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MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple
Accepted Manuscript Title: MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple Authors: Jian-Ping An, Ji-Fang Yao, Xiao-Na Wang, Chun-Xiang You, Xiao-Fei Wang, Yu-Jin Hao PII: DOI: Reference:
S0176-1617(17)30231-6 http://dx.doi.org/10.1016/j.jplph.2017.09.001 JPLPH 52654
To appear in: Received date: Revised date: Accepted date:
2-6-2017 5-9-2017 5-9-2017
Please cite this article as: An Jian-Ping, Yao Ji-Fang, Wang Xiao-Na, You ChunXiang, Wang Xiao-Fei, Hao Yu-Jin.MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2017.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MdHY5 positively regulates cold tolerance via CBF-dependent and CBFindependent pathways in apple
Running title: MdHY5 positively regulates cold tolerance Jian-Ping An1, Ji-Fang Yao1, Xiao-Na Wang2, Chun-Xiang You1, Xiao-Fei Wang1*, Yu-Jin Hao1*
1. National Key Laboratory of Crop Biology, MOA Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, 271018, Shandong, China 2. College of Life Science, Shandong Agricultural University, Tai-An, 271018, Shandong, China
*Corresponding author: Yu-Jin Hao and Xiao-Fei Wang Email address: [email protected]; [email protected]
Abstract Cold stress is a major external stimulator that affects crop quality and productivity. The CBF cold regulatory pathway has been regarded as a master regulator in the response to cold stress. In this study, we found that the apple bZIP transcription factor, MdHY5, was responsive to cold treatment both at the transcriptional and at the posttranslational levels. Moreover, overexpression of MdHY5 enhanced cold tolerance in apple calli and Arabidopsis. Subsequently, EMSA assay and transient expression assay demonstrated that MdHY5 positively regulated the transcript of MdCBF1 by binding to G-Box motif of its promoter. Furthermore, MdHY5 also regulated the expression of CBF-independent cold-regulated genes. Taken together, our data suggest that MdHY5 positively modulates plant cold tolerance through CBFdependent and CBF-independent pathways, providing a deeper understanding of MdHY5-regulated cold tolerance in apple.
Keyword: MdHY5, Cold tolerance, CBF, Apple
1. Introduction Cold temperature is one of the most important cues that affect the survivability and distribution of plants. Thus, plants have evolved precise and efficient mechanisms to sense and integrate the low-temperature signals (Chinnusamy et al., 2007). Apple tree (Malus × domestica) is an important fruit crop worldwide, and the apple quality and productivity are negatively affected when exposed to adverse low temperature (Farajzadeh et al., 2010). Therefore, it is essential to study the mechanism of the lowtemperature response improving the plants’ adaptation to cold temperature in apple. 1
Cold acclimation research has led to a number of important findings in the past decades, and there is an increased understanding of the mechanism of the lowtemperature regulatory network. In Arabidopsis, a series of genes, such as KIN1, KIN2, RD29A, and COR47, have been identified to be induced in response to low temperature (Fowler and Thomashow, 2002; Provart et al., 2003), and these coldresponsive genes contribute to an increase in cold stress tolerance (Shinozaki et al., 2003). By liberal estimates, more than 1000 genes are up-regulated during cold acclimation, and there are more than 170 genes that encode transcription factors (Thomashow, 2010). Among these cold-inducible transcription factors, the CBF (CRT binding factor) transcription factors play essential roles in regulating downstream cold-responsive genes (Gilmour et al., 1998; 2004). Three CBF genes (CBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A) have been isolated in Arabidopsis (Stockinger et al., 1997; Gilmour et al., 1998; Medina et al., 1999), which function by binding to the CRT/DRE (C-repeat/dehydration responsive element) elements of target genes. Overexpression of CBF1/2/3 in Arabidopsis results in upregulated expression of upstream cold-responsive genes and a significant increase in cold stress tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Gilmour et al., 2004). In addition to the CBF cold-response pathway, some studies have suggested that there are CBF-independent pathways involved in the cold response (Fowler and Thomashow, 2002; Zhu et al., 2004; Agarwal et al., 2006), and these cold-responsive genes are not direct targets of CBF transcription factors. The CBF-independent cold response pathway plays parallel roles in cold acclimation. In recent years, more attention has been focused on the mechanism of the lowtemperature response in apple. Five CBF transcription factors, MdCBF1 to MdCBF5, have been isolated (Wisniewski et al., 2011; 2014). In addition, overexpression of apple MbDREB1 enhances plant tolerance to low temperature (Yang et al., 2011). Moreover, the apple bHLH gene MdCIbHLH1 has been shown to to improve apple cold tolerance by binding to the promoter of MdCBF2 (Feng et al., 2012). The bZIP transcription factor HY5 plays an important role in plant cold acclimation. Previous studies have revealed that low temperature could induce the expression of HY5 (Toledo-Ortiz et al., 2014), and the stability of HY5 protein is regulated in response to low-temperature treatment (Catalá et al., 2011). In addition, HY5 has been found to play important roles in regulating cold-responsive genes and lowtemperature-induced anthocyanin accumulation in Arabidopsis (Catalá et al., 2011; Zhang et al., 2011b). Our previous study has revealed that the apple HY5 transcription factor, MdHY5, plays an important role in anthocyanin accumulation and nitrate assimilation (An et al., 2017). In this study, MdHY5 was functionally identified to play a role in cold tolerance. qRT-PCR analysis and in vitro protein degradation assay indicated that cold temperature treatment positively regulated MdHY5 both at the transcriptional and post-translational levels. Overexpression of MdHY5 increased cold stress tolerance in transgenic apple calli and Arabidopsis. Furthermore, MdHY5 positively regulated the expression of MdCBF1 gene by directly binding to its promoter. Moreover, we found that MdHY5 also regulated the expression of CBF-independent cold-responsive genes. Taken together, these results indicate that MdHY5 enhances plant cold stress tolerance through CBF-dependent and CBF-independent pathways.
2. Materials and methods 2.1. Plant material and treatment 2
Apomictic crabapple (Malus hupehensis) seedlings were used for cold treatment and gene expression analysis. The apple seedlings were grown at 24°C under longday conditions (16-h-light/8-h-dark) (photon flux density about 48 μmol·s-1·m-2). For cold treatment, seedlings were grown at 4°C under long-day conditions (16-h-light/8h-dark) (photon flux density about 48 μmol·s-1·m-2) for the indicated time. Semiquantitative RT-PCR (qRT-PCR) was performed to examine the transcript of MdHY5. The wild-type “Orin” apple calli (WT) was used for genetic transformation and phenotypic analysis. The apple calli was subcultured on MS medium supplementing 0.5 mg·L-1 IAA and 1.5 mg·L-1 6-BA at 24°C as described (An et al., 2017). Arabidopsis thaliana plants (Col-0) were used for genetic transformation and phenotypic analysis in this study. 2.2. Quantitative RT-PCR (qRT-PCR) RNA isolation and qRT-PCR analysis were performed as described previously (An et al., 2017). RNA was isolated using an RNA Plant Plus Reagent (Tiangen, Beijing, China), and the reverse transcription was carried out using a Primescript first-strand cDNA synthesis kit (Takara, Dalian, China). qRT-PCR was performed with the UltraSYBR mixture (Takara) by an ABI7500 qRT-PCR system. The concentration of cDNA was diluted to 10 ng·μl-1, and the 2-△△CT calculation method was conducted. The expression levels of MdHY5, MdCBF genes (MdCBF1, MdCBF2, and MdCBF3) (Wisniewski et al., 2014), MdCBF target genes (MdKIN1: MDP0000165526; MdRD29A: MDP0000598443; MdCOR47: MDP0000529003), and MdCBF-independent genes (MdWARK6: MDP0000935652; MdPYL6: MDP0000270731; MdSAG21 MDP0000564193; MdSOC1: MDP0000314765; MdJMT: MDP0000509245; MdESM1: MDP0000258269) were examined using specific primers (Supplementary table 1). 2.3. Apple calli and Arabidopsis transformation Plasmid construction of MdHY5-pCAMBIA1300 and MdHY5 transgenic apple calli was generated as described previously (An et al., 2017). As for the transformation of apple calli, 16-day-old wild-type apple calli were co-cultured for 20 min with Agrobacterium carrying MdHY5-pCAMBIA1300, and the apple calli were cocultured on MS medium supplementing 0.5 mg·L-1 IAA and 1.5 mg·L-1 6-BA for two days at 24°C. Then, the apple calli were washed three times with sterile water and transferred to selective media supplementing 300 mg·L-1 carbenicillin and 40 mg·L-1 hygromycin. Transgenic Arabidopsis were generated using the floral dip transformation method (Clough and Bent, 1998). 2.4. In vitro protein degradation assay of the MdHY5 protein Protein degradation assays were performed to test the post-translational regulation of MdHY5 protein in vitro. The wild-type apple calli (WT) extraction solutions were extracted by buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 4 mM PMSF). The incubations of the extraction solution and MdHY5-GST protein were conducted up to the indicated times, and the relative protein level of MdHY5 was detected by western blot with anti-GST monoclonal antibodies. 2.5. Cold stress assay 3
For low-temperature treatment of apple calli (cold tolerance of apple calli), eightday-old wild-type and transgenic apple calli were transferred to a phytotron at 4°C under long-day conditions (16-h-light/8-h-dark) (photon flux density about 48 μmol·s1 ·m-2) for 10 days. The cold stress assay of Arabidopsis seedlings (frost tolerance of Arabidopsis) was carried out as described (Shi et al., 2012; Li et al., 2017). In brief, 10-day-old Arabidopsis seedlings were cold-acclimated at 4°C under long-day conditions (16-hlight/8-h-dark) (photon flux density about 48 μmol·s-1·m-2) for three days. Subsequently, they were treated at -4°C for 0.5 h via gradient cooling, and then the seedlings were exposed to 22°C under long-day conditions (16-h-light/8-h-dark) (photon flux density about 48 μmol·s-1·m-2) for three days. All the results are based on the average of three parallel experiments. 2.6. Electrophoretic mobility shift assays (EMSA) EMSA assay was carried out using the LightShiftTM Chemiluminescent EMSA Kit (Thermo, USA). In brief, biotin-labeled probes (GTTAATGAGGCACGTGGTTCACGCGT-) were incubated in a binding buffer (2.5% glycerol, 10 mM EDTA, 5 mM MgCl2, and 50 mM KCl) with or without MdHY5GST fusion protein at 24°C for 25 min. An unlabeled probe was used for unlabeled probe competition. A mutated probe (Mut) was generated in which the 5’-CACGTG-3’ motif was replaced by 5’-CGCGCG-3’. And the free and bound DNAs were separated in an acrylamide gel. The probe sequences are listed in Supplementary Table 1. 2.7. Transient expression assay in Nicotiana benthamiana leaves Transient expression assays in N. benthamiana leaves were performed as previously described (Shang et al. 2010; An et al., 2017). The MdCBF1 promoter was amplified and cloned into pGreenII 0800-LUC vectors, which generated the reporter construct MdCBF1pro:LUC. The site-directed mutations were conducted using the TaKaRa MutanBEST kit. The effector (35Spro:MdHY5) was constructed by cloning the ORF of the MdHY5 into the pGreenII 62-SK vector. A low-light cooled CCD imaging apparatus was used to capture the LUC image and to count luminescence intensity. The leaves were sprayed with 100 mM luciferin and were placed in darkness for 3 min before detection (An et al., 2017). 2.8. Statistical analysis Statistical analysis was carried out as previously described using appropriate methods and R (3.0.2) software with the R Commander package (An et al., 2017). Differences were considered statistically significant at *P < 0.05 and **P < 0.01. The results were analyzed in triplicate.
3. Results 3.1. MdHY5 is responsive to cold temperature Previous studies have suggested that Arabidopsis HY5 is responsive to cold treatment both at the transcriptional and post-translational levels (Catalá et al., 2011; Toledo-Ortiz et al., 2014). To identify the role of MdHY5 in response to cold, the expression of MdHY5 in wild-type apple seedlings under cold treatment (4°C) was analyzed. The qRT-PCR analysis showed that MdHY5 was obviously induced in response to cold, and the expression level increased at 1 h and peaked at 3 h (Fig. 1A). 4
In addition, in vitro protein degradation assays were carried out to test the posttranslational regulation of MdHY5 in response to cold temperature. As shown in Fig. 1B, the fusion protein MdHY5-GST was unstable and rapidly degraded to an undetectable level within four hours. Furthermore, the degradation rate was significantly repressed when treating the samples with MG132, a 26S proteasomespecific inhibitor, indicating that the protein stability of MdHY5 was regulated via the 26S-proteasome pathway. Meanwhile, the fusion protein abundance of MdHY5-GST increased in response to cold temperature treatment, indicating that cold temperature was involved in the post-translational regulation of MdHY5 protein. These results suggest that MdHY5 may play an important role in the cold stress response. 3.2. MdHY5 positively regulates cold tolerance in apple calli and Arabidopsis To test whether MdHY5 regulates cold tolerance, an MdHY5-pCAMBIA1300 overexpression construct was transformed into ‘Orin’ apple calli (WT) and Arabidopsis (Col-0) through Agrobacterium-mediated genetic transformation, and two transgenic apple calli lines (MdHY5-L1 and MdHY5-L2) and three transgenic Arabidopsis lines (MdHY5#1, MdHY5#2, and MdHY5#3) were selected for further investigation (Fig. S1A-C). We next examined the cold tolerance of MdHY5 transgenic apple calli. The eightday-old wild-type and transgenic apple calli were exposed to 4°C for another 10 days. As shown in Fig. 2A-B, transgenic apple calli displayed increased cold stress tolerance compared with the wild-type control under cold conditions. Consistent with the cold stress assays in apple calli, ectopic expression of MdHY5 in Arabidopsis significantly increased survival rates in response to cold stress (Fig. 2C-D). 3.3. MdHY5 positively regulates the expression of MdCBFs To explore how MdHY5 contributes to cold tolerance, qRT-PCR was performed to test the expression of MdCBF genes in transgenic apple calli, which were considered to play major roles in configuring the low-temperature transcriptome and in regulating cold tolerance (Skinner et al., 2005; Thomashow, 2010). As shown in Fig. 3 and Fig. S3, the expression levels of MdCBF genes, including MdCBF1, MdCBF2, and MdCBF3, were substantially elevated in MdHY5 transgenic apple calli compared to the wild type under cold treatment. Consistently, the expression levels of CBF target genes, such as MdKIN1, MdRD29A, and MdCOR47, were also higher in transgenic apple calli than in the wild type. 3.4. MdHY5 binds to the MdCBF1 promoter It has been reported that MdHY5 transcription factor binds to G-Box (CACGTG) motifs in the promoters of target genes (An et al., 2017). Then, the sequences of CBFs promoter regions were analyzed and a putative G-Box motif was found in the promoter of MdCBF1 (Fig. 4A). To test whether MdHY5 directly bound to the promoter of MdCBF1 in vitro, an EMSA assay was performed. The recombinant MdHY5-GST protein was purified and the MdCBF1 promoter fragment containing the G-Box motif was prepared. As predicted, the MdHY5-GST fusion protein could bind to the promoter of MdCBF1, and no binding was tested in the GST protein (Fig. 4B). In addition, the binding was reduced by the addition of competitors, whereas no binding was observed when adding the mutated probe (Fig. 4B). In order to test whether MdHY5 could up-regulate the expression of MdCBF1, transient transactivation assays in tobacco leaves were performed. The promoter of MdCBF1 (MdCBF1pro) and mutated MdCBF1 promoter fragments (MdCBF1(M)pro) 5
were fused to the LUC gene as reporters. The MdHY5 effector construct was expressed under the 35S promoter (Fig. S2). Compared to the control (Fig. 4C, a, b), co-expression of 35Spro:MdHY5 with MdCBF1pro:LUC led to a significant increase of luminescence intensity (Fig. 4C, c), while 35Spro:MdHY5 failed to activate the expression of MdCBF1(M)pro:LUC (Fig. 4C, d; Fig. 4D). These finding reveal that MdHY5 transactivates the expression of MdCBF1 in apple. 3.5. MdHY5 regulates the expression of CBF-independent cold-regulated genes Besides the CBF-dependent cold response pathway by which MdHY5 regulates cold stress tolerance, some CBF-independent genes, including MdPYL6, MdWARK6, MdSAG21, MdSOC1, MdJMT, and MdESM1, were also selected for gene expression analysis (Robatzek and Somssich, 2001, 2002; Seo et al., 2001; Zhang et al., 2006; Cipollini, 2007; Salleh et al., 2012; Li et al., 2017). The results showed that the expression of MdPYL6, MdWARK6, and MdSAG21, positive regulatory genes of the cold stress response, were up-regulated in the MdHY5 transgenic apple calli than in the wild type under cold treatment. On the contrary, the expression of negative regulatory genes, including MdSOC1, MdJMT, and MdESM1, was consistently lower in the MdHY5 transgenic apple calli than in the wild type (Fig. 6). These results demonstrate that MdHY5 plays a dual role in regulating CBF-independent coldresponsive genes.
4. Discussion The bZIP transcription factor HY5 plays diverse roles in plant growth and development processes involved in photomorphogenesis (Ang and Deng, 1994), nutrient assimilation (Chen et al., 2016), cold stress response (Toledo-Ortiz et al., 2014), flavonol biosynthesis (Loyola et al., 2016), and ROS scavenging (Catalá et al., 2011). Among these biological responses, studying the HY5-mediated cold tolerance is of great theoretical significance and value regarding the improvement of apple production. In our previous study, the apple bZIP transcription factor, MdHY5, was cloned and functionally identified to be involved in anthocyanin accumulation and nitrate assimilation (An et al., 2017). In this study, we revealed that MdHY5 simultaneously regulates cold tolerance via CBF-dependent and CBF-independent pathways (Fig. 6). The CBF signaling pathway plays an essential role in the plant cold stress response, and plants have evolved precise mechanisms to conduct this signaling pathway (Thomashow, 1990; Chinnusamy et al., 2007). Within a short time of low-temperature exposure, the expression levels of the CBF genes and cold-responsive genes are greatly elevated in Arabidopsis (Guy et al., 1985; Gilmour et al., 1998). In apple, the transcripts of MdCBFs can also be induced by low-temperature treatment (Feng et al., 2012), and our studies showed that overexpression of MdHY5 significantly increased the transcripts of cold-responsive genes under cold stress (Fig. 3). HY5 is known for regulating the expression of target genes by binding to the GBox motifs (CACGTG) of their promoters (Zhang et al., 2011a). The synchronous expression manner of MdHY5 and MdCBFs implies that MdCBFs might be direct targets of MdHY5. Then, the EMSA assay and transient transactivation assay showed that MdHY5 directly up-regulated MdCBF1 by binding to the G-Box motif of its promoter (Fig. 4). In addition, it is noteworthy that MdHY5 transgenic apple calli also affect the expression of several CBF-independent cold-responsive genes, indicating that the CBF-independent cold response pathway modulated by MdHY5 plays an 6
essential role in MdHY5-regulated cold tolerance. It is inadvertent that MdHY5 exhibits a similar regulation pattern as Arabidopsis BZR1 in cold response (Li et al., 2017), which might be owed to the direct interaction between BZR1 and HY5 in Arabidopsis (Li et al., 2016). As a master regulator of light signaling, HY5 has been well characterized in regulating circadian rhythm and the cold stress response in Arabidopsis (Lee et al., 2007; Toledo-Ortiz et al., 2014). However, as a perennial woody plant, apple undergoes more complicated photoperiod and temperature changes, and MdHY5 might play a more complicated role in response to environmental factors. Therefore, improved comprehension of MdHY5 functions could be highly useful for producing high-quality fruits. A recent study shows that PIF3 plays a negative role in the CBF signaling pathway and cold tolerance (Jiang et al., 2017). Here, our data clearly revealed the positive contribution of the apple HY5 gene in the development of cold tolerance in apple, which provides further straightforward evidence that plants can integrate light and temperature signaling to better adapt to external stresses. In conclusion, based on previous studies and our present data, a hypothetical model for MdHY5 modulating cold tolerance in apple was proposed (Fig. 6). After exposure to the cold stimulus, MdHY5 was positively regulated both at the transcriptional and at the post-translational levels. On the one hand, MdHY5 promoted the cold tolerance by directly binding to the MdCBF1 promoter. On the other hand, the CBF-independent cold response pathway regulated by MdHY5 played an essential role in MdHY5regulated cold tolerance. Our studies provide new insight into the molecular mechanism of the MdHY5-enhanced cold tolerance in apple.
Acknowledgements This work was supported by grants from the Natural Science Foundation of China (31601742 and 31430074), the Ministry of Education of China (IRT15R42), and Shandong Province Government (SDAIT-06-03).
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Skinner, J.S., Zitzewitz, J., Szűcs, P., Marquez-Cedillo, L., Filichkin, T., Amundsen, K., Hayes, P.M., 2005. Structural, functional, and phylogenetic characterization of a large CBF gene family in barley. Plant Mol. Biol. 59 (4), 533-551. Stockinger, E.J., Gilmour, S.J., Thomashow, M.F., 1997. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the Crepeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA. 94 (3), 1035-1040. Thomashow, M. F., 2010. Molecular basis of plant cold acclimation: insights gained from studying the CBF cold response pathway. Plant Physiol. 154 (2), 571-577. Thomashow, M.F., 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann. Rev. Plant Mol. Biol. 50 (1), 571-599. Toledo-Ortiz, G., Johansson, H., Lee, K. P., Bou-Torrent, J., Stewart, K., Steel, G., Halliday, K.J., 2014. The HY5-PIF regulatory module coordinates light and temperature control of photosynthetic gene transcription. PLoS Genet. 10 (6), e1004416. Wisniewski, M., Nassuth, A., Teulieres, C., Marque, C., Rowland, J., Cao, P. B., Brown, A., 2014. Genomics of cold hardiness in woody plants. Crit. Rev. Plant Sci. 33 (2-3), 92-124. Wisniewski, M., Norelli, J., Bassett, C., Artlip, T., and Macarisin, D., 2011. Ectopic expression of a novel peach (Prunus persica) CBF transcription factor in apple (Malus × domestica) results in short-day induced dormancy and increased cold hardiness. Planta. 233 (5), 971-983. Yang, W., Liu, X.D., Chi, X.J., Wu, C.A., Li, Y.Z., Song, L.L., Liu, Y., 2011. Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways. Planta. 233 (2), 219-229. Zhang, H., He, H., Wang, X., Wang, X., Yang, X., Li, L., Deng, X.W., 2011 a. Genome-wide mapping of the HY5-mediated gene networks in Arabidopsis that involve both transcriptional and post-transcriptional regulation. Plant J. 65 (3), 346-358. Zhang, Y., Liu, Z., Liu, R., Hao, H., Bi, Y., 2011 b. Gibberellins negatively regulate low temperature-induced anthocyanin accumulation in a HY5/HYH-dependent manner. Plant Signal Behavior. 6 (5), 632-634. Zhang, Z., Ober, J.A., Kliebenstein, D.J., 2006. The gene controlling the quantitative trait locus EPITHIOSPECIFIER MODIFIER1 alters glucosinolate hydrolysis and insect resistance in Arabidopsis. Plant Cell. 18 (6), 1524-1536. Zhu, J., Shi, H., Lee, B. H., Damsz, B., Cheng, S., Stirm, V., Bressan, R.A., 2004. An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway. Proc. Natl. Acad. Sci. USA. 101 (26), 9873-9878.
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Figure legends Fig. 1 Effects of cold treatment on the gene expression and protein stability of MdHY5 (A) Expression of MdHY5 gene in the wild-type apple seedlings treated at 4°C by qRT-PCR. Apomictic crabapple (Malus hypehensis) seedlings grown at 22°C were treated at 4°C for the indicated time. The value for untreated seedlings was set to 1. (B) Protein degradation of the MdHY5-GST fusion protein and its stabilization at 4°C. The protein level at 0 h was set to 1.00.
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Fig. 2 Cold tolerance assays of MdHY5-overexpressing apple calli and Arabidopsis (A) Cold stress phenotypes of MdHY5-overexpressing apple calli under lowtemperature conditions. The wild-type (WT) and transgenic apple calli (MdHY5-L1 and MdHY5-L2) were grown on medium at room temperature for eight days and then treated at 4°C for another ten days. (B) Fresh weight of wild-type and transgenic apple calli after cold treatment. (C) Cold stress phenotypes of MdHY5-overexpressing Arabidopsis under lowtemperature conditions. The wild type (Col-0) and transgenic Arabidopsis (MdHY5#1, MdHY5#2, and MdHY5#3) were grown on MS plates at 22°C for ten days and then treated at -4°C for 0.5 h. (D) Survival rates of MdHY5-overexpressing Arabidopsis after cold treatments.
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Fig. 3 Expression analysis of MdCBF genes and MdCBF target genes in the MdHY5 transgenic apple calli Relative expression levels of MdCBF genes (MdCBF1, MdCBF2, and MdCBF3) and MdCBF target gene (MdKIN1, MdRD29A, and MdCOR47) in the wild-type (WT) and transgenic apple calli (MdHY5-L1 and MdHY5-L2). The value for WT was set to 1.
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Fig. 4 MdHY5 binds to the promoter of MdCBF1 (A) Schematic diagram of the MdCBF1 promoter showing the potential MdHY5 binding sites. The predicted G-Box (CACGTG) site and sequence are indicated with box. Mutated site (Mut) in which the 5’-CACGTG-3’ motif was replaced by 5’CGCGCG-3’. (B) EMSA assay showing that MdHY5 fusion protein directly bound to the MdCBF1 promoter at CACGTG in vitro. Biotin-labeled probes were incubated with MdHY5GST protein, and the free and bound DNAs were separated in an acrylamide gel. As indicated, unlabeled probes were used as competitors. (C) Transient expression assays showing that MdHY5 promoted the expression of MdCBF1. a: Black (empty vector); b: MdCBF1pro:LUC; c: MdCBF1pro:LUC35Spro:MdHY5; d: MdCBF1(M)pro:LUC-35Spro:MdHY5. MdCBF1(M)pro in which the 5’-CACGTG-3’ motif was replaced by 5’-CGCGCG-3’. Representative images of Nicotiana benthamiana leaves 72 h after infiltration are shown. Quantitative analysis of luminescence intensity in (D). The value for a (empty vector) was set to 1.
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Fig. 5 Expression analysis of CBF-independent genes in the MdHY5 transgenic apple calli Relative expression levels of MdPYL6, MdWRKY6, MdSAG21, MdSOC1, MdJMT, and MdESM1 in the wild-type (WT) and transgenic apple calli (MdHY5-L1 and MdHY5-L2). The value for WT was set to 1.
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Fig. 6 The proposed model of MdHY5-mediated cold tolerance in apple Cold induces the accumulation of MdHY5, and activation of MdHY5 induces the expression of MdCBF1 by binding to the G-Box motif of its promoter. MdHY5 also regulates some cold-regulated genes, including MdPYL6, MdWRKY6, MdSAG21, MdSOC1, MdJMT, and MdESM1, which are independent of CBF, to modulate plant cold tolerance.
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S0176-1617(17)30231-6 http://dx.doi.org/10.1016/j.jplph.2017.09.001 JPLPH 52654
To appear in: Received date: Revised date: Accepted date:
2-6-2017 5-9-2017 5-9-2017
Please cite this article as: An Jian-Ping, Yao Ji-Fang, Wang Xiao-Na, You ChunXiang, Wang Xiao-Fei, Hao Yu-Jin.MdHY5 positively regulates cold tolerance via CBF-dependent and CBF-independent pathways in apple.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2017.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
MdHY5 positively regulates cold tolerance via CBF-dependent and CBFindependent pathways in apple
Running title: MdHY5 positively regulates cold tolerance Jian-Ping An1, Ji-Fang Yao1, Xiao-Na Wang2, Chun-Xiang You1, Xiao-Fei Wang1*, Yu-Jin Hao1*
1. National Key Laboratory of Crop Biology, MOA Key Laboratory of Horticultural Crop Biology and Germplasm Innovation, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai-An, 271018, Shandong, China 2. College of Life Science, Shandong Agricultural University, Tai-An, 271018, Shandong, China
*Corresponding author: Yu-Jin Hao and Xiao-Fei Wang Email address: [email protected]; [email protected]
Abstract Cold stress is a major external stimulator that affects crop quality and productivity. The CBF cold regulatory pathway has been regarded as a master regulator in the response to cold stress. In this study, we found that the apple bZIP transcription factor, MdHY5, was responsive to cold treatment both at the transcriptional and at the posttranslational levels. Moreover, overexpression of MdHY5 enhanced cold tolerance in apple calli and Arabidopsis. Subsequently, EMSA assay and transient expression assay demonstrated that MdHY5 positively regulated the transcript of MdCBF1 by binding to G-Box motif of its promoter. Furthermore, MdHY5 also regulated the expression of CBF-independent cold-regulated genes. Taken together, our data suggest that MdHY5 positively modulates plant cold tolerance through CBFdependent and CBF-independent pathways, providing a deeper understanding of MdHY5-regulated cold tolerance in apple.
Keyword: MdHY5, Cold tolerance, CBF, Apple
1. Introduction Cold temperature is one of the most important cues that affect the survivability and distribution of plants. Thus, plants have evolved precise and efficient mechanisms to sense and integrate the low-temperature signals (Chinnusamy et al., 2007). Apple tree (Malus × domestica) is an important fruit crop worldwide, and the apple quality and productivity are negatively affected when exposed to adverse low temperature (Farajzadeh et al., 2010). Therefore, it is essential to study the mechanism of the lowtemperature response improving the plants’ adaptation to cold temperature in apple. 1
Cold acclimation research has led to a number of important findings in the past decades, and there is an increased understanding of the mechanism of the lowtemperature regulatory network. In Arabidopsis, a series of genes, such as KIN1, KIN2, RD29A, and COR47, have been identified to be induced in response to low temperature (Fowler and Thomashow, 2002; Provart et al., 2003), and these coldresponsive genes contribute to an increase in cold stress tolerance (Shinozaki et al., 2003). By liberal estimates, more than 1000 genes are up-regulated during cold acclimation, and there are more than 170 genes that encode transcription factors (Thomashow, 2010). Among these cold-inducible transcription factors, the CBF (CRT binding factor) transcription factors play essential roles in regulating downstream cold-responsive genes (Gilmour et al., 1998; 2004). Three CBF genes (CBF1/DREB1B, CBF2/DREB1C, and CBF3/DREB1A) have been isolated in Arabidopsis (Stockinger et al., 1997; Gilmour et al., 1998; Medina et al., 1999), which function by binding to the CRT/DRE (C-repeat/dehydration responsive element) elements of target genes. Overexpression of CBF1/2/3 in Arabidopsis results in upregulated expression of upstream cold-responsive genes and a significant increase in cold stress tolerance (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Gilmour et al., 2004). In addition to the CBF cold-response pathway, some studies have suggested that there are CBF-independent pathways involved in the cold response (Fowler and Thomashow, 2002; Zhu et al., 2004; Agarwal et al., 2006), and these cold-responsive genes are not direct targets of CBF transcription factors. The CBF-independent cold response pathway plays parallel roles in cold acclimation. In recent years, more attention has been focused on the mechanism of the lowtemperature response in apple. Five CBF transcription factors, MdCBF1 to MdCBF5, have been isolated (Wisniewski et al., 2011; 2014). In addition, overexpression of apple MbDREB1 enhances plant tolerance to low temperature (Yang et al., 2011). Moreover, the apple bHLH gene MdCIbHLH1 has been shown to to improve apple cold tolerance by binding to the promoter of MdCBF2 (Feng et al., 2012). The bZIP transcription factor HY5 plays an important role in plant cold acclimation. Previous studies have revealed that low temperature could induce the expression of HY5 (Toledo-Ortiz et al., 2014), and the stability of HY5 protein is regulated in response to low-temperature treatment (Catalá et al., 2011). In addition, HY5 has been found to play important roles in regulating cold-responsive genes and lowtemperature-induced anthocyanin accumulation in Arabidopsis (Catalá et al., 2011; Zhang et al., 2011b). Our previous study has revealed that the apple HY5 transcription factor, MdHY5, plays an important role in anthocyanin accumulation and nitrate assimilation (An et al., 2017). In this study, MdHY5 was functionally identified to play a role in cold tolerance. qRT-PCR analysis and in vitro protein degradation assay indicated that cold temperature treatment positively regulated MdHY5 both at the transcriptional and post-translational levels. Overexpression of MdHY5 increased cold stress tolerance in transgenic apple calli and Arabidopsis. Furthermore, MdHY5 positively regulated the expression of MdCBF1 gene by directly binding to its promoter. Moreover, we found that MdHY5 also regulated the expression of CBF-independent cold-responsive genes. Taken together, these results indicate that MdHY5 enhances plant cold stress tolerance through CBF-dependent and CBF-independent pathways.
2. Materials and methods 2.1. Plant material and treatment 2
Apomictic crabapple (Malus hupehensis) seedlings were used for cold treatment and gene expression analysis. The apple seedlings were grown at 24°C under longday conditions (16-h-light/8-h-dark) (photon flux density about 48 μmol·s-1·m-2). For cold treatment, seedlings were grown at 4°C under long-day conditions (16-h-light/8h-dark) (photon flux density about 48 μmol·s-1·m-2) for the indicated time. Semiquantitative RT-PCR (qRT-PCR) was performed to examine the transcript of MdHY5. The wild-type “Orin” apple calli (WT) was used for genetic transformation and phenotypic analysis. The apple calli was subcultured on MS medium supplementing 0.5 mg·L-1 IAA and 1.5 mg·L-1 6-BA at 24°C as described (An et al., 2017). Arabidopsis thaliana plants (Col-0) were used for genetic transformation and phenotypic analysis in this study. 2.2. Quantitative RT-PCR (qRT-PCR) RNA isolation and qRT-PCR analysis were performed as described previously (An et al., 2017). RNA was isolated using an RNA Plant Plus Reagent (Tiangen, Beijing, China), and the reverse transcription was carried out using a Primescript first-strand cDNA synthesis kit (Takara, Dalian, China). qRT-PCR was performed with the UltraSYBR mixture (Takara) by an ABI7500 qRT-PCR system. The concentration of cDNA was diluted to 10 ng·μl-1, and the 2-△△CT calculation method was conducted. The expression levels of MdHY5, MdCBF genes (MdCBF1, MdCBF2, and MdCBF3) (Wisniewski et al., 2014), MdCBF target genes (MdKIN1: MDP0000165526; MdRD29A: MDP0000598443; MdCOR47: MDP0000529003), and MdCBF-independent genes (MdWARK6: MDP0000935652; MdPYL6: MDP0000270731; MdSAG21 MDP0000564193; MdSOC1: MDP0000314765; MdJMT: MDP0000509245; MdESM1: MDP0000258269) were examined using specific primers (Supplementary table 1). 2.3. Apple calli and Arabidopsis transformation Plasmid construction of MdHY5-pCAMBIA1300 and MdHY5 transgenic apple calli was generated as described previously (An et al., 2017). As for the transformation of apple calli, 16-day-old wild-type apple calli were co-cultured for 20 min with Agrobacterium carrying MdHY5-pCAMBIA1300, and the apple calli were cocultured on MS medium supplementing 0.5 mg·L-1 IAA and 1.5 mg·L-1 6-BA for two days at 24°C. Then, the apple calli were washed three times with sterile water and transferred to selective media supplementing 300 mg·L-1 carbenicillin and 40 mg·L-1 hygromycin. Transgenic Arabidopsis were generated using the floral dip transformation method (Clough and Bent, 1998). 2.4. In vitro protein degradation assay of the MdHY5 protein Protein degradation assays were performed to test the post-translational regulation of MdHY5 protein in vitro. The wild-type apple calli (WT) extraction solutions were extracted by buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 5 mM DTT, 10 mM ATP, 4 mM PMSF). The incubations of the extraction solution and MdHY5-GST protein were conducted up to the indicated times, and the relative protein level of MdHY5 was detected by western blot with anti-GST monoclonal antibodies. 2.5. Cold stress assay 3
For low-temperature treatment of apple calli (cold tolerance of apple calli), eightday-old wild-type and transgenic apple calli were transferred to a phytotron at 4°C under long-day conditions (16-h-light/8-h-dark) (photon flux density about 48 μmol·s1 ·m-2) for 10 days. The cold stress assay of Arabidopsis seedlings (frost tolerance of Arabidopsis) was carried out as described (Shi et al., 2012; Li et al., 2017). In brief, 10-day-old Arabidopsis seedlings were cold-acclimated at 4°C under long-day conditions (16-hlight/8-h-dark) (photon flux density about 48 μmol·s-1·m-2) for three days. Subsequently, they were treated at -4°C for 0.5 h via gradient cooling, and then the seedlings were exposed to 22°C under long-day conditions (16-h-light/8-h-dark) (photon flux density about 48 μmol·s-1·m-2) for three days. All the results are based on the average of three parallel experiments. 2.6. Electrophoretic mobility shift assays (EMSA) EMSA assay was carried out using the LightShiftTM Chemiluminescent EMSA Kit (Thermo, USA). In brief, biotin-labeled probes (GTTAATGAGGCACGTGGTTCACGCGT-) were incubated in a binding buffer (2.5% glycerol, 10 mM EDTA, 5 mM MgCl2, and 50 mM KCl) with or without MdHY5GST fusion protein at 24°C for 25 min. An unlabeled probe was used for unlabeled probe competition. A mutated probe (Mut) was generated in which the 5’-CACGTG-3’ motif was replaced by 5’-CGCGCG-3’. And the free and bound DNAs were separated in an acrylamide gel. The probe sequences are listed in Supplementary Table 1. 2.7. Transient expression assay in Nicotiana benthamiana leaves Transient expression assays in N. benthamiana leaves were performed as previously described (Shang et al. 2010; An et al., 2017). The MdCBF1 promoter was amplified and cloned into pGreenII 0800-LUC vectors, which generated the reporter construct MdCBF1pro:LUC. The site-directed mutations were conducted using the TaKaRa MutanBEST kit. The effector (35Spro:MdHY5) was constructed by cloning the ORF of the MdHY5 into the pGreenII 62-SK vector. A low-light cooled CCD imaging apparatus was used to capture the LUC image and to count luminescence intensity. The leaves were sprayed with 100 mM luciferin and were placed in darkness for 3 min before detection (An et al., 2017). 2.8. Statistical analysis Statistical analysis was carried out as previously described using appropriate methods and R (3.0.2) software with the R Commander package (An et al., 2017). Differences were considered statistically significant at *P < 0.05 and **P < 0.01. The results were analyzed in triplicate.
3. Results 3.1. MdHY5 is responsive to cold temperature Previous studies have suggested that Arabidopsis HY5 is responsive to cold treatment both at the transcriptional and post-translational levels (Catalá et al., 2011; Toledo-Ortiz et al., 2014). To identify the role of MdHY5 in response to cold, the expression of MdHY5 in wild-type apple seedlings under cold treatment (4°C) was analyzed. The qRT-PCR analysis showed that MdHY5 was obviously induced in response to cold, and the expression level increased at 1 h and peaked at 3 h (Fig. 1A). 4
In addition, in vitro protein degradation assays were carried out to test the posttranslational regulation of MdHY5 in response to cold temperature. As shown in Fig. 1B, the fusion protein MdHY5-GST was unstable and rapidly degraded to an undetectable level within four hours. Furthermore, the degradation rate was significantly repressed when treating the samples with MG132, a 26S proteasomespecific inhibitor, indicating that the protein stability of MdHY5 was regulated via the 26S-proteasome pathway. Meanwhile, the fusion protein abundance of MdHY5-GST increased in response to cold temperature treatment, indicating that cold temperature was involved in the post-translational regulation of MdHY5 protein. These results suggest that MdHY5 may play an important role in the cold stress response. 3.2. MdHY5 positively regulates cold tolerance in apple calli and Arabidopsis To test whether MdHY5 regulates cold tolerance, an MdHY5-pCAMBIA1300 overexpression construct was transformed into ‘Orin’ apple calli (WT) and Arabidopsis (Col-0) through Agrobacterium-mediated genetic transformation, and two transgenic apple calli lines (MdHY5-L1 and MdHY5-L2) and three transgenic Arabidopsis lines (MdHY5#1, MdHY5#2, and MdHY5#3) were selected for further investigation (Fig. S1A-C). We next examined the cold tolerance of MdHY5 transgenic apple calli. The eightday-old wild-type and transgenic apple calli were exposed to 4°C for another 10 days. As shown in Fig. 2A-B, transgenic apple calli displayed increased cold stress tolerance compared with the wild-type control under cold conditions. Consistent with the cold stress assays in apple calli, ectopic expression of MdHY5 in Arabidopsis significantly increased survival rates in response to cold stress (Fig. 2C-D). 3.3. MdHY5 positively regulates the expression of MdCBFs To explore how MdHY5 contributes to cold tolerance, qRT-PCR was performed to test the expression of MdCBF genes in transgenic apple calli, which were considered to play major roles in configuring the low-temperature transcriptome and in regulating cold tolerance (Skinner et al., 2005; Thomashow, 2010). As shown in Fig. 3 and Fig. S3, the expression levels of MdCBF genes, including MdCBF1, MdCBF2, and MdCBF3, were substantially elevated in MdHY5 transgenic apple calli compared to the wild type under cold treatment. Consistently, the expression levels of CBF target genes, such as MdKIN1, MdRD29A, and MdCOR47, were also higher in transgenic apple calli than in the wild type. 3.4. MdHY5 binds to the MdCBF1 promoter It has been reported that MdHY5 transcription factor binds to G-Box (CACGTG) motifs in the promoters of target genes (An et al., 2017). Then, the sequences of CBFs promoter regions were analyzed and a putative G-Box motif was found in the promoter of MdCBF1 (Fig. 4A). To test whether MdHY5 directly bound to the promoter of MdCBF1 in vitro, an EMSA assay was performed. The recombinant MdHY5-GST protein was purified and the MdCBF1 promoter fragment containing the G-Box motif was prepared. As predicted, the MdHY5-GST fusion protein could bind to the promoter of MdCBF1, and no binding was tested in the GST protein (Fig. 4B). In addition, the binding was reduced by the addition of competitors, whereas no binding was observed when adding the mutated probe (Fig. 4B). In order to test whether MdHY5 could up-regulate the expression of MdCBF1, transient transactivation assays in tobacco leaves were performed. The promoter of MdCBF1 (MdCBF1pro) and mutated MdCBF1 promoter fragments (MdCBF1(M)pro) 5
were fused to the LUC gene as reporters. The MdHY5 effector construct was expressed under the 35S promoter (Fig. S2). Compared to the control (Fig. 4C, a, b), co-expression of 35Spro:MdHY5 with MdCBF1pro:LUC led to a significant increase of luminescence intensity (Fig. 4C, c), while 35Spro:MdHY5 failed to activate the expression of MdCBF1(M)pro:LUC (Fig. 4C, d; Fig. 4D). These finding reveal that MdHY5 transactivates the expression of MdCBF1 in apple. 3.5. MdHY5 regulates the expression of CBF-independent cold-regulated genes Besides the CBF-dependent cold response pathway by which MdHY5 regulates cold stress tolerance, some CBF-independent genes, including MdPYL6, MdWARK6, MdSAG21, MdSOC1, MdJMT, and MdESM1, were also selected for gene expression analysis (Robatzek and Somssich, 2001, 2002; Seo et al., 2001; Zhang et al., 2006; Cipollini, 2007; Salleh et al., 2012; Li et al., 2017). The results showed that the expression of MdPYL6, MdWARK6, and MdSAG21, positive regulatory genes of the cold stress response, were up-regulated in the MdHY5 transgenic apple calli than in the wild type under cold treatment. On the contrary, the expression of negative regulatory genes, including MdSOC1, MdJMT, and MdESM1, was consistently lower in the MdHY5 transgenic apple calli than in the wild type (Fig. 6). These results demonstrate that MdHY5 plays a dual role in regulating CBF-independent coldresponsive genes.
4. Discussion The bZIP transcription factor HY5 plays diverse roles in plant growth and development processes involved in photomorphogenesis (Ang and Deng, 1994), nutrient assimilation (Chen et al., 2016), cold stress response (Toledo-Ortiz et al., 2014), flavonol biosynthesis (Loyola et al., 2016), and ROS scavenging (Catalá et al., 2011). Among these biological responses, studying the HY5-mediated cold tolerance is of great theoretical significance and value regarding the improvement of apple production. In our previous study, the apple bZIP transcription factor, MdHY5, was cloned and functionally identified to be involved in anthocyanin accumulation and nitrate assimilation (An et al., 2017). In this study, we revealed that MdHY5 simultaneously regulates cold tolerance via CBF-dependent and CBF-independent pathways (Fig. 6). The CBF signaling pathway plays an essential role in the plant cold stress response, and plants have evolved precise mechanisms to conduct this signaling pathway (Thomashow, 1990; Chinnusamy et al., 2007). Within a short time of low-temperature exposure, the expression levels of the CBF genes and cold-responsive genes are greatly elevated in Arabidopsis (Guy et al., 1985; Gilmour et al., 1998). In apple, the transcripts of MdCBFs can also be induced by low-temperature treatment (Feng et al., 2012), and our studies showed that overexpression of MdHY5 significantly increased the transcripts of cold-responsive genes under cold stress (Fig. 3). HY5 is known for regulating the expression of target genes by binding to the GBox motifs (CACGTG) of their promoters (Zhang et al., 2011a). The synchronous expression manner of MdHY5 and MdCBFs implies that MdCBFs might be direct targets of MdHY5. Then, the EMSA assay and transient transactivation assay showed that MdHY5 directly up-regulated MdCBF1 by binding to the G-Box motif of its promoter (Fig. 4). In addition, it is noteworthy that MdHY5 transgenic apple calli also affect the expression of several CBF-independent cold-responsive genes, indicating that the CBF-independent cold response pathway modulated by MdHY5 plays an 6
essential role in MdHY5-regulated cold tolerance. It is inadvertent that MdHY5 exhibits a similar regulation pattern as Arabidopsis BZR1 in cold response (Li et al., 2017), which might be owed to the direct interaction between BZR1 and HY5 in Arabidopsis (Li et al., 2016). As a master regulator of light signaling, HY5 has been well characterized in regulating circadian rhythm and the cold stress response in Arabidopsis (Lee et al., 2007; Toledo-Ortiz et al., 2014). However, as a perennial woody plant, apple undergoes more complicated photoperiod and temperature changes, and MdHY5 might play a more complicated role in response to environmental factors. Therefore, improved comprehension of MdHY5 functions could be highly useful for producing high-quality fruits. A recent study shows that PIF3 plays a negative role in the CBF signaling pathway and cold tolerance (Jiang et al., 2017). Here, our data clearly revealed the positive contribution of the apple HY5 gene in the development of cold tolerance in apple, which provides further straightforward evidence that plants can integrate light and temperature signaling to better adapt to external stresses. In conclusion, based on previous studies and our present data, a hypothetical model for MdHY5 modulating cold tolerance in apple was proposed (Fig. 6). After exposure to the cold stimulus, MdHY5 was positively regulated both at the transcriptional and at the post-translational levels. On the one hand, MdHY5 promoted the cold tolerance by directly binding to the MdCBF1 promoter. On the other hand, the CBF-independent cold response pathway regulated by MdHY5 played an essential role in MdHY5regulated cold tolerance. Our studies provide new insight into the molecular mechanism of the MdHY5-enhanced cold tolerance in apple.
Acknowledgements This work was supported by grants from the Natural Science Foundation of China (31601742 and 31430074), the Ministry of Education of China (IRT15R42), and Shandong Province Government (SDAIT-06-03).
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Figure legends Fig. 1 Effects of cold treatment on the gene expression and protein stability of MdHY5 (A) Expression of MdHY5 gene in the wild-type apple seedlings treated at 4°C by qRT-PCR. Apomictic crabapple (Malus hypehensis) seedlings grown at 22°C were treated at 4°C for the indicated time. The value for untreated seedlings was set to 1. (B) Protein degradation of the MdHY5-GST fusion protein and its stabilization at 4°C. The protein level at 0 h was set to 1.00.
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Fig. 2 Cold tolerance assays of MdHY5-overexpressing apple calli and Arabidopsis (A) Cold stress phenotypes of MdHY5-overexpressing apple calli under lowtemperature conditions. The wild-type (WT) and transgenic apple calli (MdHY5-L1 and MdHY5-L2) were grown on medium at room temperature for eight days and then treated at 4°C for another ten days. (B) Fresh weight of wild-type and transgenic apple calli after cold treatment. (C) Cold stress phenotypes of MdHY5-overexpressing Arabidopsis under lowtemperature conditions. The wild type (Col-0) and transgenic Arabidopsis (MdHY5#1, MdHY5#2, and MdHY5#3) were grown on MS plates at 22°C for ten days and then treated at -4°C for 0.5 h. (D) Survival rates of MdHY5-overexpressing Arabidopsis after cold treatments.
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Fig. 3 Expression analysis of MdCBF genes and MdCBF target genes in the MdHY5 transgenic apple calli Relative expression levels of MdCBF genes (MdCBF1, MdCBF2, and MdCBF3) and MdCBF target gene (MdKIN1, MdRD29A, and MdCOR47) in the wild-type (WT) and transgenic apple calli (MdHY5-L1 and MdHY5-L2). The value for WT was set to 1.
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Fig. 4 MdHY5 binds to the promoter of MdCBF1 (A) Schematic diagram of the MdCBF1 promoter showing the potential MdHY5 binding sites. The predicted G-Box (CACGTG) site and sequence are indicated with box. Mutated site (Mut) in which the 5’-CACGTG-3’ motif was replaced by 5’CGCGCG-3’. (B) EMSA assay showing that MdHY5 fusion protein directly bound to the MdCBF1 promoter at CACGTG in vitro. Biotin-labeled probes were incubated with MdHY5GST protein, and the free and bound DNAs were separated in an acrylamide gel. As indicated, unlabeled probes were used as competitors. (C) Transient expression assays showing that MdHY5 promoted the expression of MdCBF1. a: Black (empty vector); b: MdCBF1pro:LUC; c: MdCBF1pro:LUC35Spro:MdHY5; d: MdCBF1(M)pro:LUC-35Spro:MdHY5. MdCBF1(M)pro in which the 5’-CACGTG-3’ motif was replaced by 5’-CGCGCG-3’. Representative images of Nicotiana benthamiana leaves 72 h after infiltration are shown. Quantitative analysis of luminescence intensity in (D). The value for a (empty vector) was set to 1.
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Fig. 5 Expression analysis of CBF-independent genes in the MdHY5 transgenic apple calli Relative expression levels of MdPYL6, MdWRKY6, MdSAG21, MdSOC1, MdJMT, and MdESM1 in the wild-type (WT) and transgenic apple calli (MdHY5-L1 and MdHY5-L2). The value for WT was set to 1.
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Fig. 6 The proposed model of MdHY5-mediated cold tolerance in apple Cold induces the accumulation of MdHY5, and activation of MdHY5 induces the expression of MdCBF1 by binding to the G-Box motif of its promoter. MdHY5 also regulates some cold-regulated genes, including MdPYL6, MdWRKY6, MdSAG21, MdSOC1, MdJMT, and MdESM1, which are independent of CBF, to modulate plant cold tolerance.
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