Cold-inducible expression of an Arabidopsis thaliana AP2 transcription factor gene, AtCRAP2, promotes flowering under unsuitable low-temperatures in chrysanthemum

Plant Physiology and Biochemistry 146 (2020) 220–230

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Research article

Cold-inducible expression of an Arabidopsis thaliana AP2 transcription factor gene, AtCRAP2, promotes flowering under unsuitable low-temperatures in chrysanthemum

T

Chang Luoa, Hua Liua, Junan Renb, Dongliang Chena, Xi Chenga, Wei Sunc, Bo Hongd, Conglin Huanga,∗ a Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing Engineering Research Center of Functional Floriculture, Beijing Key Laboratory of Agricultural Genetic Resources and Biotechnology, Beijing, 100097, China b Beijing Industrial Technology Research Institute, Beijing, 101111, China c Yuquan School of the Capital Normal University, Beijing, 100195, China d Department of Ornamental Horticulture, China Agricultural University, Beijing, 100193, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Chrysanthemum DREB1A Flowering Low temperature Short-day plant SOC1

Flowering time is regulated by biotic and abiotic stresses and affected by the ambient temperature. For chrysanthemum, a low ambient growth temperature can cause a flowering delay, which limits the annual commercial production. Therefore, it is important to improve the low-temperature flowering capability of chrysanthemum through genetic modifications. Here, we isolated a natural variation of a CRT/DRE-binding factor (CBF/DREB) 3 gene, CRAP2, from the Arabidopsis thaliana accession Condara (190AV) that encodes a stop codon at position 151 of the CBF3 protein. Unlike AtCBF3, the overexpression AtCRAP2 in Arabidopsis did not cause detectable growth retardation nor delayed flowering and it conferred cold tolerance. The cold-inducible expression of AtCRAP2 in chrysanthemum promoted flowering under short-day conditions with a low 15 °C nighttime temperature. RNAsequencing of rd29A:AtCRAP2 and qRT-PCR assays of flowering time-related genes and AtCRAP2 expressed at an ambient temperature revealed that AtCRAP2 positively affected SOC1 and FTL3, thereby promoting flowering under low temperature stress and short-day conditions. These results indicate that DREB genes can be used in the genetic engineering of crop plants without accompanying negative effects by modifying the encoded proteins’ C termini.

1. Introduction Flowering is a critical event during the transition from vegetative to reproductive phases in plant life cycles. Flowering time is regulated mainly by four genetic pathways: photoperiod, vernalization, gibberellin (GA) biosynthesis and aging. Signals from these pathways are recruited by the so-called flowering pathway integrators FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and LEAFY, whose expression levels determine flowering time (Song et al., 2013). The expression levels of the integrators are regulated positively by a photoperiod pathway core component CONSTAN (CO) and negatively regulated by FLOWERING LOCUS C (FLC), a MADS-box transcription factor that plays a key role in the autonomous/vernalization pathway (Lee et al., 2000). Their regulatory mechanisms have been well studied and elucidated. Recently, the link between flowering and stress tolerance has ∗

received increased attention (Kazan and Lyons, 2016). In addition to the above four pathways, flowering time is also regulated by biotic and abiotic stresses, and plants have evolved varied mechanisms to regulate flowering to cope with the effects of diverse stress factors. For example, ambient temperature affects plant flowering times. Low ambient temperatures cause flowering delays through the thermosensory pathway but do not influence normal plant growth and development (Lee et al., 2007). In contrast, high ambient temperatures promote flowering through increased FT expression activated by PHYTOCHROME INTERACTING FACTOR4 (Kumar et al., 2012). Plants have evolved different pathways to regulate flowering in response to different levels of cold stress. Plants accelerate flowering by silencing the floral repressor FLC through the vernalization pathway when exposed to long-term non-freezing cold temperatures (Michaels and Amasino, 2001). In contrast, flowering is delayed by activating FLC when exposed to short-term cold or the overexpression of cold-

Corresponding author. E-mail address: [email protected] (C. Huang).

https://doi.org/10.1016/j.plaphy.2019.11.022 Received 27 August 2019; Received in revised form 22 October 2019; Accepted 14 November 2019 Available online 17 November 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved.

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2. Materials and methods

responsive genes (Seo et al., 2009). While the two signal pathways’ influence on flowering is mediated primarily by FLC, they function independently (Bond et al., 2011). Plants improve their tolerance to cold stress by expressing cold-responsive genes. Many cold-regulated (COR) genes possess the C-repeat/ dehydration responsive element (CRT/DRE) DNA-regulatory motif in their promoters. CRT/DRE-binding factors (CBF/DREB) play key roles in cold-responsive pathways in Arabidopsis thaliana (Thomashow, 1999). The linearly clustered CBF1, -2 and -3 genes, also known as DREB1b, DREB1c and DREB1a, respectively, have been identified; however, the constitutive overexpression of these three genes leads to undesirable phenotypic changes, including late flowering and growth retardation, in different transgenic plant species (Gilmour et al., 2004; Yamaguchi-shinozaki et al., 1999). Yamaguchi-shinozaki et al. (1999) showed that the overexpression of Arabidopsis CBF3, driven by either the Cauliflower mosaic virus (CaMV) 35S or rd29A promoter, led to a marked increase in tolerances to freezing, water stress and salinity stress. However, the overexpression of AtCBF3 also resulted in severe growth retardation under normal growth conditions. Similar observations were also noted by Gilmour et al. (2000) in transgenic Arabidopsis plants overexpressing CBF3 (DREB1a). After growing at a normal temperature, the leaf size and overall dimensions of the CBF3-expressing plants were considerably less than those of the control plants. Additionally, delayed flowering was observed in the CBF3-expressing plants. These undesirable phenotypic changes restrict the practical applications of CBF genes in crop and ornamental plants. Cross-talk occurs between the cold-responsive and flowering time regulatory pathways in plants. CBFs, FLC and SOC1 form a feedback loop to adjust the flowering time of Arabidopsis based on temperature fluctuations. In this loop flowering is delayed by the increase in FLC at low ambient temperatures, which represses cold-inducible genes through SOC1 when floral induction occurs. The CBFs upstream and positive regulator ICE1, similarly use cold signals in the flowering pathways by directly inducing the gene encoding FLC to delay flowering (Seo et al., 2009). This was inhibited by SOC1 under floral-promoting conditions and accompanied by a reduction in freezing tolerance. Chrysanthemum (Chrysanthemum morifolium), an important ornamental species in commercial production, is a short-day (SD) plant. Therefore, their year-round cut-flower production mainly depends on the artificial regulation of day length. In addition, temperature is important for flowering. The reported optimal temperature for C. morifolium flowering is ~20 °C. Thus, when the ambient temperature is less than 18 °C, flower bud differential is severely retarded in chrysanthemum variety ‘Jimba’, even under SD (9-h light/15-h dark) conditions. In northern China, maintaining the optimal temperature for flowering during winter and early spring is a major production problem. Therefore, it is important to increase the flowering-related cold tolerance of chrysanthemum through genetic modifications. Natural variations in CBF gene sequences, gene expression levels and freezing tolerance exist in the Versailles core collection, a set of 48 Arabidopsis thaliana accessions (Mckhann et al., 2008). Notably, one accession has a stop codon at position 151 of the CBF3 protein (Condara; 190 AV), but a detailed molecular characterization has not yet been performed. In this study, we isolated a cold resistance-related AP2 transcription factor gene, AtCRAP2 (DQ415923), from the Arabidopsis accession Condara (190 AV). The overexpression of this gene in transgenic Arabidopsis conferred cold tolerance without causing dwarf phenotypes or late flowering. In addition, we introduced AtCRAP2 into the chrysanthemum variety ‘Jimba’, and the cold-inducible expression of AtCRAP2 promoted flowering under unsuitable low-temperature conditions.

2.1. Plant materials and growth conditions Arabidopsis thaliana (Condara 190 AV and Col-0 186 AV) seeds were sterilized in 70% ethanol for 1 min and then 10% NaClO for 10 min. They were further washed with distilled water and incubated on Murashige and Skoog (MS) medium containing 50 μL ml−1 kanamycin (Km) for transgene screening. To investigate the phenotypical changes of Arabidopsis, T3 seeds were first incubated in 1% agarose for 3 d at 4 °C and were then grown on a soil of mixture 1:1 (v/v) peat:vermiculite under normal conditions (22 °C, 70% relative humidity and 100 μmol m−2 s−1 illumination by fluorescent lamps) under a long-day (LD) cycle (16-h light/8-h dark) until the six-leaf stage to measure flowering time and biomass. For the cold treatment, at least 60 samples from six-leaf stage plants of each line were placed at −6 °C for 4, 6 and 8 h. The survival rates of these plants were determined after 7 d of recovery under normal growth conditions (see above). A popular cut-flower, Chrysanthemum morifolium ‘Jimba’, having white flowers, was used in this study and propagated by in vitro culturing. Chrysanthemum shoots, without leaves, including the first and second nodes, were cultured on 1/2 MS medium for 25 d under a LD cycle (16-h light/8-h dark). Then, they were transplanted into 9-cm diameter pots containing a mixture of 1:1 (v/v) peat:vermiculite and grown in a growth chamber (MLR-352H, Panasonic, Tokyo, Japan) at 22 ± 1 °C at 60% relative humidity, and 150 μmol m−2 s−1 illumination with fluorescent lamps under a LD cycle (16-h light/8-h dark). For the intermittent cold treatments, 10-d-old plants at the vegetative stage were transferred to a growth chamber at 22 °C with an SD cycle (8-h light/16-h dark) and exposed to 4 °C for the first 6 h of every light period. For the flowering under low night-temperature treatments, 20- d-old plants were transferred to a growth chamber with low night temperatures under SD conditions (8-h light at 23 °C/16-h dark at 15 °C and 70% relative humidity). Third leaves from the top of plants were harvested for PCR analysis, and the plants were photographed to record their phenotypes. Three biological replicates were used at each of the sampling points. 2.2. Phenotypic characterization of transgenic chrysanthemum The plants were considered to be entering the flowering stage when at least 50% of the ray flowers on at least one inflorescence were reflexed. The times of first visible flower buds and first flower openings were recorded. 2.2.1. AtCRAP2 gene isolation Genomic DNA was extracted from Arabidopsis accessions Col-0 (186 AV) and Condara (190 AV) using the CTAB method. The full length of AtCRAP2 was obtained using gene-specific primers (Supplementary Table S1) designed by Oligo 6.0 based on the CBF3 (At4g25480) genomic sequence The PCR product was cloned into the pGEM-T Easy Vector (Promega) and then purified for sequencing. Sequence alignments were constructed using ClustalW (http://www.ch.embnet.org/ software/ClustalW.html) and BioEdit (http://www.mbio.ncsu.edu/ BioEdit/bioedit.html). The primary structures of the AtCRAP2 protein sequences were analyzed using NCBI's Structure tool (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi). The alignment of the two protein sequences was conducted using DNAMAN 8. 2.2.2. Arabidopsis transformation To construct the overexpression vector for Arabidopsis, the open reading frame (ORF) sequences of AtCRAP2 and CBF3 were first independently cloned into the PUC18 vector containing DHA and then the sequences of the target gene, DHA, and the CaMV 35S promoter were subcloned into the pGreen0029 vector for transformation. The PCR primers are listed in Supplementary Table S1. The overexpression 221

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the edgeR package (http://www.r-project.org/) was used. We identified genes with a fold change ≥2 and a false discovery rate < 0.05 in a comparison as significant differentially expressed genes.

vectors of AtCRAP2 and CBF3 were introduced into Agrobacterium tumefaciens strain GV3101, which was then used to transform Arabidopsis by the floral dip method (Clough and Bent, 1998; Hasbún et al., 2008). Independent transformants were screened on MS basal medium containing 50 mg L−1 kanamycin (Km). The homozygotic T3 plants selected by segregation analysis were used for further analysis in this study.

2.7. Quantitative RT-PCR analysis Total RNA samples were extracted from chrysanthemum and Arabidopsis leaves using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I (Promega). First-strand cDNAs were synthesized from 1 μg total RNA using an oligo (dT) primer and the PrimeScript™ RT Master Mix system according to the manufacturer's instructions (TaKaRa). qRT-PCR reactions (20 μL volume containing 1 μL cDNA as the template) were run using the StepOne Plus Real-Time PCR System (Applied Biosystems) in standard mode with the SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa). The chrysanthemum Actin (GenBank accession number: AB205087) and Arabidopsis Actin2 (GenBank accession number: NM_112764) genes were used as internal controls. Relative transcript abundances were calculated using the 2−△△Ct method. All the reactions were performed with three independent biological replicates. PCR primers are listed in Supplementary Table S3.

2.3. Chrysanthemum transformation To construct the cold-inducible vector for chrysanthemum, the ORF sequence of AtCRAP2 and the rd29A promoter containing HindIII and BamHI were amplified and digested with corresponding restriction enzymes and inserted into the PUC18 vector. Then, sequences containing AtCRAP2 and the rd29A promoter were subcloned into the pBI121 vector for transformation. The resulting plasmids were introduced into A. tumefaciens strain EHA 105 and transformed into chrysanthemum by Agrobacteriummediated transformation (Bo et al., 2006). Leaf explants pre-cultured for 2 d were infected by Agrobacterium that had reached an OD 600 of 0.5 after 10 min. Co-cultivation was performed with somatic embryos on regeneration medium (MS + 3.0 mg L−1 2,4-dichloro phenoxyacetic acid + 3.0 mg L−1 6-benzyladenine) at 25 °C in the dark for 2 d, and then, the explants were transferred onto medium supplemented with 300 mg L−1 carbenicillin and 8 mg L−1 Km to select putatively transformed cells. The Km-resistant primary transformants were screened by PCR to confirm that the plants contained the transgene.

2.8. Electrolyte leakage Electrolyte leakage in leaves was determined according to the method of Leopold et al. (1981), with some modifications. A leaf sample from the middle position of six-leaf-stage plants of each line was taken after 4 h at −6 °C. Leaf samples (0.1 g) were placed in tubes containing 5 mL deionized water and then shaken gently for 3 h. To obtain a value for 100% electrolyte leakage, the samples were boiled for 15 min. The conductivity of the resulting solutions were measured using a conductance meter.

2.4. RNA sequencing (RNA-seq) analysis Two groups of seedlings, wild type (WT) and rd29A:AtCRAP2, were subjected to intermittent cold treatments. Three biologically replicated leaves were harvested from WT and rd29A:AtCRAP2 plants before (RT) and 10 d after the intermittent cold treatment under SD conditions (CT). Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN). The OD260/280 of the RNAs lay between 1.8 and 2.2, and the OD260/230 was > 1.8. According to the method of Singh et al. (2013), a 3-μg pool of RNA was formed by combining 1 μg from each biological replicate used for library construction. The four RNA-seq libraries were constructed as described by Ren et al. (2014).

2.9. Superoxide dismutase (SOD) activity After adding 5 mL 50 nmol (pH 7.8) to leaf samples (0.2 g), they were ground in an ice bath and centrifuged at 14,000 ×g for 20 min. The supernatant was used as a crude enzyme extract for further measurements. The soluble protein content was measured according to the method of Bradford (1976). SOD activity was measured using a spectrophotometer based on the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT). A glass containing a 3-mL reaction solution [75 μM NBT, 2 μM riboflavin, 13 μM methionine, 100 μM EDTA, 50 mM NaH2PO4/Na2HPO4 (pH 7.8)] and 50 μL crude leaf extract was illuminated at a 75 μmol m−2 s−1 light intensity. One unit of SOD was defined as the quantity of enzyme in the 3-mL reaction required to inhibit the reduction of NBT by 50%.

2.5. Transcriptome assembly and gene annotation Transcriptome sequencing was performed using an Illumina HiSeq™ 4000 by Gene Denovo Biotechnology Co. (Guangzhou, China; http:// www.genedenovo.com). The clean data were obtained by removing reads containing adapter and low quality bases, which affected the following assembly and analysis. The transcriptome de novo assembly was carried out using the short reads assembling program Trinity based on clean data of high quality. To annotate the unigenes, we used the BLASTx program (http://www.ncbi.nlm.nih.gov/BLAST/) with an Evalue threshold of 1e-5 against the NCBI non-redundant protein (Nr) database (http://www.ncbi. nlm.nih.gov), the Swiss-Prot protein database (http://www.expasy.ch/sprot), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg) and the COG/KOG database (http://www.ncbi.nlm.nih.gov/COG). Protein functional annotations were then obtained according to the best alignment results. Gene ontology (GO) functional annotations of unigenes were obtained from the Nr annotation results. GO annotations of unigenes were analyzed using Blast2GO software (Conesa and Gotz, 2005). Functional classifications of unigenes were performed using WEGO software (Ye and Fang, 2006).

2.10. Determination of the endogenous GA content After transplantation, the rd29A:AtCRAP2 and WT plants were grown under SD conditions for 10 d and then underwent an intermittent cold treatment for 3 d. Three leaves from WT, representing biological replicates, and three replicated leaf samples from each rd29A:AtCRAP2 line were collected before treatments were initiated and after 3 d of treatments for measurement. At the time of sampling, WT and rd29A:AtCRAP2 plants were at the vegetative stage. The leaf materials were powdered by grinding in the presence of liquid nitrogen. Then, 100 mg fresh weights of samples were extracted with 1.0 mL 80% methanol (v/v) at 4 °C and centrifuged (10,000 ×g at 4 °C for 20 min). As internal standards, GA1 (1.0 ng/g), GA3 (1.0 ng/g) and GA4 (1.0 ng/ g) were added to the samples after grinding. After centrifugation, the supernatants were passed through a tandem C18 SPE cartridge (Waters, MA, USA) and dried under nitrogen gas. Then, samples were dissolved in 200 μL water/acetonitrile (90/10, v/v) for analysis using a QTRAP® 5500 LC/MS/MS (AB/SCIEX).

2.6. Analysis of differentially expressed genes To identify differentially expressed genes across samples or groups, 222

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Fig. 1. Identification of the AtCRAP2 gene.

3. Results

F. Flowering times of WT and transgenic plants grown under normal conditions; G. Epidermal cells of six-leaf-stage WT and transgenic plants. 9, 20 and 22: three independent AtCRAP2-overexpression lines; 4, 5 and 7: three independent CBF3-overexpression lines. Three independent experiments were performed, and error bars indicate standard deviations. The letters above the bars indicate significant differences as assessed using Duncan's multiple range test (P < 0.05). Bars, 50 μm.

3.1. Isolation and identification of AtCRAP2 from Arabidopsis To investigate the function of the mutant CBF3 gene in Arabidopsis Condara (190 AV) (Mckhann et al., 2008), we isolated the gene from Condara (190 AV) and named it AtCRAP2 (DQ415923). We identified AtCRAP2, a cold resistance-related AP2 transcription factor and predicted a 453-bp open reading frame encoding 150 amino acids. PCRbased sequencing revealed a stop codon at position 151 of the CBF3 protein (NM_118680; Fig. 1A and B). A BLASTP algorithm-based search indicated that the highly conserved DNA-binding domain AP2/ERF of AtCRAP2 was to the same as that of AtDREB1A, but the 60-aa Cterminal region lacked an acidic domain when compared with AtDREB1A (Supplementary Fig. S1). A. Amino acid changes in the AtDREB1A gene. B. Comparison of the nucleotide and deduced amino acid sequences of AtCRAP2 with those of AtDREB1A. The arrowhead indicates the position of the single base change. An asterisk indicates the termination of translation.

3.3. Overexpression of AtCRAP2 confers cold tolerance To investigate the freezing-stress tolerance of 35S::AtCRAP2 and 35S::CBF3 transgenic plants, we subjected the six-leaf-stage seedlings to treatments of −6 °C for 4, 6 and 8 h. As shown in Fig. 3A, after the −6 °C for 4 h treatment and a 7 d recovery period, WT plants had a 34.3% survival rate, while 35S::AtCRAP2 and 35S::CBF3 plants had over 90% survival rates. After the −6 °C for 6 h treatment, the survival rate of WT plants dropped to 18.7%, while those of the other two lines were maintained at over 90%. We further treated the plants at −6 °C for 8 h, and there were still over 65% and 85% survival rates for 35S::AtCRAP2 and 35S::CBF3 plants, respectively. Thus, AtCRAP2 overexpression enhanced the low-temperature tolerance of Arabidopsis, but the enhancement was not as strong as that supplied by the expression of the CBF3 gene when exposed to −6 °C for 6–8 h, indicating that its overexpression has milder effects on plants than CBF3. Electrolyte leakage assays are usually employed to determine any increased cold or freezing tolerance imparted to the cell by the numerous biochemical changes that occur during acclimation (Gilmour et al., 2000). In this study, the relative electrolyte leakage values of WT and transgenic plants after the −6 °C treatments were measured. The 35S::CBF3 and 35S::AtCRAP2 plants had lower electrolyte leakage values and smaller reductions after treatments compared with WT and transgenic control plants. There were no significant differences between 35S::CBF3 and 35S::AtCRAP2 transgenic plants (Fig. 3B). Cold-inducible freezing tolerance is consistent with high activity levels of antioxidant enzymes. In this study, 35S::CBF3 and 35S::AtCRAP2 plants exhibited significantly higher SOD activity levels than WT plants, but no significant differences were found between 35S::CBF3 and 35S::AtCRAP2 plants (Fig. 3C). The results indicate that the freezing tolerance in 35S::CBF3 and 35S::AtCRAP2 plants is greater than that in WT. A. Representative plants exposed to −6 °C for 4, 6 and 8 h followed by a 7-d recovery period; SR, survival rate; 9, 20 and 22: three independent AtCRAP2-overexpression lines; 4, 5 and 7: three independent CBF3-overexpression lines. B. Effects of cold treatment on electrolyte leakage of wild type and transgenic plants. C. Effects of cold treatment on SOD activities of wild type and transgenic plants. In B and C, three independent experiments were performed, and error bars indicate standard deviations. The letters above the bars indicate significant differences as assessed using Duncan's multiple range test (P < 0.05). As a CRT/DRE-binding factor, CBF3 can specifically bind to the CRT/DRE sequences in the promoters of COR genes and activate their transcription (Thomashow, 1999). The overexpression of CBF3/ DREB1A can induce the expression of the downstream CRT/DRE

3.2. Overexpression of AtCRAP2 and AtCBF3 in Arabidopsis To assess the phenotypes of AtCRAP2 and AtCBF3 transgenic plants, the two genes, under the control of the CaMV 35S promoter, were independently introduced into WT Arabidopsis. We screened transgenic lines for each of the constructs using Km selection, followed by PCR. In total, 11 T0 lines of 35S:CBF3 and 14 T0 lines of 35S:AtCRAP2 were obtained. Their T3 homozygous seeds were collected for functional gene analyses (Supplementary Fig. S2). Under normal growth conditions, the 35S::AtCRAP2 lines did not present any obvious abnormalities compared with the WT plants. However, the leaf sizes, fresh weights and root lengths of the 35S::CBF3 lines were significantly different from the other two lines. The biomasses of WT and transgenic plants at the 12-leaf stage were evaluated, and the fresh weights of 35S::CBF3 plants were significantly less than those of WT and 35S::AtCRAP2 plants (Fig. 2A and B). The root lengths of 35S::CBF3 lines were shorter compared with WT and 35S::AtCRAP2 plants at the four-leaf stage, while there was no difference in root length between WT and 35S::AtCRAP2 plants (Fig. 2C and D). Microscopic observations indicated that the epidermal cell size in 35S::CBF3 plants was much smaller than in 35S::AtCRAP2 and WT (Fig. 2G). Consistent with these differences, the overexpression of AtCBF3 in transgenic Arabidopsis lead to growth retardation and slower growth compared with WT plants under normal growth conditions, which did not occur in 35S::AtCRAP2 lines. In addition, the flowering times of the 35S::CBF3 lines were delayed for ~12 d compared with 35S::AtCRAP2 and WT plants (Fig. 2E and F). Unlike AtCBF3, the overexpression AtCRAP2 in transgenic plants did not cause detectable growth retardation or delay flowering in Arabidopsis. A. Phenotypic changes of transgenic Arabidopsis plants (T3); B. Fresh weights of 12-leaf-stage WT and transgenic plants under normal growth conditions; C, D. Root lengths of four-leaf-stage WT and transgenic plants; E. Growth and development of WT and transgenic plants; 223

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Fig. 2. Different phenotypes were observed among wild type (WT) and transgenic plants.

Fig. 3. Freezing tolerance of the 35S::CBF3 and 35::AtCRAP2 plants. 224

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conditions. Three independent experiments were performed, and error bars indicate standard deviations. Asterisks indicate significant differences according to Student's t-test (*P < 0.01).

contained in COR target genes (Gilmour et al., 2000; Yamaguchi-shinozaki et al., 1999). To investigate whether the AtCRAP2 transcription factor could also induce the expression of downstream COR genes, we analyzed the expression levels of RD29A, COR15A and KINII (Supplementary Fig. S3). Under normal conditions, the expression levels of RD29A, COR15A and KINII were slightly induced in 35S::CBF3 and 35S::AtCRAP2 plants. After cold treatments, the RD29A, COR15A and KINII transcript levels were substantially induced both in 35S::CBF3 and 35S::AtCRAP2 plants, with no significant differences between the two types of transgenic plants. Thus, we concluded that AtCRAP2 had a similar role as the CBF3 gene in modulating the expression of COR genes to control plant responses to low temperatures.

3.5. RNA-seq analysis of AtCRAP2 and flowering-related genes at the ambient temperature To examine the expression patterns of flowering-related genes in WT and transgenic ‘Jimba’ plants during the intermittent cold (daily for 6 h after exposure to light) treatment, we performed an RNA-seq analysis of WT and transgenic plants at vegetative stages before and after 10 d of SD conditions with intermittent cold treatments. Four cDNA libraries were assembled (WT-RT, WT-CT, T-RT and T-CT) and sequenced using Illumina HiSeq™ 4000. The total number of raw reads obtained from the WT-RT, WT-CT, T-RT and T-CT libraries were 74,255,970, 85,765,528, 77,259,516 and 65,777,124, respectively. After data cleaning and the removal of invalid reads, we obtained 297 × 106 clean reads (44.31 × 109 nt). The raw sequence reads were deposited into the National Center for Biotechnology Information SRA database under the accession number SRP189692. The average Q20 and GC percentages were96.68% and 43.82%, respectively (Supplementary Table S2). After assembly, 148,356 non-redundant unigenes were identified; these had an average length of 683 nt and were associated with an N50 (genome splicing quality) of 1073 nt. To acquire expression and functional annotations of the assembled unigenes, the assembled Unigene sequences were aligned against the Nr, Swiss-Prot, COG/KOG and KEGG protein databases. Of the 148,357 unigenes, 65,871 were successfully matched to homologous sequences in at least one of the searched databases. Among them, 59,974, 49,747, 41,371 and 25,570 unigenes were found in Nr, Swiss-Prot, COG/KOG and KEGG, respectively. The Nr database produced the largest number of annotations (Supplementary Table S3). Compared with other species, Jimba showed the most matches to Vitis vinifera (4,231), followed by Sesamum indicum (3,574) and Theobroma cacao (2,752) (Supplementary Fig. S5A). A COG analysis divided the unigenes into 25 categories (Supplementary Fig. S5B). A GO analysis identified the unigenes as being involved in 21 biological processes, 16 cellular components and 12 molecular functions (Supplementary Fig. S5C). Based on the annotations, we selected several flowering-associated genes involved in mediating the cold-stress responses. These genes play vital roles in the photoperiod pathway, GA biosynthesis, GA signaling,

3.4. Cold-inducible expression of AtCRAP2 enhances the low-temperature tolerance of flowering in ‘Jimba’ To improve low-temperature tolerance of flower bud formation in ‘Jimba’, transgenic experiments were performed. AtCRAP2 under the control of the cold-inducible promoter of rd29A was transformed into ‘Jimba’. In total, 11 putative transgenic plants were obtained through Km-resistance screening. However, only four transgenic lines were confirmed by PCR (Supplementary Fig. S4). Long-term cold can promote flowering in plants that are sensitive to vernalization through the vernalization pathway, while the short-term cold-induced overexpression of cold-responsive genes leads to flowering delay by activating FLC. ‘Jimba’ is unable to flower when the ambient temperature is lower than 18 °C. To investigate the effects of the expression AtCRAP2 on the flowering of ‘Jimba’, WT and rd29A:AtCRAP2 transgenic plants were grown under LD conditions at room temperature for 25 d and then transferred to SD conditions with a 15 °C nighttime temperature during the dark period. The plants' development was surveyed. Visible capitula were observed among rd29A:AtCRAP2 plants after 20 d of 15 °C nighttime temperatures under SD conditions, while the WT plants did not show any signs of capitulum development until 50 d (Fig. 4A and B), which indicated that AtCRAP2 enhanced the low-temperature tolerance of flowering in ‘Jimba’. A. Phenotypes of wild type (WT) and transgenic plants grown at 15 °C during the night under SD conditions were recorded and photographed at different time points (d after treatments). #5 and #7: two independent AtCRAP2 transgenic lines. B. Flower bud emergence time in WT and transgenic plants grown at 15 °C during the night under SD

Fig. 4. Flowering time of rd29A:AtCRAP2 ‘Jimba’ plants exposed to 15 °C night temperatures and SD conditions. 225

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Table 1 Transcripts related to flowering time-related genes detected in chrysanthemum ‘Jimba’ wild type and transgenic plants subjected to intermittent cold treatments under short-day conditions. Gene

Annotation

GA biosynthesis and signaling Unigene0050713 Gibberellin 20-oxidase Unigene0020889 Gibberellin 3-oxidase 1 Unigene0051240 Gibberellin 2-oxidase1 Flowering integrators Unigene0009677 CmFTL3 Unigene0058963 FLC Unigene0047732 SOC1 Photoperiod pathway Unigene0042085 CmTFL1 family protein Unigene0069020 constans-like protein Unigene0056963 gigantea-like DREB1 Unigene0009562 AtCRAP2

WILD TYPE

rd29A:AtCRAP2

P-value

WT-RT

WT-CT

Log2 Ratio (WT-CT/WT-RT)

P-value

T-RT

T-CT

Log2 Ratio (T-CT/T-RT)

1.88 1.54 1.63

0.01 0.12 6.16

−10.88 −3.66 1.92

8.88E-53 1.67E-16 1.02E-40

7.46 0.46 1.04

0.61 0.08 4.74

−3.61 −2.59 2.20

1.09E-121 1.55E-03 1.03E-35

2.91 0.47 25.32

14.22 2.09 11.52

2.29 2.16 −1.14

1.83E-243 9.26E-06 1.39E-98

3.72 0.35 18.49

11.67 2.67 19.59

1.65 2.94 0.08

2.36E-123 6.05E-09 0.13

15.09 93.71 4.91

0.17 609.46 106.10

−6.47 2.70 4.43

4.84E-190 0.00E+00 0.00E+00

11.97 144.39 5.18

0.32 664.39 126.37

−5.23 2.20 4.61

2.20E-120 0.00E+00 0.00E+00

0.00

0.00

CNC

0.00E+00

2.13

25.57

3.59

0.00E+00

CNC indicates values that could not be calculated. Data in columns are RPKM values. WT, wild type; T, transgenic plants; RT, plants grown at room temperature; CT, plants after intermittent cold treatments. Bold letters indicate differentially regulated transcripts between WT and transgenic plants. Significant differences were determined at P < 0.05.

and other flowering-related processes (Table 1). CO and GIGANTEA (GI) encode proteins involved in the photoperiod pathway, and their expression levels were up-regulated after the SD treatment in WT and rd29A:AtCRAP2 plants. GA3-oxidase (ox) and GA20-ox are crucial in the GA-synthesis pathway, while GA2-oxes deactivate bioactive GAs. After low temperature treatments, GA20ox expression was downregulated in both WT and rd29A:AtCRAP2 plants, while GA2ox was upregulated. As a result, the endogenous GA concentration decreased in WT and rd29A:AtCRAP2 plants after the intermittent cold treatment under SD conditions, and the GA level in rd29A:AtCRAP2 plants was slightly lower than in WT plants before and after the cold treatment (Supplementary Fig. S6). For the flowering pathway integrators FT and SOC1, the expression level change of FT was not consistent with those of CO and GI, which were higher in WT and rd29A:AtCRAP2 plants after treatment. In contrast, SOC1 expression was down-regulated by 1.14fold in WT plants, but up-regulated by 0.08-fold in transgenic plants. In addition, no AtCRAP2 transcripts were detected in WT plants, while AtCRAP2 expression was significantly up-regulated in rd29A:AtCRAP2 plants. These results further validated the expression of AtCRAP2 in rd29A:AtCRAP2 plants and suggested the positive influence of AtCRAP2 on SOC1. To validate the data from the RNA-seq digital expression analysis, we performed qRT-PCR assays of seven flowering-associated genes. The trends of gene expression changes detected by the two different approaches were generally consistent (Supplementary Fig. S7A), resulting in a significantly high correlation (R2 = 0.8323) (Supplementary Fig. S7B) that indicated the good consistency between the two analysis techniques.

under the control of the rd29A promoter. A. Comparison of the expression of AtCRAP2 in rd29A:AtCRAP2 and wild type (WT) chrysanthemum ‘Jimba’ plants. The 30-d-old seedlings were transferred to a growth chamber at 4 °C for 6 h under SD conditions. Leaves harvested at Zeitgeber Time 6 were used for RNA isolation. The expression levels of AtCRAP2 were assessed using qRT-PCR. For qRT-PCR assays, the mean was calculated from three biological replicates. Error bars indicate standard deviations. The letters above the bars indicate significant differences as assessed by Duncan's multiple range test (P < 0.05). B. Daily rhythms of AtCRAP2 in ‘Jimba’ under SD conditions with or without cold treatments. Leaves of 30-d-old seedlings grown at normal conditions or 4 °C for 6 h under SD conditions were harvested for RNA isolation every 3 h for 1 d, and three replicate samples were taken. The expression level of AtCRAP2 was assessed using qRT-PCR. For the qRT-PCR assay, the means were calculated from three biological replicates of each line. Open and filled horizontal bars represent non-cold and cold, respectively. Grey vertical areas represent dark periods. Error bars indicate standard deviations. To determine the effects of the low temperature on flowering timerelated genes, we further analyzed the expression levels of these genes in WT and rd29A::AtCRAP2 plants. After being exposed to 4 °C for 6 h, the expression of the flowering integrator SOC1 was significantly upregulated in transgenic plants compared with in WT plants. COL, GI, FTL3, FLC and GA2ox showed similar expression patterns in both WT and rd29A:AtCRAP2 plants. They were all clearly upregulated after the cold treatment and had higher expression levels in rd29A:AtCRAP2 plants than in WT plants (Fig. 6A and B). The expression level of FLC in rd29A:AtCRAP2 was higher than in WT plants after treatments, which suggested that cold stress and the expression of AtCRAP2 increased the transcript level of FLC. This was in agreement with the CBFs’ positive regulation of FLC expression (Seo et al., 2009). In contrast, the expression of AtCRAP2 and the cold stress repressed GA20ox and GA3ox expression levels. GI, a circadian clock gene, which is involved in mediating cold-stress responses, increased in WT and rd29A:AtCRAP2 plants after the cold treatment. The expression patterns of FLC, GA20ox, GA3ox and GA2ox under the low-temperature conditions were in agreement with previous studies in Arabidopsis, and the expression levels of GI, COL, FTL3 and SOC1 exhibited a sensitivity to the cold. SOC1 showed different patterns between WT and transgenic plants under cold and non-cold conditions. Intermittent cold treatments cause flowering delays by upregulating FLC (Kim et al., 2004). To investigate the expression patterns of flowering time-related genes, we examined their expression patterns in WT

3.6. qRT-PCR assays of flowering time-related genes and AtCRAP2 at the ambient temperature To determine whether the expression of AtCRAP2 controlled by rd29A was subjected to circadian regulation, we examined its diurnal expression in ‘Jimba’ under 23 °C and SD conditions. rd29A induced AtCRAP2's consistent expression in plants at warm temperatures, and the expression peaked at ZT9 (Zeitgeber time 9 from light), but decreased after ZT12. When exposed to cold (4 °C) for 6 h, the highest expression peak was observed at ZT6, which was consistent with the time of the cold treatment and was significantly higher than without the cold treatment. Then, it declined at ZT12. (Fig. 5A and B). Thus, the rd29A-promoted AtCRAP2 expression was under the regulation of a circadian clock, and the expression of AtCRAP2 was cold sensitive 226

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Fig. 5. Relative expression of AtCRAP2 transcripts.

flower bud differentiation in rd29A:AtCRAP2 plants under low-night temperatures, we examined the expression levels of key genes involved in the flowering pathway before and after flower bud formation under 15 °C nighttime conditions (Fig. 7B). The qRT-PCR results showed that during the SD and low-temperature period the expression levels of FTL3 and SOC1 exhibited significant upregulations in rd29A:AtCRAP2 plants compared with in WT plants. GI, CO and FLC expression levels were elevated before flower buds emerged and then decreased, and GI and COL expression levels were at the same relatively high levels in rd29A:AtCRAP2 plants compared with WT plants. TERMINAL FLOWER 1, the floral repressor, was downregulated in rd29A:AtCRAP2 plants after flower bud emergence and the WT plants did not flower, indicating that the repressor was not upregulated after bud emergence but was upregulated after 15 d. The expression pattern of the GA biosynthetic gene GA3ox was similar in rd29A:AtCRAP2 transgenic and WT plants. GA2ox expression was upregulated in transgenic plants but downregulated in WT plants. These results were consistent with the flower bud emergence of WT and rd29A:AtCRAP2 plants exposed to 15 °C during the nighttime under SD conditions. Thus, the data further demonstrated that AtCRAP2 increased the low-temperature tolerance of flowering initiation in rd29A:AtCRAP2 and indicated that there are correlations with SOC1 and FTL3.

and rd29A:AtCRAP2 plants under intermittent cold conditions (Fig. 7A). The AtCRAP2 expression level was highest after 2 d of the intermittent cold treatment and then decreased. Similar expression patterns were observed for COL, GI and FLC genes in WT and rd29A:AtCRAP2 plants, indicating that the flowering time-related genes were sensitive to the cold. The expression of GA20ox was suppressed after 2 d of the intermittent cold treatment in transgenic plants and increased from 3 d. It was negatively regulated by AtCRAP2. Interestingly, of these genes, only the expression levels of SOC1, FTL3 and GA2ox exhibited were significantly higher in rd29A:AtCRAP2 than in WT plants. Based on data from the RNA-seq (Table 1) and flowering time-related gene expression levels after a 6-h cold treatment (Fig. 6), we hypothesized that SOC1 and FTL3 play positive roles in promoting flowering in transgenic plants under low-temperature conditions. The optimal flowering temperature for C. morifolium is ~20 °C. For ‘Jimba’, when the ambient temperature is lower than 18 °C, flower bud differentiation is severely retarded, even under SD conditions. At a 15 °C nighttime temperature, flower buds cannot form. To assess whether rd29A:AtCRAP2 plants could form flower buds under low-night temperatures, we treated rd29A:AtCRAP2 and WT plants under 15 °C night temperature and SD conditions. After 20 d of treatment, flower buds were first observed on the rd29A:AtCRAP2 plants but not on the WT (Fig. 4). To identify the potential causative genes responsible for

Fig. 6. Comparisons of expression profiles of flowering time-related genes in rd29A:AtCRAP2 and wild type (WT) chrysanthemum ‘Jimba’ plants as measured by RNA-seq and qRT-PCR. A. Heatmap showing the expression profiles of flowering time-related genes (RPKM). T, transgenic plants; RT, plants grown at room temperature; CT, plants after intermittent cold treatments. B. Comparison of the expression profiles of flowering time-related genes in rd29A:AtCRAP2 and WT chrysanthemum ‘Jimba’ plants under cold conditions. The 30-d-old seedlings were transferred to a growth chamber at 4 °C for 6 h under SD conditions. Leaves harvested at Zeitgeber Time 6 were used for RNA isolation, and three replicate samples were taken. The expression levels of flowering time-related genes were assessed using qRT-PCR. For the qRT-PCR assay, the means were calculated from three biological replicates. Correlations between RT-PCR and RNA-seq expression levels were calculated and their associated coefficient values are indicated. Error bars indicate standard deviations. The letters above the bars indicate significant differences as assessed by Duncan's multiple range test (P < 0.05). 227

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Fig. 7. Expression profiles of AtCRAP2 and flowering time-related genes in rd29A:AtCRAP2 and wild type (WT) chrysanthemum ‘Jimba’ plants exposed to intermittent cold or low-night temperature treatments and SD conditions. A. Expression analysis of flowering time-related genes in rd29A:AtCRAP2 and WT chrysanthemum ‘Jimba’ plants under intermittent cold treatments and SD conditions as determined by qRT-PCR. Intermittent cold treatments lasted daily for 6 h after exposure to light. Samples were collected at Zeitgeber Time 6 at 3-d intervals, and three replicate samples were taken. For the qRT-PCR assay, the means were calculated from three biological replicates. Error bars indicate standard deviations. B. Expression analysis of flowering time-related genes in rd29A:AtCRAP2 and WT chrysanthemum ‘Jimba’ plants under low-night temperature and SD conditions as determined by qRT-PCR. Leaves were harvest from WT and transgenic plants grown at 12 °C during the nighttime under SD conditions, and three replicate samples were taken. For the qRT-PCR assay, the means were calculated from three biological replicates. Error bars indicate standard deviations.

4. Discussion

of the C-terminal region are also conserved in diverse plant species, which indicates their important functional roles. All of the DREB/CBF genes possess a carboxyl terminal acidic region. This region contains a transcriptional activator motif (Stockinger et al., 1997). In the present study, a bioinformatics analysis showed that the AtCRAP2 and CBF3 proteins have core functional AP2 domains, but the domain in AtCRAP2 is one helix shorter at the C-terminal region than the domain in CBF3. The overexpression of AtCRAP2 and CBF3 in transgenic Arabidopsis significantly increased the plants’ freezing tolerance, but plants expressing AtCRAP2 are not as robust as those expressing CBF3 when exposed to −6 °C for 8 h. These results were in agreement with a previous study, in which Col-0 (186AV) accessions expressing CBF3 showed higher degrees of freezing tolerance than Condara (190AV) (Mckhann et al., 2008). The expression of both genes increased the expression levels of upregulated downstream COR genes, including RD29A, COR15A and KINII, which indicated that their core functions of binding DRE elements had not changed. No significant morphological changes were observed in transgenic plants overexpressing AtCRAP2 compared with WT. We hypothesized that there is a trade-off between increasing the level of abiotic stress tolerance and the associated negative effects on plants. The defect in the C-terminal region of AtCRAP2

4.1. Overexpression of AtCRAP2 in Arabidopsis increased cold tolerance without growth retardation or delayed flowering The negative effects of DREB/CBF overexpression on transgenic plant development have limited the application of DREB genes in genetic engineering. In the majority of studies, constitutive DREB overexpression affected plant morphology and reproduction. For example, the expression of DREB1A/CBF3 controlled by the strong constitutive CaMV 35S promoter in transgenic Arabidopsis plants enhances tolerance to freezing temperatures compared with WT plants (Liu et al., 1998). However, severe growth retardation was observed in the mature transgenic plants. In addition, delayed flowering is also a common phenomenon in transgenic Arabidopsis that are overexpressing DREB1 genes (Liu et al., 1998). In the present work, the overexpression of CBF3 in transgenic Arabidopsis led to severe growth retardation and delayed flowering compared with WT and 35S::AtCRAP2 lines. The conserved AP2 domain in DREB/CBF genes plays a central role in increasing cold tolerance by recognizing and binding the DRE elements. The DSAW motif at the end of the AP2/ERF domain and the LWSY motif at the end 228

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GA is biosynthesized by the activities of GA20-oxes and GA3-oxes but reduced by an accumulation of GA2oxes. The activation of the GA2ox gene when the DREB/CBF1-type transcription factor was overexpressed in transgenic lines negatively affects GA synthesis, leading to the dwarf phenotype (Achard et al., 2008). The overexpression of GA2ox7 significantly decreased GA levels (Zhou et al., 2017). GA's influence on flowering time depends on day length. In Arabidopsis, endogenous GA acts on FT signaling under LD conditions, and GA acts as a second floral signal (Tamotsu and King, 2008). In chrysanthemum, the GA pathway plays a subsidiary role in regulating floral induction under SD conditions, but plays a predominant role in flowering under LD conditions, operating through SOC1 and LEAFY (Dong et al., 2017). The suppression of Cm-BBX24 significantly increases the endogenous GA concentration and accelerates flowering by activating CmFTL3 (Yang et al., 2014). In this study, the transcriptomic and qRT-PCR data showed that CmGA20ox was downregulated after the cold treatment in WT and rd29A:AtCRAP2 plants. Similarly, with intermittent cold and low night-temperature treatments, the expression level of CmGA20ox was down-regulated in the rd29A:AtCRAP2 transgenic lines compared with in WT plants. However, the expression level of CmGA2ox was much higher in the rd29A:AtCRAP2 transgenic lines than in WT plants, indicating that the increase in AtCRAP2 repressed GA accumulation. As a result, the endogenous GA concentration was significantly reduced in WT and rd29A:AtCRAP2 plants after the intermittent cold treatment under SD conditions, and the GA level in rd29A:AtCRAP2 plants was slightly lower than in WT plants before and after the cold treatment (Supplementary Fig. S7), which suggested that the low temperature resulted in retarded plant growth. Thus, we hypothesize that the GA level's changes in WT and rd29A:AtCRAP2 plants did not contribute to the latter's flowering under the low night-temperature conditions. When coupled with the lack of delayed flowering in Arabidopsis overexpressing AtCRAP2, these results led us to hypothesize that the defect in AtCRAP2 relative to CBF3 affected its ability to participate in the flowering pathway and weakened the protection against premature flowering that could result from harmful environmental conditions. This benefits the application of AtCRAP2 genes in crop improvement.

compared with the CBF3 protein may be responsible for the attenuation of the negative effects of DREB/CBF overexpression on plant development. 4.2. Cold-induced expression of AtCRAP2 enhanced the low-temperature tolerance of flower bud differentiation in ‘Jimba’ The overexpression of AtDREB1A under the rd29A promoter can increase drought and freezing tolerance levels in transgenic Arabidopsis and tobacco plants without retarding growth (Kasuga et al., 2004). The overexpression of DREB1C controlled by the CaMV 35S promoter in Medicago truncatula enhanced the freezing tolerance but led to the retardation of shoot growth. In contrast, the same gene driven by the rd29A promoter in China rose (Rosa chinensis Jacq.) did not produce a negative phenotype under optimal growth conditions and enhanced freezing tolerance under stress conditions (Chen et al., 2010). The constitutive expression of DREB2 from Sorghum beticola, driven by the CaMV 35S promoter, in transgenic rice plants limits plant growth and yield. However, the use of the rd29A promoter improved the transgenic rice plants' drought tolerance without reducing seed set (Bihani et al., 2011). Moreover, in chrysanthemum (Bo et al., 2006) transformed with DREB1 genes driven by the rd29A promoter, plants are more tolerant to abiotic stresses than those in which the gene is driven by the CaMV 35S promoter. In the present study, we transformed AtCRAP2, driven by the rd29A promoter, into chrysanthemum ‘Jimba’. The RNA-seq and RTPCR analyses showed that AtCRAP2 was expressed under non-cold conditions in rd29A:AtCRAP2 plant lines and exhibited diurnal oscillation. Under low-temperature stress, the expression of AtCRAP2 was elevated in rd29A:AtCRAP2 plants (Fig. 5). Notably, the inducible rd29A promoter responded to cold and conferred the maximum freezing tolerance. As a floral promoter, SOC1 integrates signals from the floweringrelated pathways, photoperiod, vernalization, GA biosynthesis and aging. SOC1 and FLC are the key regulators of crosstalk between cold response and flowering-time regulation. During intermittent cold treatments, SOC1 expression and flowering are repressed (Seo et al., 2009). SOC1 also acts as a transcriptional repressor of the CBF genes, which positively regulate FLC expression, and then, FLC represses flowering pathway integrators to delay flowering (Seo et al., 2009). Here, with the intermittent cold (4 °C or 15 °C at night) treatments under SD conditions, the SOC1 expression was higher in rd29:AtCRAP2 transgenic plants than in WT plants (Fig. 7A and B). Similarly, FLC exhibited an increasing tendency in rd29A:AtCRAP2 plants and WT plants, which suggested it was positively regulated by the cold. Unlike a previous study in which CBF3 was overexpressed in Arabidopsis, in this study, the overexpression of AtCRAP2 in Arabidopsis and the cold-inducible expression of AtCRAP2 in ‘Jimba’ did not result in delayed flowering compared with WT plants. We also observed several other genes involved in the flowering pathway that were affected by the intermittent cold-sensing pathway in chrysanthemum. In chrysanthemum, FTL3 is a common product of day length- (Oda et al., 2012) and temperature- (Nakano et al., 2013) sensing pathways, and it causes chrysanthemums to flower in autumn. Under 15 °C nighttime and SD conditions, the CmFTL3 transcripts significantly accumulated during the flower-transition process, and it promoted flowering in rd29A:AtCRAP2 plants. In contrast, the lownight temperature caused a severed repression of CmFTL3 in WT plants, which led to flowering retardation. GI, the photoperiodic flowering time-related gene, promotes flowering through the photoperiod and circadian pathways, and is involved in responses to cold in Arabidopsis. The expression of GI increases five-to eight-fold in cold-treated Arabidopsis plants (Fowler and Thomashow, 2002). In the present study, the flower bud initiation of the rd29A:AtCRAP2 transgenic chrysanthemum exposed to 15 °C at night was consistent with the upregulated expression of GI. However, whether a correlation exists between AtCRAP2 and GI in chrysanthemum requires further investigation.

5. Conclusions In this study, we identified the AtCRAP2 gene and showed that its overexpression in transgenic Arabidopsis conferred cold tolerance. In comparison with 35S:CBF3-containing plants, the 35S:AtCRAP2-containing plants did not undergo growth retardation or delayed flowering. In addition, we demonstrated that AtCRAP2 under the control of a coldinducible promoter in transgenic ‘Jimba’ can enhance flower bud formation-related low-temperature tolerance by positively regulating the expression levels of SOC1 and FTL3. AtCRAP2 will be useful in generating chrysanthemum germplasms that are more tolerant to unsuitable low-temperatures that negatively affect flowering during yearround cut-flower production.

Funding This research was mainly supported by The Project of Science and Technology of Beijing Academy of Agriculture and Forestry Sciences (KJCX20170108, KJCX20170203 and JNKST201610) and the Project of Beijing Municipal Science & Technology Commission (D161100001916004).

Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. 229

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Author contributions

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