Chrysanthemum (Chrysanthemum morifolium) CmICE2 conferred freezing tolerance in Arabidopsis

Journal Pre-proof Chrysanthemum (Chrysanthemum morifolium) CmICE2 conferred freezing tolerance in Arabidopsis Zhaohe Zhang, Lu Zhu, Aiping Song, Haibin Wang, Sumei Chen, Jiafu Jiang, Fadi Chen PII:

S0981-9428(19)30450-4

DOI:

https://doi.org/10.1016/j.plaphy.2019.10.041

Reference:

PLAPHY 5914

To appear in:

Plant Physiology and Biochemistry

Received Date: 5 April 2019 Revised Date:

30 October 2019

Accepted Date: 31 October 2019

Please cite this article as: Z. Zhang, L. Zhu, A. Song, H. Wang, S. Chen, J. Jiang, F. Chen, Chrysanthemum (Chrysanthemum morifolium) CmICE2 conferred freezing tolerance in Arabidopsis, Plant Physiology et Biochemistry (2019), doi: https://doi.org/10.1016/j.plaphy.2019.10.041. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Masson SAS.

Contributions Fadi Chen, Sumei Chen and Jiafu Jiang conceived and designed research, Zhaohe Zhang, Lu Zhu, Aiping Song and Haibin Wang conducted experiments, Zhaohe Zhang and Lu Zhu analyzed data, Zhaohe Zhang， Sumei Chen and Jiafu Jiang worte the manuscript. All authors read and approved the final manuscript.

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Title: Chrysanthemum (Chrysanthemum morifolium)

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CmICE2 conferred freezing tolerance in Arabidopsis

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Authors: Zhaohe Zhang1, Lu Zhu1, Aiping Song1, Haibin Wang1, Sumei

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Chen1, Jiafu Jiang1, Fadi Chen1, *

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Affiliation:

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State Key Laboratory of Crop Genetics and Germplasm Enhancement,

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Key Laboratory of Landscaping, Ministry of Agriculture and Rural

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Affairs, College of Horticulture, Nanjing Agricultural University, Nanjing

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210095, China

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Corresponding author: Fadi Chen

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Address: College of Horticulture, Nanjing Agricultural University,

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Nanjing 210095,

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China

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Tel: +86-025-84396579

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Fax: +86-025-84395266

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E-mail: [email protected]

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Zhaohe Zhang, [email protected]

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Lu Zhu, [email protected]

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Aiping Song, [email protected]

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Haibin Wang, [email protected] 1

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Sumei Chen, [email protected]

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Jiafu Jiang, [email protected]

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Abstract

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Genes of the ICE (Inducer of CBF Expression) family play a key role in cold and freezing stresses response via the CBF regulatory pathway. In this work, we identified the ICE family gene, CmICE2, from Chrysanthemum morifolium ‘Jinba’. CmICE2 encodes a 451-amino acid protein with a conserved nuclear localization domain, a bHLH domain and ACT domain. CmICE2 is expressed in abundance in leaves and flowers, and the expression of CmICE2 is induced by freezing and drought stresses. CmICE2 localized to the nucleus, and has transcriptional activity in yeast cells. After a 24-hour 4 acclimation, Arabidopsis plants overexpressing CmICE2 were more tolerant to freezing stress (-9 for 6 h) than the Col-0. When exposed to -9 for 6 h, the expression levels of genes such as AtCBF1, AtCBF2, AtCBF4, AtCOR6.6A, AtCOR414 and AtKIN1 were up-regulated significantly in CmICE2 overexpression plant lines compared to wild type. The proline content, activities of superoxide dismutase (SOD), peroxidase (POD) and catalase(CAT) were also increased in plants overexpressing CmICE2. In summary, CmICE2 confers to plant response to freezing stress. Keywords: ICE, Chrysanthemum morifolium, freezing stress, transgenic Arabidopsis, cold acclimation 1. Introduction The growth and development of plants depends to a great extent on their environment. The abiotic stresses, such as salt, drought and low temperature have negative effects for plant growth and development (Zhu, 2002). The effects of low temperatures on plants can be divided into two types, cold acclimation or freezing stress. Cold acclimation, being exposed to low but not-freezing temperatures, is a positive process for plants to acquire more tolerance to freezing stress (Thomashow, 1999). Thousands of genes in complex physiological and biochemical pathways are involved in cold acclimation, with the C-repeat-binding factor (CBF) pathway playing a key role in this process (Chinnusamy et al., 2007; Medina et al., 2011). Once Arabidopsis thaliana was exposed to cold acclimation temperature, CBF genes which belong to the AP2/ERF (APETALA2/ethylene-responsive factor) factor subfamily bind to the promoters of its downstream cold-responsive (COR) genes and activate their expression, which confers freezing tolerance (Gilmour et al., 1998; Stockinger et al., 1997; Liu et al., 1998; Jia et al., 2016; Zhao et al., 2016). CBF is regulated by several transcription factors, among which Inducer of CBF (ICE) is considered as a major regulator of CBF (Chinnusamy et al., 2003). ICE1 encodes a MYC-like basic-helix-loop-helix transcription factor and binds to the MYC-recognition motif in the promoter of CBF (Chinnusamy et al., 2003; Kim et al., 2015). ICE1, a transcription factor activates the expression of CBF, regulated by the cold response through post-translational mechanisms. HOS1 encodes a RING-type ubiquitin E3 ligase that is involved in the cold-induced degradation process of ICE1 3

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(Dong et al., 2006; Lee et al., 2001). SIZ1 mediates the sumoylation of ICE1, which stabilizes the ICE1 protein and thus enhances freezing tolerance via its downstream CBF network (Miura et al., 2007). MYB15 was proved to interact with ICE1 protein and enhanced the expression of downstream CBF genes (Agarwal, et al. 2006). The transcriptional activity of ICE1 could be repressed by the interacting with JAZ1 and JAZ4. Overexpression of JAZ1 or JAZ4 led to the decrease of freezing stress responses in Arabidopsis (Hu et al., 2013). OST1, a Ser/Thr protein kinase, contributes to the stability of ICE1 by phosphorylating ICE1 as well (Ding et al., 2015). Recently, MAPKs have been found to be involved in the cold response. MPK3/MPK6 phosphorylate and destabilize ICE1, leading to the degradation of the ICE1 protein (Li et al., 2017; Zhao et al., 2017). ICE2 is a paralog of ICE1, and the two proteins share highly conserved motifs and domains. Previous studies showed that ICE2 has a similar function to ICE1 in promoting the freezing tolerance of Arabidopsis (Fursova et al., 2009). Chrysanthemum morifolium is one of the most popular ornamental plants in the world. Previously one Chrysanthemum ICE gene, CdICE1, was identified to enhance freezing tolerance via miR398 in Arabidopsis (Chen et al., 2012; Chen et al., 2013). However, many other species were proved to have two or more ICE genes, such as AtICE1/AtICE2 in Arabidopsis (Chinnusamy et al., 2007), OsICE1/OsICE2 in rice (Nakamura et al., 2011) and TaICE41/TaICE87 in wheat (Badawi et al., 2008). Whether another ICE gene existing in Chrysanthemum remains to be explored. To further elucidate the function of ICE gene family in chrysanthemum, a new ICE family homologue gene CmICE2 was separated from C. morifolium. Overexpression of CmICE2 in Arabidopsis indicated that CmICE2 conferred freezing tolerance to Arabidopsis via CBFs pathway and ROS pathway.

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2.1 Plant materials and growth conditions The C. morifolium ‘Jinba’ was obtained from the Chrysanthemum Germplasm Resource Preserving Centre, Nanjing Agricultural University, China. Seedlings of six to eight leaves at uniform stages were planted in a 1:3 (v/v) mixture of soil and vermiculite for cold and ABA treatment, and the seedlings were cultivated in nutrient solution for salt and dehydration treatments. All the seedlings were grown in a greenhouse under a 14 h light/10 h dark photoperiod at 25°C/18 °C with a relative humidity of 70%, light intensity: 120 µmol·m−2·s−1.

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2.2 Isolation and bioinformation analysis of CmICE2 To amplify the full-length sequence of CmICE2, primers Full-F/R (Supplementary Table S1) were designed using the Primer version 5.0 software according to the transcriptome data of Chrysanthemum nankingense (Ren et al., 2014). The full-length CmICE2 cDNA sequence was obtained using Pfu DNA polymerase (TaKaRa Ex Taq®).

2. Materials and methods

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The coding sequence (CDS) of CmICE2 (GenBank accession no.MN241451) was identified using the BioXM version 2.6 software. The amino acid of CmICE2 homologs were obtained from Genbank (www.ncbi.nlm.nih.gov/genbank/), and the phylogenetic tree was constructed based on the neighbor-joining method using MEGA6.0 software. 1000 of bootstrap replicates were set to test the phylogeny.

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2.3 Intracellular localization analysis and transcriptional activity analysis The CDS of CmICE2 was amplified using primers Location-F/R (Supplementary Table S1), and introduced into the BamH I/Not I cloning site of the pENTRTM1A vector. The plasmid pENTRTM1A-CmICE2 was confirmed by DNA sequencing and was subsequently used to construct the green fluorescent protein (GFP) fusion vector pMDC43-CmICE2 by LR reaction (as described in Gateway® Technology with Clonase® II). The confirmed plasmid pMDC43-CmICE2 was transformed into onion (Allium cepa) epidermal cells by a gene gun (PDS-1000; Bio-Rad, Hercules, CA, United States). The 35S::D53-RFP construct was co-transformed as a nuclear marker (Zhang et al., 2017). The epidermal cells were incubated in the dark for 16-20h, the GFP signals were observed by confocal laser scanning microscopy at 488 nm (Zeiss, Germany) (Wan et al., 2008). For transcriptional activity analysis, the amplified CmICE2 CDS was cloned into pGBKT7 via EcoR I/BamH I site to generate the yeast expression vector pGBKT7-CmICE2. The fragments CmICE2-132, which lacked the front 132 amino acids, was amplified with primer pair Activity-132-F/R (Supplementary Table S1) and inserted into pGBKT7 via the same restriction sites to obtain the plasmids pGBKT7-CmICE2-132. Afterwards, the two yeast expression vectors above, pCL1 (positive control) and pGBKT7 (negative control) were transformed into the yeast cells Y2HGold.The yeast cells were spread on SD/His-Ade- medium with or without X-α-gal supplementation.

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2.4 Transcriptional patterns of CmICE2 For the transcription profile analysis of CmICE2, the root, stem, leaves, tubular flowers and ray flowers of the C. morifolium ‘Jinba’ were harvested at the flowering stage. Chrysanthemum seedlings at six to eight leave stage were employed for stress treatment. For the cold treatment, the chrysanthemum seedlings were exposed to 4°C. The ABA treatment was performed by spraying 100 µM ABA solution on the seedlings. In addition, the solution containing 200 mM NaCl and 20% PEG were applied to the seedlings for salt and dehydration treatment, separately. All the seedlings were cultivated under a 14 h light/10 h dark photoperiod with a relative humidity of 70%, light intensity: 120 µmol·m−2·s−1. The samples were collected at 0, 1, 3, 6, 12, and 24 h after each treatment. 2.5 Regeneration of CmICE2 overexpressing Arabidopsis To further identify the function of CmICE2, the plasmid pMDC43-CmICE2 was transformed into ArabidopsisCol-0 plants with Agrobacterium strain EHA105 (Höfgen and Willmitzer, 1988) by the floral-dip method. The hygromycin 5

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(Hyg)-resistant plants were confirmed by the PCR analysis using the primers Hyg-F/R (Supplementary Table S1). In addition, the overexpression of CmICE2 was validated by Semiquantitative RT-PCR with the primer pair RT-F/R (Supplementary Table S1), and the gene AtActin was used as the internal reference with the primer pair ACTIN-F/R (Supplementary Table S1).

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2.6 Freezing tolerance analysis of CmICE2 overexpression in Arabidopsis The wild type and CmICE2 overexpressing Arabidopsis seedlings were planted in a 1 : 3 (v/v) mixture of soil and vermiculite in a greenhouse under a 14 h light/10 h dark photoperiod at 25°C with a relative humidity of 70%, light intensity: 120 µmol·m−2·s−1. Freezing assay were performed as described previously, with slight modifications (Cai et al., 2018; Ren et al, 2018). Seedlings at 3 week old stages were exposed to 4°C acclimation for 24h in an incubator (Sanyo, MIR-154). The acclimated seedlings were then subjected to freezing stress at -9°C for 6h. Freezing stress treated seedlings underwent a recovery growth period at 25°C for 14d before the survival rates were calculated. Every experiment included three replicates, and each replicate contained nine seedlings. After the 6 h freezing treatment, seedlings were sampled immediately. All samples were snap-frozen in liquid nitrogen, and kept at -80°C. Superoxide dismutase (SOD) activity was detected using the protocol from (Yin et al. 2009), peroxidase (POD) activity using the protocol given by (Pan et al. 2006), and the activity of catalase (CAT) was assessed using the protocol (Fatima et al. 2011). Moreover, the proline content was assayed as previously described (Liu et al., 2005). Each assay included three replicates from three different plants per line per time point. 2.7 Quantification analysis of gene expression profiles Total RNA was isolated using the TRIzol reagent (Invitrogen, Germany) in accordance with the manufacturer’s instructions. The first cDNA strand was synthesized using the TaKaRa reverse transcription system (Japan) following the manufacturer’s protocol and 500 ng·μL−1 RNA was used for cDNA synthesis (Liu et al., 2018). Real-time quantitative PCR (qPCR) assays were performed to analyze the expression patterns of CmICE2 and its downstream genes, using the SYBR Green master mix (SYBR Premix Ex TaqTM II, TaKaRa Bio) and the corresponding primer pairs (Gao J. et al., 2016). For Chrysanthemum, CmEF1α (GenBank accession no.AB548817.1) was used as the reference gene, and AtActin was used as the reference gene in Arabidopsis. All of the sequences of these primers is given in Supplementary Table S1. Relative expression levels were calculated by the 2-∆∆Ct method (Kenneth and Livak, 2001). 2.8 Statistical analysis The statistical analyses were carried out using the SPSS v17.0 software (SPSS Inc, Chicago, IL). Student’s t-test (P = 0.05) was employed to test for statistical significance.

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3. Result 3.1 Identification and characterization of CmICE2 Here, CmICE2 was isolated from C. morifolium ‘Jinba’. It contains an open reading frame (ORF) of 1353 bp, which encodes a 451 amino acid-protein. Sequence alignment reveals that CmICE2 has a conversed nuclear localization domain and a bHLH motif with only 3 amino acids different between ICE1 and ICE2 in Arabidopsis. The deduced peptide sequence of CmICE2 also has an ACT domain highly similar to AtICE1 and AtICE2. However, the serine-rich region is absent in CmICE2 compared to homologs of Arabidopsis ICEs (Figure 1A). In addition, the phylogenetic analysis showed that CmICE2 is of highest similarity to CdICE1 (Figure 1B).

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3.3 Transcriptional profiling of CmICE2 in the chrysanthemum plant The relative expressions of CmICE2 in leaves, tubular flowers and ray flowers were about 6 times higher than in roots and stems (Figure 3A). After 3h cold treatment, the expression level of CmICE2 began to increase, and increased constantly up to 12 h after cold treatment commenced, and remained at a high expression level until 24 h after the treatment. In response to the exogenous ABA treatment, the expression level of CmICE2 decreased slightly at 1h but increased at 3h. The expression of CmICE2 was down-regulated at 6, 12, and 24h after ABA treatment (Figure 3B). In addition, the expression of CmICE2 was constantly up-regulated in dehydrated conditions up to 12 hours. At 24h, the expression level of CmICE2 was lower than that at 12h, but still maintained a significant higher expression level compared to the control. However, the expression level of CmICE2 was less affected by NaCl treatment (Figure 3C).

3.2 Nucleus subcellular localization of CmICE2 and transcriptional activity assay Subcellular localization analysis showed that the CmICE2 protein is localized to the nucleus. This result was further confirmed by the co-transformation of 35S::D53-RFP construct, which produced RFP signal co-localized to the nucleus. No GFP signals could be observed in cytoplasm and plasma membrane of the onion epidermal cell (Figure 2A). To test the transcriptional activity of CmICE2, a yeast assay were performed. We found that CmICE2 had transcriptional activation activity in yeast cells. Whereas deletion of 132 amino acids at the N-termination of the CmICE2 protein caused a loss of transcriptional activation activity of CmICE2 (Figure 2B).

3.4 CmICE2 overexpression improves the resistance of transgenic Arabidopsis to freezing temperature under acclimation condition Given the conversed bHLH domain of ICE genes and inducible expression by 4℃, we hypothesized that CmICE2 might play a role in regulating cold and freezing tolerance. To validate the hypothesis, CmICE2 was introduced into Col-0 via floral dip transformation. Six transgenic lines were confirmed by PCR using Hyg-F/R primers 7

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(Figure 4A).Further the transcript abundances of six lines was detected via a semi-quantitative PCR and quantitative real time RT-PCR (Figure 4B, C). Two overexpression lines of high expression levels OE1 and OE2 were selected, and the freezing tolerance of the T3 generation homozygous OE1 and OE2 lines was assayed. Cold acclimation (CA) could significantly induce a plant positive response to freezing tolerance. Therefore, we performed a 24-hour 4℃ cold acclimation period before the 6-hour freezing stress at -9℃. When acclimated plants were subjected to freezing stress of -9℃ for 6h, both the Col-0 and the transgenic Arabidopsis survived the constant freezing treatment, with no differences in appearance identified. After a 2-week period of recovery growth, far more than half of the two transgenic lines, OE1 and OE2, had recovered from the stress, but only the half Col-0 plant lines survived after the recovery (Figure 5A). OE1 and OE2 maintained a remarkably high level of survival rate (77% and 81%) compared to Col-0 survival rate (51%) (Figure 5B).

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3.5 CmICE2 overexpression improves proline content and ROS scavenging capability of transgenic Arabidopsis under freezing stess The activities of antioxidant enzymes and contents of proline in plants under non-stress conditions or -9℃ for 6h were compared. The results show that there was no obvious difference in enzyme activities (SOD, POD, CAT) in both transgenic lines and wild type plants under 25℃. After freezing treatment, the whole three antioxidant enzymes activities increased significantly both in Col-0 plants and CmICE2 OE lines, with the activities of SOD, POD and CAT enzymes being much higher in the CmICE2 OE lines than in the Col-0 (Figure 6A). Ion leakage assays indicate that OE1 and OE2 suffered less damage than Col-0. After freezing treatment Col-0 plants had a significantly higher ion leakage (73%) than OE lines (61%/59%) (Figure 6B). There are no significant differences in contents of proline between Col-0 and CmICE2 OE lines under non-stressed conditions, however, sharp increases in proline content were observed in OE1 and OE2 after freezing treatments and they were much higher than that in Col-0(Figure 6C).

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3.6 CmICE2 overexpression in transgenic Arabidopsis up-regulates the expression of cold-responsive genes To further explore the possible molecular mechanism of CmICE2 in improving freezing tolerance, the expression profiles of a set of stress responsive genes were investigated. Given the downstream CBF genes regulated by ICEs, real-time quantitative PCR assays were performed to analyze the expression of Arabidopsis CBFs genes: AtCBF1, AtCBF2, AtCBF3, AtCBF4 and AtCBF5. Under non-stressed conditions, five Arabidopsis CBFs in Col-0 plants was comparable to those in CmICE2 OE lines. When exposed to -9℃ for 6h, the relative expression of AtCBF1, AtCBF2, AtCBF3, and AtCBF4 increased significantly in both OE lines and Col-0 plants, with the transcript abundance in OE lines being much more than Col-0 in AtCBF1, AtCBF2 and AtCBF4. Nevertheless, there was no significant change in the expression of AtCBF5, neither at 25℃ nor after the freezing stress. The abundance of AtCBF5 transcripts in OE1 and OE2 plants just mirrored the abundance in Col-0 8

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plants (Figure 7). Regulated by AtCBFs, six downstream COR (COLD RESPONSIVE) genes: COR6.6A, COR15B, COR78, COR414, KIN1 and NCED3 were further investigated. The expression of all these genes increased several times after the treatment, suggesting that the whole CBF-dependent pathway in plants was up-regulated through 24-hour cold acclimation and freezing stress. In addition, there was no significant change in the expression of the six Arabidopsis COR genes in either Col-0 plants or OE lines at 25℃. After the 6 hour freezing treatment however, a remarkable enhancement in expression levels of COR6.6A, COR414 and KIN1 were observed in OE1 and OE2, compared to Col-0. The other three genes (COR15B, COR78 and NCED3) show no apparent differences. These results indicate that CmICE2 succeed in up-regulating AtCBF1, AtCBF2, AtCBF4 and the downstream COR6.6A, COR414, KIN1 genes, in which way conferred freeing tolerance in Arabidopsis (Figure 8). 4. Discussion In Arabidopsis, two paralogous ICE genes ICE1 and ICE2 have been isolated and both play roles in the cold regulatory network at the transcription level (Zhu, 2016). ICE1 binds to the promotor of CBF3 and ICE2 up-regulates the expression of CBF1 (Chinnusamy et al., 2003; Fursova et al., 2009). Recently, more and more studies of ICE1 and its post-translational mechanisms have been published (Liu et al., 2017; Liu and Zhou, 2018). The role of ICE1 in the cold regulating network and phosphorylation modification are being uncovered gradually (Ramirez and Poppenberger, 2017). It has been noticed that several serine sites are of great importance in the phosphorylation of ICE1. Ser403 is involved in MPK3/MPK6-meidated ICE1 phosphorylation (Miura et al., 2011; Zhao et al., 2017). Ser278 stabilized the ICE1 protein and enhanced tolerance of Arabidopsis to freezing (Ding et al., 2015). In our study, we identified the second ICE family gene CmICE2 in chrysanthemum. The expression of CmICE2 is cold and drought inducible (Figure 3). However, expression of CdICE1 increased after cold, salt and ABA treatments but not in 20% polyethylene glycol (PEG) dehydration (Chen et al., 2013), suggesting that CdICE1 and CmICE2 might play different roles in the ABA and salt response. Arabidopsis AtICE2 activates the expression of CBF1 (Chinnusamy et al., 2003; Fursova et al., 2009). Unlike AtICE1 and AtICE2 which share an absolutely identical bHLH domain (Budhagatapalli et al., 2015), CmICE2 and CdICE1 proteins have two amino acids different in their bHLH domains. The difference in this conserved sequence lead to the diverse combination with downstream genes. The lack of a serine-rich region distinguishes CmICE2 from CmICE1 and ICE proteins in other plants. Whether the lack of a serine-rich region might influence post transcriptional modifications of the CmICE2 protein remains to be investigated further. In addition, the ICE proteins, also known as SCREAM and SCREAM2, participate in the process of stomatal development, and together interact with three core basic-helix-loop-helix proteins i.e., SPCH, MUTE and FAMA, in the sequential (Hofmann, 2008; Kanaoka et al., 2008). 9

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Therefore, more experiment is to perform to dig into the nature relationship between CdICE1 and CmICE2. In order to identify the function of CmICE2, CmICE2 overexpression Arabidopsis lines were generated and the overexpression plant lines were found to enhance the tolerance of Arabidopsis to freezing, suggested that CmICE2 assisted in freezing tolerance. To further dissect the putative mechanisms involved in freezing tolerance, the transcript abundance of a set of genes in response to freezing treatment were analyzed (An et al., 2018; Yao et al., 2018). The transcript abundance of AtICEs remained a normal level at room temperature, while AtICEs were cold-induced expressors (Chinnusamy et al., 2003). Therefore, a 24-hour cold accumulation was performed before the freezing treatment. After the 6-hour freezing stress, CmICE2 activated expression of CBF (AtCBF1, AtCBF2, AtCBF4) genes remarkably, and the downstream COR (COR6.6A, COR414 and KIN1) genes had an apparent inducible increase in transgenic lines. Both ICEs and CBFs were transcription factors and responded to induction in a very short time (Baird et al., 2018). The expression levels of CBF and COR genes at -9 were at least five times that at 25 through a 24h cold acclimation (Figure7, 8). Nevertheless, in CmICE2 overexpressing plants there was no noticeable difference in the expression of AtCBF5 compared to Col-0. In Arabidopsis, two ICE genes have a precise regulating network that AtICE1 induced AtCBF3 while AtICE2 induced AtCBF1. At present, all the two Chrysanthemum ICE genes have been transformed into Arabidopsis. The previous research illustrated that CdICE1 increased the expression level of down-stream AtCBF1, AtCBF2 and AtCBF3 (Chen et al., 2013). However, CmICE2 up-regulated AtCBF1, AtCBF2 and AtCBF4 (Figure 8). These results indicate CmICE2 participates in a particular response to freezing stress different from CdICE1. The accurate mechanism of CmICE2 involved in freezing tolerance need to be further elucidated. The ROS system acts as a damage indicator in plants. Upon exposure to abiotic stresses, ROS will increase significantly (Miller et al., 2010; He et al., 2018), an efficient elimination of ROS accumulation is known to confer tolerance to stresses and plants will express a number of enzymes such as SOD, POD, and CAT to cope with ROS scavengers. In this present study, after the -9 freezing stress, the activities of antioxidant enzymes (SOD, POD and CAT) increased and ion leakage decreased correspondingly in both Col-0 and transgenic Arabidopsis, suggested that elevated enzyme activities might confer membrane integrity. Since the plants acquired freezing tolerance through cold acclimation, both Col-0 and the two overexpressing lines were able to reduce some irreversible freezing damage (Zhu, 2016). Through these gene regulating network and ROS scavenging mechanisms, CmICE2 conferred freeing tolerance in Arabidopsis

Conclusion In summary, our study demonstrates that CmICE2 is a cold-related transcription factor, which functions as a positive regulatory role in enhancing freezing stress. Under cold acclimation condition, overexpression of CmICE2 conferred the response of 10

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transgenic Arabidopsis to freezing stress by up-regulating the expression of CBF and COR genes, increasing the activities of antioxidant enzymes of SOD, POD and CAT, and the accumulation of prolines. Taken together, these findings lay a foundation for dissecting the roles of CmICE2 in stress responses in chrysanthemum. Contributions Fadi Chen, Sumei Chen and Jiafu Jiang conceived and designed research, Zhaohe Zhang, Lu Zhu, Aiping Song and Haibin Wang conducted experiments, Zhaohe Zhang and Lu Zhu analyzed data, Zhaohe Zhang, Sumei Chen and Jiafu Jiang worte the manuscript. All authors read and approved the final manuscript. Acknowledgements This work is supported by the National Science Fund for Distinguished Young Scholars (31425022), the National Natural Science Foundation of China (31700620),the Fundamental Research Funds for the Central Universities (KYZ201507), the Natural Science Fund of Jiangsu Province (BK20151429, BK20170722). Conflicts of interest The authors declare that they have no conflicts of interest.

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Figure Legends Figure 1 Characterization of the CmICE2 polypeptide sequence. (A) Alignment of the deduced polypeptide sequences of CmICE2 with those of other plant ICEs. Black lines indicate the conserved motifs and domains. (B) A phylogenetic analysis of the CmICE2 sequence. Phylogenetic tree was constructed using the Neighbor-Joining method with 1000 bootstrap replicates. All sequences of ICE homologs were obtained from Genebank database, and their accession numbers were as follow. Arabidopsis thaliana AtICE1: AT3G26744, AtICE2: AT1G12860; Glycine max GmICE1: 100217332, GmICE4: NP_001238707.1, GmICEb: ADV36253.1, GmICEd: ADV36254.1; Zea mays ZmICE2: ONM34888.1; Vitis vinifera VvICE1: AHM24953.1; Vitis amurensis VaICE2: AGP04218.1; Raphanus sativus RsICE1: ADY68771.1; Brassica juncea BjICE: AEB97375.2; Camellia sinensis CsICE1: AFP25102.1; Lactuca sativa LsICE1: ADX86750.1 and Chrysanthemum dichroum CdICE1: AEO50748.1. Figure 2 Subcellular localization and transcriptional activity of CmICE2. (A) Subcellular localization of CmICE2 in onion epidermal cells. 35S::D53-RFP was used as a nuclear marker. Bars: 50 µm. (B) Transcriptional activation assays of CmICE2 via yeast one-hybrid assay. The yeast cells were spread on SD/His-Ademedium with or without X-α-gal supplemetation. pCL1 and an empty pGBKT7 were used as the positive and negative controls, respectively. Figure 3 Transcriptional profiling of CmICE2 in C. morifolium ‘Jinba’. (A) Transcript abundances of CmICE2 in different plant organs. CmICE2 expression was induced by cold (B), dehydration stress and NaCl treatment (C). Total RNA samples were obtained from Chrysanthemum seedlings at six to eight leaf stage treated with 4°C, 100 µM ABA, 200 mM NaCl and 20% PEG at indicated time point. All the seedlings were cultivated under a 14 h light/10 h dark photoperiod with a relative humidity of 70%, light intensity: 120 µmol·m−2·s−1. Error bars indicate standard deviations of three independent experiments. Each experiment contained three seedlings. Value of P<0.05 was considered to be statistically significant. Figure 4 Validation of transgenic plants. (A) PCR analysis of genomic DNA extracted from hygromycin-resistant selected plant lines. (B) Transcription levels of CmICE2 in Col-0 and transgenic lines determined by RT-PCR. AtActin was used as the reference gene. (C) Transcription levels of CmICE2 in Col-0 and transgenic lines determined by qRT-PCR. OE7 exhibited the lowest transcript abundance and were defined as "1" for 15

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calculating relative transcript abundances in the other OE lines. Error bars indicate standard deviations of three independent experiments. Each experiment contained three seedlings. The figure was spliced and grouped using Photoshop software. Figure 5 Overexpression CmICE2 in Arabidopsis improve survival rate after freezing tolerance. (A) Phenotype of plants under freezing stress after recovery. (B) Survival rate after 2-week recovery. Error bars indicate standard deviations of three independent experiments. Each experiment contained nine seedlings. Value of P<0.05 was considered to be statistically significant. Figure 6 Antioxidant enzymes activities, ion leakage and proline (Pro) contents in Col-0 and CmICE2 overexpression plants after freezing treatment. (A) The activities of SOD, POD, and CAT in transgenic lines and control plants. (B) Ion leakage of transgenic lines and control plants. (C) The proline content of transgenic lines and control plants. Statistical significant differences were compared with Col-0 plants under different conditions. Value of P<0.05 was considered to be statistically significant. Figure 7 The expression level of AtCBF genes in CmICE2 overexpressing lines and Col-0 plants after freezing treatment. The Arabidopsis AtActin2 gene was used as the reference gene. The 2−∆∆CT method was used to evaluate the relative expression, and the expression levels of genes in the Col-0 plants at 25℃ were defined as "1". Each value is the average of three replicates, and error bars represent ± SD. *, Statistical significant differences were compared with Col-0 plants under different conditions. Value of P < 0.05 was considered to be statistically significant. Figure 8 Analysis of cold-responsive genes in transgenic lines and Col-0 plants after freezing treatment. The total RNA was extracted from transgenic and Col-0 Arabidopsis after freezing treatments. The Arabidopsis Actin2 gene was used as the reference gene. The 2−∆∆CT method was used to evaluate the relative expression, and the expression levels of genes in the Col-0 plants at 25℃ were defined as "1". Each value is the average of three replicates, and error bars represent ± SD. *, Statistical significant differences compared with Col-0 plants under different conditions. Value of P < 0.05 was considered to be statistically significant.

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Supplementary data Table S1. The PCR Primers used in this study. Figure S1. Alignment of the peptide sequences of CmICE2 and CnICE2. Figure S2. Scheme for the construction of the expression vector pMDC43-CmICE2. Figure S3. Phenotype of plants under different freezing temperature after recovery.

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1.This study describes the isolation of an ICE family transcription factor CmICE2 from Chrysanthemum. 2.Heterologous expression studies revealed that CmICE2 involved in freezing and drought stresses response. 3.qRT-PCR analysis revealed overexpression of CmICE2 in Arabidopsis up-regulated some AtCBF and AtCOR genes.

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The authors declare that they have no conflicts of interest.