Overexpression of the CpCOR413PM1 Gene from Wintersweet ( Chimonanthus praecox ) Enhances Cold and Drought Tolerance in Arabidopsis

: Wintersweet ( Chimonanthus praecox (L.) Link) is a commercial significance woody plant. As a rare winter-blooming plant, C. praecox is widely distributed and highly adaptable to various climates, especially low temperatures. In this study, we identified the COR413 plasma membrane gene CpCOR413PM1 in wintersweet. CpCOR413PM1 was expressed in all tissues of the plant, with the highest expression observed in the root and floral parts. Cultivation under 4 ◦ C and with treatment of ABA led to the up-regulated expression of CpCOR413PM1 . The expression of recombinant CpCOR413PM1 protein in Escherichia coli resulted in the tissues’ increased resilience to cold and drought stress. In vitro enzyme activity assays confirmed the protective impact of the CpCOR413PM1 protein on superoxide dismutase under low-temperature stress. Furthermore, the overexpression of CpCOR413PM1 in Arabidopsis thaliana resulted in increased cold and drought tolerance and ABA inhibited transgenic Arabidopsis seed germination. The CpCOR413PM1 gene promoter can influence expression of the GUS reporter gene under conditions of 4 ◦ C, 42 ◦ C and abscisic acid. Overall, our study demonstrates that CpCOR413PM1 plays a significate role in cold and drought stress. Our findings strengthen the knowledge of the molecular mechanisms underlying wintersweet’s tolerance to stress and lay the groundwork for the future investigation of the functions of the COR gene family.


Introduction
Wintersweet (Chimonanthus praecox (L.) Link) is a winter-flowering, woody ornamental plant in China and is also an important industrial crop.As a rare winter-blooming plant, wintersweet is highly adaptable to low-temperature climates owing to the presence of cold-resistance genes, such as cold-regulated (COR) genes.COR413 is a family of lowtemperature responsive genes unique to plants.The COR413 protein is classified into three forms based on its localization in cells: COR413PM in the plasma membrane, COR413TM in the inner capsule membrane, and COR413IM in the chloroplast inner membrane [1].
In this study, we have identified CpCOR413PM1 as a member of the COR413 family found in wintersweet.The expression pattern of CpCOR413PM1 was analyzed under various organs and different abiotic stresses.Furthermore, the cold and drought stress resistance of Escherichia coli overexpressing CpCOR413PM1 was analyzed.CpCOR413PM1 protein can protect superoxide dismutase (SOD) during low temperatures and drought stresses.Transgenic Arabidopsis expressing CpCOR413PM1 was evaluated under lowtemperature and drought stress conditions.Furthermore, the upstream promoter fragment of CpCOR413PM1 was cloned with the aim of exploring the function and mechanism of this gene contributing to the acquisition of cold and drought resistance.Overall, our findings enhance our understanding of CpCOR413PM1 regarding the cold and drought stress resistance of wintersweet.

RNA Extraction and qRT-PCR Analysis
To isolate total RNA, Trizol (Invitrogen, Carlsbad, CA, USA) was used.For cDNAs synthesis, a Primescript RT reagent kit (Takara, Dalian, China) was used.Bio-Rad equipment with the Ssofast EvaGreen Supermix (BIO-RAD, Hercules, CA, USA) was used for real-time quantitative polymerase chain reaction (qRT-PCR).CpActin and CpTublin were chosen as the reference genes for wintersweet [11].AtActin was chosen as the reference gene for Arabidopsis [12].The PCR program comprised preheating at 95 • C for 30 s, followed by 40 cycles of denaturation at 95 • C for 5 s, annealing at 60 • C for 5 s, and extension at 72 • C for 5 s.There were three biological replicates for each experiment and three technical replicates for each sample.The PubMed online website (https://pubmed.ncbi.nlm.nih.gov/,(accessed on 2 October 2021)) was used to design qRT-PCR primers specific to the target Cpcor413pm1 gene and the internal reference gene, and the most appropriate primer pairs were chosen from the primer sequences provided on the website based on the target.

Protein Expression, Purification, and Concentration
The pET32(a)+-CpCOR413PM1 plasmids were transformed into E. coli BL21 (DE3) (ShangHai WEIDI Biotechnology Co., Ltd., Shanghai, China) for expression.Bacterial cultures were grown in Luria-Bertani (LB) medium (10 g L −1 tryptone, 10 g L −1 NaCl, and 5 g L −1 yeast extract) containing ampicillin (Amp, 50 mg L −1 ) at 37 • C in an orbital shaker at 180 rpm until the density at 600 nm reached 0.6.The cells were then induced with 1.0 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) at 37 • C for 6 h.The fusion proteins were purified using the His-tag protein purification kit (P2226, Beyotime Biotechnology, Shanghai, China), according to the manufacturer's instructions.The recombinant protein was purified and concentrated by an ultrafiltration centrifuge tube (Millipore, Billerica, MA, USA).The PAGE Gel Rapid Preparation Kit (Shanghai Epizyme Biomedical Technology Co., Ltd., Shanghai, China) was used to prepare protein electrophoresis.

Resistance
Analysis of E. coli E. coli BL21 (DE3)-containing pET32a(+) and pET32a(+)-CpCOR413PM1 plasmids were subjected to low-temperature stress treatment at 4 • C and drought stress.The bacterial fluids were inoculated at a 1:100 (v v −1 ) ratio into 100 mL of LB liquid medium containing Amp (50 mg L −1 ), spread onto LB plates to observe the survival rate, and incubated in a 37 • C oven.Every 1 h, 3 mL of bacterial liquid was sampled to determine the OD 600 value, and statistics were recorded.In order to simulate drought stress, the bacterial solution was diluted 100 times, and 10 µL was aspirated and distributed onto both regular LB and 15% PEG6000 plates, respectively.The experiment was carried out three times.

Plant Material and Growth Conditions
Arabidopsis ecotype Columbia-0 (Col-0) was used for genetic transformation.All Arabidopsis plants were grown at 25 ± 1 • C under a 16 h/8 h (light/dark, respectively) photoperiod with 55 µmol•m −2 •s −1 fluorescent illumination.Murashige and Skoog (MS) medium supplemented with kanamycin (100 mg L −1 ) was used to screen for transformants.Individual, adult wintersweet plants were planted in a nursery at Southwest University (Chongqing, China).The 'Suxin' wintersweet cultivar was used, whose seeds were collected from the campus of Southwest University and sown in the Floriculture Laboratory at Southwest University, China.
The plasmids were transformed into Arabidopsis (Col-0) using the floral-dip method [13] mediated by Agrobacterium tumefaciens strain GV3101 cells.The transgenic plants were screened on 1/2 strength MS medium containing 50 mg L −1 of kanamycin or 25 mg L −1 of hygromycin B as selective agents.

Subcellular Localization of CpCOR413PM1
The recombinant plasmid 35S::CpCOR413PM1 green fluorescent protein (GFP) and the empty vector 35S::GFP were separately introduced in the A. tumefaciens strain GV3101.The young leaves of tobacco (Nicotiana benthamiana) were injected with A. tumefaciens containing35S::GFP and 35S::CpCOR413PM1-GFP. LTI6b red fluorescent protein (RFP) was used as a reporter gene for plasma membrane localization [14].Confocal laser microscopy (Carl Zeiss, Jena, Germany) was used to observe GFP/RFP fluorescence.

Stress Experimental Conditions
Select consistent and healthy four-leaf-stage wintersweet seedlings for the experiment.Place the seedlings in a growth chamber set at 4 • C and spray the other seedlings with 50 µM of abscisic acid (ABA, until all leaves are covered with water droplets).Each treatment will have five time points: samples will be taken at 0 h as the control, then at 2 h, 6 h, 12 h, and 24 h after treatment.Three biological replicates will be included for each treatment.Seeds of wild-type (WT) and the third generation (T 3 ) of CpCOR413PM1 transgenic Arabidopsis were surface-sterilized and germinated on 1/2 MS medium for 7 d in an illuminated incubation chamber at 25 • C, with a 16 h/8 h (light/dark, respectively) cycle and relative humidity of 70%.The seedlings were then transplanted into rectangular flowerpots filled with a mixture of soil and sand (1:1).For the freezing tolerance assay, 6-week-old WT and transgenic Arabidopsis seedlings were treated at 4 • C for 12 h.Plants were moved to room temperature (22 • C) and allowed to recover for 3 d.For the drought tolerance assay, WT and transgenic Arabidopsis plants were grown without watering for 10 d and then re-watered for recovery.A total of 6 plants were used in each biological replicate and 3 analytical replicates were there for the biochemical assessment.
The CpCOR413PM1pro transgenic Arabidopsis were treated with 50 µM of ABA, 1-Aminocyclopropanecarboxylic Acid (ACC), 4 • C, and 42 • C, and treated with water as the control.Samples were taken for qRT-PCR analysis after 6 h.

Measurement of Physiological Indicators
To assess the effect of CpCOR413PM1 on the physiological indicators of cold stress, rosette leaves were sampled from the transgenic and WT plants (each sample comprised 0.1 g).The malondialdehyde (MDA) content was estimated using the procedure described by Heath and Packer [15].The total chlorophyll content was extracted by ethanol and acetone extraction (1:1) [16], and total chlorophyll levels were quantified by the absorbance value measured with a Varioskan Flash multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 663 nm and 646 nm.SOD activity was assayed according to the method of Beauchamp and Fridovich [17].Each measurement was replicated three times.

Cloning and Analysis of the Upstream Sequence of CpCOR413PM1
Genomic DNA was extracted from the leaves of young wintersweet seedlings using the cetyltrimethylammonium bromide method [18].The leaves were mechanically broken, and the DNA was dissolved by adding a CTAB separation buffer.The protein was removed by chloroform-isoamyl alcohol extraction, and after the addition of ice-cold alcohol, the DNA was precipitated out of the solution.The white precipitate was collected by spinning it in a centrifuge, where it settled to the bottom of the tube.After washing and re-suspending it in a buffer solution, the extracted DNA can finally be used in the research.

Statistical Analysis
All statistical analyses were performed using SPSS 17.0 (IBM SPSS Inc., Chicago, IL, USA).Statistical significance was evaluated using Student's t-tests; p-values < 0.05 or <0.01 were used as the thresholds for significant and extremely significant differences, respectively.

Cloning and Sequence Analysis of CpCOR413PM1
The complete ORF of the cold-adapted protein gene from C. praecox, named Cp-COR413PM1 (GenBank access number: DQ359747), was successfully amplified.The gene has a 606 bp ORF that encodes 201 amino acids with no introns.The protein calculated molecular weight is 22.69 kDa and the estimated isoelectric point is 9.55.The phylogenetic tree showed that CpCOR413PM1 was most closely related to OeCOR413PM2 (XP_022861037.1) from Olea europaea var.sylvestris, with an amino acid sequence identity of 73% (Figure 1A).The primary structural characteristics of these proteins included a poorly conserved N-terminal, five transmembrane domains, and one possible glycosylphosphatidylinositol anchor site.The full COR413 (also known as WCOR413 [20]) protein's conserved structuralfunctional domain (amino acids 5-190) was found in CpCOR413PM1 based on a conserved domain analysis (Figure 1B).
A subcellular localization website (Plant-mPloc) predicted that CpCOR413PM1 is primarily present in the plasma membrane.Indeed, confocal microscopic analysis revealed fluorescent signals of CpCOR413PM1-GFP in the plasma membrane of tobacco leaf cells (Figure 2), supporting the assignment of CpCOR413PM1 to the COR413-PM subclade of the COR413 family of proteins.

Tissue-Specific Expression of CpCOR413PM1
The analysis of qRT-PCR revealed that CpCOR413PM1 was expressed in all analyzed tissues, including the roots, stems, cotyledons, young leaves, old leaves (mature leaves), outer petals, middle petals, inner petals, stamens, and pistils.However, the relative expression levels showed tissue-specific variation (Figure 3A).The highest expression levels were detected in the roots and flowers, followed by the leaves and fruits, and the lowest expression was detected in the stems.
flower development, the expression level was the lowest at the flower-bud stage peaked during flower senescence.Specifically, the expression level in the flo senescence stage was more than 5.8-times greater than that at the flower-bud stage (F 3C).CpCOR413PM1 expression was significantly increased under low-temperature ( and ABA treatments (Figure 4A,B).Among the various floral organs, the relative expression level of CpCOR413PM1 was higher in the stamens and pistils than in the petals (Figure 3B).Correspondingly, during flower development, the expression level was the lowest at the flower-bud stage and peaked during flower senescence.Specifically, the expression level in the flower-senescence stage was more than 5.8-times greater than that at the flower-bud stage (Figure 3C).Cp-COR413PM1 expression was significantly increased under low-temperature (4 • C) and ABA treatments (Figure 4A,B).
outer petals, middle petals, inner petals, stamens, and pistils.However, the relative expression levels showed tissue-specific variation (Figure 3A).The highest expression levels were detected in the roots and flowers, followed by the leaves and fruits, and the lowest expression was detected in the stems.
Among the various floral organs, the relative expression level of CpCOR413PM1 was higher in the stamens and pistils than in the petals (Figure 3B).Correspondingly, during flower development, the expression level was the lowest at the flower-bud stage and peaked during flower senescence.Specifically, the expression level in the flowersenescence stage was more than 5.8-times greater than that at the flower-bud stage (Figure 3C).CpCOR413PM1 expression was significantly increased under low-temperature (4 °C) and ABA treatments (Figure 4A,B).

Resistance of E. coli Overexpressing the CpCOR413PM1 Gene
To further explore the function of CpCOR413PM1, the protein was expressed and purified in E. coli BL21(DE3).The result shows a single pure band of a molecular mass of approximately 41 kDa containing recombinant tags.The molecular weight of the recombinant tags (His6-Tag, S-Tag, and trxATag) in the prokaryotic expression vector was approximately 18 kDa, thus indicating the theoretical molecular weight (22.69 kDa) of the CpCOR413PM1 protein.The protective effect of the CpCOR413PM1 protein on SOD activity in wintersweet under low-temperature and drought stress conditions was determined in vitro (Figure 5A,B).We next investigated the function of the expressed CpCOR413PM1 fusion protein under cold and drought stress conditions in E. coli.Under low-temperature and drought stress conditions, the logarithmic growth phase of E. coli containing the empty plasmid pET32a (+) was significantly delayed compared with that of E. coli containing the recombinant plasmid pET32a (+)-CpCOR413PM1 (Figure 5C,D).After low-temperature treatments and polyethylene glycol (PEG) treatments, the plaque density of the E. coli with the CpCOR413PM1 recombinant plasmid was significantly higher than that of the control (Figure 5E).
activity in wintersweet under low-temperature and drought stress conditions was determined in vitro (Figure 5A,B).We next investigated the function of the expressed CpCOR413PM1 fusion protein under cold and drought stress conditions in E. coli.Under low-temperature and drought stress conditions, the logarithmic growth phase of E. coli containing the empty plasmid pET32a (+) was significantly delayed compared with that of E. coli containing the recombinant plasmid pET32a (+)-CpCOR413PM1 (Figure 5C,D).After low-temperature treatments and polyethylene glycol (PEG) treatments, the plaque density of the E. coli with the CpCOR413PM1 recombinant plasmid was significantly higher than that of the control (Figure 5E).

Effects of CpCOR413PM1 Overexpression on Stress Tolerance in Arabidopsis
Three transgenic Arabidopsis lines with high (OE-8), medium (OE-5), and low (OE-1) CpCOR413PM1 expression levels (Figure 6A) were selected by qRT-PCR for further experiments.The growth and development of Arabidopsis plants were not significantly affected by the overexpression of CpCOR413PM1.Nonetheless, transgenic CpCOR413PM1 Arabidopsis showed greater resistance to cold and drought than the wild type (WT).The results highly suggested that the membrane damage of WT plants is more severe than that of the overexpression lines under low temperatures; thus, tolerance to lowtemperature stress is significantly higher for the transgenic plants than for the WT.The survival rates of the OE-8 (76.10%),OE-5 (73.30%), and OE-1 (52.40%) overexpression lines were significantly higher than those of the WT (16.30%) (p < 0.05) (Figure 6B).The expression levels of two cold-regulated genes (AtCor15A and AtCor15B) in the transgenic and wild-type plants were compared under cold stress.Under cold treatment, the expression levels of AtCor15A and AtCor15B in the transgenic line were evidently higher than those in wild-type plants (Figure 6E,F).The leaves of WT plants turned yellow and then died (Figure 6G), whereas the overexpression transgenic Arabidopsis continued to grow normally.On MS media without ABA, the germination rate and sprouting speed of OE-1, which had the highest expression, were significantly lower than those of the other strains under 0.8 µM ABA (Figure 7).Under drought stress, specifically, the survival rates of the transgenic lines 8, 5, 1 were 60.5%, 48.2%, and 36.8%,respectively, while the WT Arabidopsis had the low survival rate of 28.5% (Figure 8A).The chlorophyll content of CpCOR413PM1 transge Arabidopsis was higher than that of the WT (Figure 8B), whereas the MDA increas amount and content were lower in transgenic Arabidopsis than in the WT (Figure 8 These results indicate that the CpCOR413PM1 gene improves the survival rate Arabidopsis under drought stress to a certain extent.After approximately 10 days of w deprivation, the phenotypic observation showed that all plants were damaged to differ degrees (Figure 8D).Even five days after recovery, some Arabidopsis plants were una to be revived due to severe water loss.However, the transgenic plants exhibited be growth and a higher survival rate compared to those of the WT Arabidopsis.Under drought stress, specifically, the survival rates of the transgenic lines 8, 5, and 1 were 60.5%, 48.2%, and 36.8%,respectively, while the WT Arabidopsis had the lowest survival rate of 28.5% (Figure 8A).The chlorophyll content of CpCOR413PM1 transgenic Arabidopsis was higher than that of the WT (Figure 8B), whereas the MDA increasing amount and content were lower in transgenic Arabidopsis than in the WT (Figure 8C).These results indicate that the CpCOR413PM1 gene improves the survival rate of Arabidopsis under drought stress to a certain extent.After approximately 10 days of water deprivation, the phenotypic observation showed that all plants were damaged to different degrees (Figure 8D).Even five days after recovery, some Arabidopsis plants were unable to be revived due to severe water loss.However, the transgenic plants exhibited better growth and a higher survival rate compared to those of the WT Arabidopsis.

Functional Characterization of the CpCOR413PM1 Promoter
The 1815bp upstream fragment of CpCOR413PM1 was obtained, which was named CpCOR413PM1pro.Bioinformatics analysis showed the presence of known abiotic stress and hormone response elements, such as MYB, ABA-responsive element (ABRE), and ERE (Figure 9A), in the promoter sequence (Table 1).Under 4 • C and ABA treatments, GUS gene expression and enzyme activity increased compared with those of the control (Figure 9B).The expression of GUS was detected in all organs of transgenic plants (T 3 ), with the highest GUS activity found in the roots, followed by the stem, leaf, fruit, and flower (Figure 9C,D), indicating that CpCOR413PM1pro was active in all organs of the plants and acts as a constitutive promoter.
These results indicate that the CpCOR413PM1 gene improves the survival rate of Arabidopsis under drought stress to a certain extent.After approximately 10 days of water deprivation, the phenotypic observation showed that all plants were damaged to different degrees (Figure 8D).Even five days after recovery, some Arabidopsis plants were unable to be revived due to severe water loss.However, the transgenic plants exhibited better growth and a higher survival rate compared to those of the WT Arabidopsis.

Discussion
A cold-climate stress environment is an important non-biological environmental stress that directly affects the sustained growth of plants and the economic yield of crops.COR genes, as a class of cold-inducible genes in plants, can be rapidly expressed under

Discussion
A cold-climate stress environment is an important non-biological environmental stress that directly affects the sustained growth of plants and the economic yield of crops.COR genes, as a class of cold-inducible genes in plants, can be rapidly expressed under low-temperature-induced conditions.In addition to this, COR proteins are able to play important roles in a variety of environmental stress processes, such as the drought stress studied in this paper.
The lack of introns in COR413 allows for the rapid production of COR proteins to support the organism's quick reaction to signals of adverse conditions (such as low temperatures).Subcellular localization analysis confirmed that CpCOR413PM1 is localized to the plasma membrane, belonging to the COR413-PM (COR413-plasma membrane proteins) subclade of the COR413 protein family.CpCOR413PM1 expression increased gradually under low-temperature stress.Notably, CpCOR413PM1 was expressed in the floral organs of wintersweet.In particular, the expression levels of stamens were significantly higher than those of the petals; these studies demonstrated that the cold sensitivity of each part of the floral organs varies.Previous studies focusing on the cold tolerance of floral organs have predominantly investigated non-winter-flowering plants.Specifically, the stamens and pistils were most vulnerable to low-temperature chilling, while the sepals and petals were more cold-tolerant [21].Thus, the higher expression of CpCOR413PM1 in the wintersweet stamens may imply its association to the preservation of wintersweet flowers from cold damage under low-temperature conditions in winter to ensure proper flowering.The wintersweet flower undergoes severe dehydration upon wilting.Since the polypeptide encoded by the COR gene is highly hydrophilic, the structure of the cell membrane composed of the polypeptide will be more stable, thus reducing dehydrationinduced cell damage [22][23][24][25].We found that CpCOR413PM1 expression was the highest in the flower-senescence stage and was the lowest in the flower-bud stage.Under open-field cultivation conditions, CpCOR413PM1 gene expression gradually increased with the decrease of ambient temperature.Therefore, our findings indicate that CpCOR413PM1 may be involved in the regulation of wintersweet flowers during the winter, contributing to their growth and development.
Further analysis of CpCOR413PM1 expression under various stress treatments of wintersweet demonstrated that its function is closely related to cold regulation.Currently, the most extensively studied low-temperature regulatory network is the inducer of the CBF expression (ICE)-CBF-COR signal transduction model.For instance, the expression of COR in Aegilops-Triticum composite group was found to be low, whereas the expression levels of ICE and CBF genes increased gradually and the expression of the COR gene increased significantly in the later stage of low-temperature treatment [26].The expression of COR genes can be divided into ABA-dependent and ABA-independent responses [27].Our findings reveal that CpCOR413PM1 may be involved in ABA-dependent stress response pathways, necessitating further analysis.Some CORs may be the protective, functional proteins of cells, including playing a protective role under low temperatures [28].Ma [29] found that the tomato SlCOR413IM1 gene could maintain high SOD activity under low temperatures and drought stress.Our results also demonstrate that the CpCOR413PM1 protein has a strong protective effect on SOD enzyme activity under both low temperatures and drought stresses, with more significant effects detected under low-temperature stress.Previous studies have demonstrated that introducing plant stress-tolerance-related genes into E. coli cells can boost the bacterial survival rate under abiotic stress.Liu [30] transferred CCOR1 and CCOR2 expression vectors into E. coli, which effectively improved salt resistance.Consistently, we found that expressing the CpCOR413PM1 protein in E. coli enhanced survival under low temperature and drought.
Previous studies have shown that the heterologous overexpression of PsCOR413PM2 and PsCOR413IM1 from Phlox subulata may enhance cold tolerance in Arabidopsis [9,31].The overexpression of rice OsCOR413TM1 resulted in drought tolerance in rice, and the OsCOR413TM1 protein could be activated by the ABRE-binding factor (OsABF1) and thus participate in the ABA-dependent pathway in drought stress regulation [10].Stress from low temperatures will reduce the amount of chlorophyll in plant leaves and damage nearly all important photosynthetic components [32].In our study, under low-temperature and drought stress, the levels of chlorophyll in the overexpression transgenic Arabidopsis lines increased significantly and the MDA increasing amount and content were lower than those in the WT.These results suggest the involvement of CpCOR413PM1 in resisting low-temperature and drought stress.
Previous studies demonstrated that the cold tolerance of tea trees (Camellia sinensis (L.) O. Ktze.) and corn (Zea mays L.) was significantly improved after ABA treatment [33][34][35].We found that external ABA treatment decreased the expression of downstream genes and down-regulated GUS gene expression and enzyme activity in transgenic CpCOR413PM1pro Arabidopsis, suggesting that ABA suppressed promoter activity, which has been shown to enhance the tolerance of plants to adverse conditions [36].Therefore, it is hypothesized that CpCOR413PM1pro controls downstream gene expression in plants under cold stress by acting on the ABA pathway, as it may be inhibited by ABA.

Conclusions
In summary, we have identified and characterized a new COR gene, CpCOR413PM1, and its promoter in wintersweet.Biochemical and genetic evidence suggests that Cp-COR413PM1 plays important roles in cold and drought stress responses.CpCOR413PM1 can be induced by ABA and protects plants from damage under low-temperature and drought stress by regulating chlorophyll and MDA contents.Our study makes it possible to gain a better understanding of how the CpCOR413PM1 gene functions, contributes to the theory of plant-flowering cold resistance, opens up new avenues for research on flowering regulation and frost resistance in woody plants, and generates genetic resources and theoretical references for the development of agroforestry crop-flowering frost defense in practice.

Figure 1 .
Figure 1.Characterization of CpCOR413PM1.(A) Phylogenetic analysis of COR413 p 6 software with the neighbor-joining method (1000 bootstrap repeats) was used to phylogenetic tree.(B) Multiple sequence alignment of CpCOR413PM1 and oth proteins.Identical and similar amino acids are shaded in blue and light blue, re triangle indicates the phosphorylation sites.The triangle represents the phosphoryl

Figure 1 .
Figure 1.Characterization of CpCOR413PM1.(A) Phylogenetic analysis of COR413 proteins.MEGA 6 software with the neighbor-joining method (1000 bootstrap repeats) was used to reconstruct the phylogenetic tree.(B) Multiple sequence alignment of CpCOR413PM1 and other COR413PM proteins.Identical and similar amino acids are shaded in blue and light blue, respectively.The triangle indicates the phosphorylation sites.The triangle represents the phosphorylation site.

Figure 1 .
Figure 1.Characterization of CpCOR413PM1.(A) Phylogenetic analysis of COR413 proteins.MEG 6 software with the neighbor-joining method (1000 bootstrap repeats) was used to reconstruct t phylogenetic tree.(B) Multiple sequence alignment of CpCOR413PM1 and other COR413P proteins.Identical and similar amino acids are shaded in blue and light blue, respectively.T triangle indicates the phosphorylation sites.The triangle represents the phosphorylation site.

Figure 2 .
Figure 2. Subcellular localization of 35S::CpCOR413PM1-GFP in tobacco leaf epidermal cells.Transient expression of 35S::GFP and 35S::CpCOR413PM1-GFP fusion constructs in tobacco leaf epidermal cells.LTI6b-RFP served as a marker for plasma membrane localization [14].Images were taken under bright-and fluorescence-field views.Merged images are shown in the right panel.Scale bars = 20 µm.

Figure 3 .
Figure 3. Expression pattern of CpCOR413PM1 determined by quantitative real-time polym chain reaction in different (A) organs, (B) floral organs, and (C) flowering stages of winters Different letters indicate significant differences and p value < 0.05 or better.

Figure 3 .
Figure 3. Expression pattern of CpCOR413PM1 determined by quantitative real-time polymerase chain reaction in different (A) organs, (B) floral organs, and (C) flowering stages of wintersweet.Different letters indicate significant differences and p value < 0.05 or better.

Figure 3 .
Figure 3. Expression pattern of CpCOR413PM1 determined by quantitative real-time polymerase chain reaction in different (A) organs, (B) floral organs, and (C) flowering stages of wintersweet.Different letters indicate significant differences and p value < 0.05 or better.

Figure 5 .
Figure 5. Escherichia coli in vitro activity analysis.Protective effect of the CpCOR413PM1 protein on superoxide dismutase (SOD) activity under freezing (A) or 15% PEG6000 (B) treatment (the stress assay was performed three times).The growth curve of E. coli pET32a(+)-CpCOR413PM1 and

Figure 6 .
Figure 6.CpCOR413PM1-expressing transgenic Arabidopsis plants (T3) are more tolerant to cold stress than wild-type (WT) plants.(A) Relative expression levels of CpCOR413PM1 in CpCOR413PM1-transformed Arabidopsis.(B) Quantitative analysis of the survival rates.(C) Measurement of chlorophyll content of the transgenic and WT plants under a low temperature.(D) Measurement of malondialdehyde (MDA) concentration of the transgenic and WT plants under a low temperature.(E,F) Analysis of the expressions of AtCor15A and AtCor15B in the wild-type (WT) and CpCOR413PM1 overexpressing Arabidopsis plants under cold stress.(G) Performance of the CpCOR413PM1 transgenic plants under low-temperature stress: before (control); freezing for 12 h; recovery.Different letters indicate significant differences and p value < 0.05 or better."**" indicates p < 0.01

Figure 6 .
Figure 6.CpCOR413PM1-expressing transgenic Arabidopsis plants (T 3 ) are more tolerant to cold stress than wild-type (WT) plants.(A) Relative expression levels of CpCOR413PM1 in CpCOR413PM1transformed Arabidopsis.(B) Quantitative analysis of the survival rates.(C) Measurement of chlorophyll content of the transgenic and WT plants under a low temperature.(D) Measurement of malondialdehyde (MDA) concentration of the transgenic and WT plants under a low temperature.(E,F) Analysis of the expressions of AtCor15A and AtCor15B in the wild-type (WT) and CpCOR413PM1 overexpressing Arabidopsis plants under cold stress.(G) Performance of the CpCOR413PM1 transgenic plants under low-temperature stress: before (control); freezing for 12 h; recovery.Different letters indicate significant differences and p value < 0.05 or better."**" indicates p < 0.01.

Figure 7 .
Figure 7. Germination rates of WT and overexpression lines of CpCOR413PM1 under MS (A) and 0.8 µM ABA treatment (B).

Figure 8 .Figure 8 .
Figure 8. Expression of CpCOR413PM1 confers enhanced tolerance to drought stress in transgenic Arabidopsis (T3).(A) Quantitative analysis of the survival rates.(B) Chlorophyll content of the transgenic and wild-type (WT) plants under drought stress.(C) Malondialdehyde (MDA) concentration of the transgenic and WT plants under drought stress.(D) Performance of the Figure 8. Expression of CpCOR413PM1 confers enhanced tolerance to drought stress in transgenic Arabidopsis (T 3 ).(A) Quantitative analysis of the survival rates.(B) Chlorophyll content of the transgenic and wild-type (WT) plants under drought stress.(C) Malondialdehyde (MDA) concentration of the transgenic and WT plants under drought stress.(D) Performance of the CpCOR413PM1 transgenic plants under drought stress: before (control), drought for 10 d, and recovery for 5 d.Different letters indicate significant differences and p value < 0.05 or better.

Figure 9 .
Figure 9. (A) Cis-acting regulatory elements predicted in the promoter region of CpCOR413PM1.(B) Expression analysis of GUS under different stresses of transgenic Arabidopsis with CpCOR413PM1pro.(C) Expression analysis of GUS in transgenic Arabidopsis organs with CpCOR413PM1pro.(D) CpCOR413PM1pro transgenic Arabidopsis histochemical staining.Different letters indicate significant differences and p value < 0.05 or better.

Figure 9 .
Figure 9. (A) Cis-acting regulatory elements predicted in the promoter region of CpCOR413PM1.(B) Expression analysis of GUS under different stresses of transgenic Arabidopsis with Cp-COR413PM1pro.(C) Expression analysis of GUS in transgenic Arabidopsis organs with Cp-COR413PM1pro.(D) CpCOR413PM1pro transgenic Arabidopsis histochemical staining.Different letters indicate significant differences and p value < 0.05 or better.

Table 1 .
Cis-acting regulatory elements predicted in the promoter region of CpCOR413PM1.