h. Representative picture is shown in the left panel, and luciferase activity is shown in the right panel. Data are the means ± SE of three independent experiments, each of which had eight technical repeats. **P < 0.01, two-tailed t-test. Interaction of OST1 and BTF3L detected by BiFC analysis. The construct combinations were co-transformed into N. benthamiana leaves and expressed for 48 h. The signal was detected by confocal microscopy. Scale bar: 50 μm. Co-IP assay showing the interaction of OST1 with BTF3L in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted and immunoprecipitated with anti-Myc agarose beads. The proteins were detected with anti-Myc and anti-GFP antibodies. Source data are available online for this figure. Source Data for Figure 1 [embj201798228-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint We previously reported that OST1 interacted with ICE1 (Ding et al, 2015), and then, we tested whether ICE1 interacted with BTF3 proteins in yeast and failed to detect their interaction (Appendix Fig S1F). Characterization of BTF3 genes To determine the expression patterns of BTF3 and BTF3L genes, we performed a histochemical β-glucuronidase (GUS) gene reporter assay. Genomic fragments, 1.5-kb in length, upstream of the BTF3 and BTF3L translational start codon were amplified and cloned into a pCAMBIA1381-GUS vector. The GUS signals for BTF3 and BTF3L were detected in leaves, hypocotyls, and roots, including in root and leaf vascular tissues (Fig 2A and Appendix Fig S2A). OST1 was shown to be expressed in guard cells and vascular tissues (Mustilli et al, 2002). These results suggest that the expression pattern of BTF3 and BTF3L partially overlaps with OST1. Figure 2. Characterization of BTF3L GUS staining of 10-day-old BTF3L:GUS transgenic plants. Gene expression of BTF3L. Two-week-old seedlings were grown at 22°C or 4°C for the time indicated and subjected to qRT–PCR analysis. Relative expression in untreated WT plants was set to 1. Data are means ± SE of three independent experiments. Differences are not significant, two-tailed t-test. Localization of BTF3L in the root of BTF3L:BTF3L-GFP transgenic plant. Two-week-old seedlings were grown at 22°C or 4°C for 3 h. Scale bar: 50 μm. Subcellular fractionation analysis of BTF3L. Proteins were prepared from 2-week-old BTF3L:BTF3L-GFP transgenic plants grown at 22°C or 4°C for 3 h. BTF3L was detected with anti-GFP antibody. Anti-H3 and anti-PEPC antibodies were used to detect nuclear and cytosolic proteins, respectively. T: total, S: soluble, N: nuclear. Immunoblot assay of BTF3L protein. Total proteins were extracted from 2-week-old plants grown at 22°C or 4°C for the time indicated. BTF3L protein was detected by anti-GFP antibody. RuBisCO large subunit was used as a loading control. BTF3L binds to plant ribosomes in vitro. Purified MBP-His-BTF3L protein was incubated with ribosome at 30°C for 30 min. Unbound MBP-His-BTF3L in supernatant (S) was separated from ribosome-associated BTF3L in pellet (P) by centrifugation through a sucrose cushion. The fractions were separated by SDS–PAGE with Coomassie Brilliant Blue staining (top panel). BTF3L protein was detected with anti-MBP antibody (bottom panel). Source data are available online for this figure. Source Data for Figure 2 [embj201798228-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint To further understand the functions of BTF3s in plant cold responses, we analyzed the gene expression pattern of BTF3s under normal and cold conditions. Quantitative real-time PCR (qRT–PCR) analysis showed that expression of both BTF3 and BTF3L was not affected by low temperature (Fig 2B and Appendix Fig S2B). Next, we examined the subcellular localization of BTF3 proteins. BTF3:BTF3-GFP or BTF3L:BTF3L-GFP constructs harboring BTF3 or BTF3L genomic DNA fragments, including their native promoters, were transformed into wild-type plants to obtain transgenic plants. The GFP signals of both BTF3 and BTF3L were detected in the cytosol and nuclei of root cells at 22°C, and cold treatment did not influence their localization (Fig 2C and Appendix Fig S2C). Cell fractionation assays further demonstrated that BTF3 proteins were localized at the cytosol and nucleus of root cells under normal and cold conditions (Fig 2D and Appendix Fig S2D). In addition, immunoblot blot assays showed that the protein levels of BTF3 and BTF3L kept unchanged before or after cold treatment (Fig 2E and Appendix Fig S2E). Nascent polypeptide-associated complex was found to be required for RNA polymerase-dependent transcriptional initiation in mammalian cells (Rospert et al, 2002). We then examined the transcriptional activities of BTF3 and BTF3L protein. However, yeast harboring BTF3 and BTF3L on selection medium failed to show any transcriptional activities (Appendix Fig S2F). Since NAC can bind ribosomes as translation initiation factors in yeast and mammals (Rospert et al, 2002), we asked whether Arabidopsis NAC could bind to ribosome. Interestingly, there is a conserved ribosome binding motif (RRKKK), which mediates ribosome binding (Wegrzyn et al, 2006), in the N-terminus of BTF3L protein (Fig 2F). To examine whether BTF3s bound to ribosome, we performed ribosome binding assay (Wegrzyn et al, 2006). Purified MBP-His-BTF3L fusion protein or MBP-His was incubated with plant ribosomes at 30°C for 30 min, and the mixture was loaded onto a sucrose cushion which was separated by ultracentrifugation. MBP-His-BTF3L was detected in both supernatant (S) and pellet (P), whereas MBP-His was only detected in supernatant (S) (Fig 2F). This result suggests that BTF3L may function in translation initiation with binding to Arabidopsis ribosomes. Mutation of BTF3L alleviates plant freezing tolerance To investigate the role of NAC proteins in a plant's response to cold stress, we isolated T-DNA insertion mutants of BTF3L, named btf3l-1, btf3l-2, and btf3l-3 (Fig 3A and B) and subjected seedlings of these mutants to freezing tolerance assays. All of the btf3l mutants were sensitive to freezing stress under both non-acclimated (NA) and cold-acclimated (CA) conditions (Fig 3C and D, and Appendix Fig S3). Ion leakage reflects plasma membrane injury caused by freezing stress, and we found that the ion leakage was much higher in btf3l mutants than in the wild type (Fig 3E and Appendix Fig S3). The BTF3L genomic fragment fully complemented the freezing sensitivity of btf3l (Fig 3C–E). Thus, the impaired freezing tolerance is indeed caused by mutation of BTF3L. We also generated two btf3 mutant alleles (btf3-1, btf3-2) by CRISPR/Cas9 technology. A base “T” was added into BTF3 coding region before the 64th base “A” and caused a stop codon “TAG” in btf3-1 mutant. Two bases “G” and “C” were added before the 45th base “T” and the 64th base “A”, respectively, in the coding region of BTF3, which results in a premature termination of BTF3 protein in NAC domain (Fig EV1A). These btf3 mutants showed decreased freezing tolerance than the wild type (Fig EV1B and C). Consistently, ion leakage was higher than in the wild type (Fig EV1D and E). In consideration of function redundancy of BTF3 and BTF3L genes, we obtained btf3 btf3l double mutant by CRISPR/Cas9 technology. A base “C” was added before the 45th base “T” in the coding region of BTF3, and 4-bp deletion was found in the coding region of BTF3L (Fig EV1F). Moreover, freezing tolerance assay showed that btf3 btf3l double mutant was much more sensitive to freezing stress than btf3 or btf3l single mutant (Fig 3F–H). Figure 3. Mutation of BTF3L results in enhanced freezing sensitivity A. Diagram of the genomic fragment of BTF3L. Exons are represented by dark blue boxes, UTRs by light blue boxes, and introns by black lines. T-DNA positions of btf3l-1 (SALK_043673C), btf3l-2 (GK-181D04), and btf3l-3 (GK-208H06) are denoted with arrowheads. B. Gene expression of BTF3L in wild-type (WT) plants, the btf3l-1, btf3l-2, and btf3l-3 mutants, and the BTF3/btf3-1 complementation line. C–E. Freezing phenotype (C), survival rate (D), and ion leakage (E) of WT, btf3l-1, and the BTF3L/btf3l-1 complementation line. Two-week-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 0.5 h; CA, −8°C, 1 h). F–H. Freezing phenotype (F), survival rate (G), and ion leakage (H) of WT, btf3, btf3l, and btf3 btf3l double mutants. 16-day-old seedlings were subjected to freezing temperatures (NA, −5°C, 0.5 h; CA, −8°C, 0.5 h). I–K. Expression of CBF and COR genes in different plant genotypes. Two-week-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (I, K) or 24 h (J), and total RNAs were extracted and subjected to qRT–PCR analysis. Relative expression in untreated WT plants was set to 1. Data information: In (D, E, G–K), each bar represents the mean ± SE of three independent experiments, each of which had three technical repeats. *P < 0.5, **P < 0.01 (two-tailed t-test). Source data are available online for this figure. Source Data for Figure 3 [embj201798228-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. BTF3 genes positively regulate plant freezing tolerance A. SANGER sequencing chromatography showing the mutations in btf3-1 and btf3-2 mutants. B, C. Representative pictures showing the freezing sensitive phenotypes of btf3-1 (B) and bft3-2 (C) under NA (−5°C, 0.5 h) and CA (−8°C, 1 h) conditions. D, E. Survival rates and ion leakage of btf3-1 (D) and btf3-2 (E) after freezing treatments. Each bar represents the mean ± SE of three independent experiments, each of which had three plates at the same freezing temperature. Asterisks indicate significant differences compared to the wild type with the same treatment (**P < 0.01, two-tailed t-test). F. SANGER sequencing chromatography showing the mutation in btf3 btf3l double mutants. Download figure Download PowerPoint To further explore the biological function of BTF3 genes, we examined the expression of CBF genes and their target genes, including COR15A and KIN1, in btf3l-1 mutants with or without cold treatment. The expression of CBF genes in btf3l-1 was lower than that in wild-type plants at 22°C. After cold treatment, the cold induction of CBFs in btf3l-1 mutants was slightly decreased compared with the wild type (Fig 3I). Intriguingly, cold induction of COR15A and KIN1 in btf3l-1 mutants was significantly lower than that in the wild type (Fig 3J). BTF3L genomic fragment could fully rescue the basal and cold-induced expression of CBFs and their targets in btf3l mutant (Fig 3I and J). Next, we determined the expression of CBF genes in btf3 btf3l double mutant. Consistent with significantly decreased freezing tolerance, cold-induced expression of CBFs was much lower than that in btf3, btf3l single mutants, and the wild type (Fig 3K). Based on these findings, we conclude that BTF3s are positive regulators of plant freezing tolerance at least partially via CBF-dependent signaling pathway. Overexpression of BTF3 genes enhances plant freezing tolerance To further assess the role of BTF3 genes in plant freezing tolerance, we generated transgenic plants that overexpressed BTF3 or BTF3L, tagged with Myc driven by a constitutive promoter (BTF3/BTF3L-OE) (Figs 4A and EV2A). These transgenic plants showed consistently enhanced freezing tolerance compared with the wild type (Figs 4B and C, and EV2B and C). Ion leakage assay further indicated that freezing caused less membrane injury in BTF3- and BTF3L-overexpressing plants than in the wild type (Figs 4D and EV2D). Figure 4. Overexpression of BTF3L enhances freezing tolerance A. Immunoblot assay of BTF3L protein in BTF3L-Myc overexpressing (BTF3L-OE) plants. Total proteins extracted from 2-week-old seedlings were subjected to immunoblot analysis. BTF3L-Myc was detected with anti-Myc antibody. HSP90 was used as an equal loading control. B–D. Freezing phenotype (B), survival rate (C), and ion leakage (D) of WT and BTF3L-overexpressing transgenic plants. 16-day-old seedlings were exposed to freezing temperatures (NA, −5°C, 0.5 h; CA, −8°C, 1 h). E, F. Expression of CBFs (E) and COR genes (F) in BTF3L-overexpressing plants. Two-week-old plants grown at 22°C were placed at 4°C for 3 h (E) or 24 h (F), and subjected to qRT–PCR analysis. Relative expression in untreated WT plants was set to 1. Data information: In (C–F), each bar represents the mean ± SE of three independent experiments, each of which had three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (**P < 0.01, two-tailed t-test). Source data are available online for this figure. Source Data for Figure 4 [embj201798228-sup-0006-SDataFig4.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Overexpression of BTF3 results in enhanced freezing tolerance A. Immunoblot analysis of BTF3 protein in BTF3-overexpressing (BTF3-OE) plants. Total proteins were extracted from 2-week-old WT, BTF3-OE (1# and 2#) plants, and subjected to immunoblot analysis. BTF3L-Myc was detected by anti-Myc antibody. HSP90 was used as an equal loading control. B–D. Freezing phenotype (B), survival rate (C), and ion leakage (D) in BTF3-overexpressing transgenic plants. Seedlings were grown on MS medium for 16 days and exposed to freezing temperatures (NA, −5°C, 0.5 h; CA, −8°C, 1 h). Each bar represents the mean ± SE of three independent experiments, each of which had three plates at the same freezing temperature. Asterisks indicate significant differences compared to the wild type with the same treatment (**P < 0.01, two-tailed t-test). E, F. Expression of CBFs (E) and COR genes (F) in BTF3-overexpressing plants under normal and cold conditions. Two-week-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (E) or 24 h (F), and total RNAs were extracted and subjected to qRT–PCR analysis. Relative expression in untreated WT plants was set to 1. Each bar represents the mean ± SE of three independent experiments, each of which had three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test). Source data are available online for this figure. Download figure Download PowerPoint Next, we tested whether overexpression of BTF3 or BTF3L affected CBF and COR gene expression. The basal and cold-induced expression of CBFs was higher in BTF3- and BTF3L-overexpressing transgenic plants than in the wild type (Figs 4E and EV2E). Accordingly, expression of CBF target genes was also much higher in these plants than in the wild type with or without cold treatment (Figs 4F and EV2F). These data collectively support that BTF3s are positive regulators of the CBF signaling pathway. OST1 phosphorylates BTF3s under cold stress Since OST1 kinase interacts with BTF3 and BTF3L, it is possible that OST1 phosphorylates BTF3 proteins. To test this hypothesis, we performed an in vitro kinase assay, in which His-OST1 or MBP-His was incubated with MBP-His-BTF3L for 30 min at 30°C in the presence of 1 μCi [γ-32P]ATP. In addition to being strongly auto-phosphorylated, His-OST1 phosphorylated MBP-His-BTF3L (Fig 5A). We also found that OST1 phosphorylated BTF3, but not MBP-His (Fig EV3A). These results suggest that OST1 phosphorylates BTF3s in vitro. Figure 5. OST1 phosphorylates BTF3L under cold stress A. OST1 phosphorylates BTF3L in vitro. Recombinant purified His-OST1 was incubated with MBP-His-BTF3L or MBP-His-BTF3LS50A in kinase reaction buffer with 1 μCi [γ-32P]ATP for 30 min at 30°C. The proteins were separated by SDS–PAGE. Top, autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining. B, C. Phosphorylation assay of BTF3L (B) and BTF3LS50A (C) in planta under cold stress. Total proteins were extracted from BTF3L-Myc overexpressing plants of WT and ost1 mutant backgrounds exposed to 4°C for the indicated period. The proteins were immunoprecipitated with anti-Myc agarose beads and separated on SDS
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