PsCor413pm2, a Plasma Membrane-Localized, Cold-Regulated Protein from Phlox subulata, Confers Low Temperature Tolerance in Arabidopsis

Low temperature stress adversely affects plant growth and development. Isolation and characterization of cold response genes from cold-tolerant plants help to understand the mechanism underlying low temperature tolerance. In this study, PsCor413pm2, a cold-regulated (COR) gene isolated from Phlox subulata, was transferred to Arabidopsis plants to investigate its function. Real-time quantitative PCR analysis revealed that PsCor413pm2 expression was induced by cold. Subcellular localization revealed that the PsCor413pm2-green fluorescent protein (GFP) fusion protein localized to the plasma membrane in tobacco and Arabidopsis plants. Furthermore, overexpression of PsCor413pm2 in Arabidopsis plants enhanced tolerance to low temperature stress. Transgenic Arabidopsis roots had more influx of Ca2+ after a cold shock than wild-type plants, as shown using non-invasive micro-test technology (NMT). Moreover, the transcription abundance of five COR and two C-repeat (CRT) binding factor (CBF) genes in transgenic Arabidopsis plants was higher than that in the wild-type plants under cold stress. Taken together, our results suggest that overexpression of PsCor413pm2 enhances low temperature tolerance in Arabidopsis plants by affecting Ca2+ flux and the expression of stress-related COR and CBF genes.


Introduction
Low temperatures adversely affect plant growth, development, and crop productivity [1]. During the process of long-term adaptive evolution, some temperate plant species have evolved a series of physiological and molecular mechanisms to acclimate to low temperatures. Among these mechanisms, cold signaling pathways dependent on C-repeat binding factors (CBFs) are the best-understood regulatory pathways involved in cold acclimation [2,3].
The Cor413 family of proteins has been divided into two distinct groups, including Cor413-plasma membrane (Cor413pm) and Cor413-inner membrane (Cor413im) proteins [13]. Studies have shown that Arabidopsis AtCor413im localizes to the inner membrane of chloroplasts and may stabilize the chloroplast membrane under cold stress [20,21]. However, Cor413pm localization to the plasma membrane and its functions in response to cold stress have not been reported. Recently, we characterized the functions of PsCor413im1 from Phlox subulata, which are cold-tolerant plants.
Overexpression of PsCor413im1 enhanced cold tolerance in Arabidopsis plants [22]. Here, we identified PsCor413pm2 as another member of the Cor413 family and conducted a preliminary evaluation of its functions using transgenic Arabidopsis plants. Subcellular localization of PsCor413pm2 was performed using GFP as a marker. Using non-invasive micro-test technology (NMT), we then measured the effects of PsCor413pm2 overexpression on Ca 2+ flux in Arabidopsis roots under cold shock. Our study helps to further characterize the function of Cor413 proteins.

Sequence Analysis of PsCor413pm2
The full-length PsCor413pm2 cDNA contained a 609-bp open reading frame (ORF), which was predicted to encode a protein of 202 amino acids with a molecular mass of 22.07 kDa. Multiple sequence alignment and phylogenetic analyses revealed that PsCor413pm2 is highly homologous to two members of the Cor413pm protein family from other plant species (>77.11% identity), including Arabidopsis thaliana, Nicotiana tomentosiformis, Ipomoea nil, Tarenaya hassleriana, Jatropha curcas, and Gossypium hirsutum ( Figure 1A,B). PsCor413pm2 was predicted to have five transmembrane domains using the TMHMM algorithm ( Figure 1A,C). qPCR analysis showed that PsCor413pm2 expression was obviously induced by cold (4 • C) and mannitol (300 mM) stress and slightly by NaCl (150 mM) and abscisic acid (ABA) (100 µM) ( Figure 2). We add it, please confirm. Cor78, and Cor413 have been identified in Arabidopsis plants [11][12][13]. Among these genes, AtCor15 and AtCor78 are the most characterized. Reports have shown that AtCor15 is a chloroplast stromal protein that protects chloroplast membranes during freezing stress [14][15][16]. Further, AtCor78, also known as AtRD29A (responsive to dehydration, RD), is involved in the response to cold, dehydration, and salt stressors [17][18][19]. The Cor413 family of proteins has been divided into two distinct groups, including Cor413plasma membrane (Cor413pm) and Cor413-inner membrane (Cor413im) proteins [13]. Studies have shown that Arabidopsis AtCor413im localizes to the inner membrane of chloroplasts and may stabilize the chloroplast membrane under cold stress [20,21]. However, Cor413pm localization to the plasma membrane and its functions in response to cold stress have not been reported. Recently, we characterized the functions of PsCor413im1 from Phlox subulata, which are cold-tolerant plants.
Overexpression of PsCor413im1 enhanced cold tolerance in Arabidopsis plants [22]. Here, we identified PsCor413pm2 as another member of the Cor413 family and conducted a preliminary evaluation of its functions using transgenic Arabidopsis plants. Subcellular localization of PsCor413pm2 was performed using GFP as a marker. Using non-invasive micro-test technology (NMT), we then measured the effects of PsCor413pm2 overexpression on Ca 2+ flux in Arabidopsis roots under cold shock. Our study helps to further characterize the function of Cor413 proteins.

Sequence Analysis of PsCor413pm2
The full-length PsCor413pm2 cDNA contained a 609-bp open reading frame (ORF), which was predicted to encode a protein of 202 amino acids with a molecular mass of 22.07 kDa. Multiple sequence alignment and phylogenetic analyses revealed that PsCor413pm2 is highly homologous to two members of the Cor413pm protein family from other plant species (>77.11% identity), including Arabidopsis thaliana, Nicotiana tomentosiformis, Ipomoea nil, Tarenaya hassleriana, Jatropha curcas, and Gossypium hirsutum ( Figure 1A,B). PsCor413pm2 was predicted to have five transmembrane domains using the TMHMM algorithm ( Figure 1A,C). qPCR analysis showed that PsCor413pm2 expression was obviously induced by cold (4 °C) and mannitol (300 mM) stress and slightly by NaCl (150 mM) and abscisic acid (ABA) (100 μM) ( Figure 2). We add it, please confirm.

Subcellular Localization of PsCor413pm2
To confirm whether PsCor413pm2 is a membrane protein, localization of PsCor413pm2 in plant cells was examined using GFP as a fusion protein marker. In tobacco leaf epidermis cells transiently expressing PsCor413pm2-GFP, GFP signals were localized to the cell periphery, which is similar to the plasma membrane ( Figure 3A). In Arabidopsis root and root hair cells stably expressing PsCor413pm2-GFP, the GFP signals were also observed in the cell periphery and co-localized with the plasma membrane marker dye FM4-64 (after staining for 1 min) ( Figure 3B). However, when GFP alone was used as a control, it localized to the cytoplasm in tobacco leaf epidermis and Arabidopsis root hair cells ( Figure 3A,B). These results suggest that PsCor413pm2-GFP localized to the plasma membrane.

Subcellular Localization of PsCor413pm2
To confirm whether PsCor413pm2 is a membrane protein, localization of PsCor413pm2 in plant cells was examined using GFP as a fusion protein marker. In tobacco leaf epidermis cells transiently expressing PsCor413pm2-GFP, GFP signals were localized to the cell periphery, which is similar to the plasma membrane ( Figure 3A). In Arabidopsis root and root hair cells stably expressing PsCor413pm2-GFP, the GFP signals were also observed in the cell periphery and co-localized with the plasma membrane marker dye FM4-64 (after staining for 1 min) ( Figure 3B). However, when GFP alone was used as a control, it localized to the cytoplasm in tobacco leaf epidermis and Arabidopsis root hair cells ( Figure 3A,B). These results suggest that PsCor413pm2-GFP localized to the plasma membrane.

Subcellular Localization of PsCor413pm2
To confirm whether PsCor413pm2 is a membrane protein, localization of PsCor413pm2 in plant cells was examined using GFP as a fusion protein marker. In tobacco leaf epidermis cells transiently expressing PsCor413pm2-GFP, GFP signals were localized to the cell periphery, which is similar to the plasma membrane ( Figure 3A). In Arabidopsis root and root hair cells stably expressing PsCor413pm2-GFP, the GFP signals were also observed in the cell periphery and co-localized with the plasma membrane marker dye FM4-64 (after staining for 1 min) ( Figure 3B). However, when GFP alone was used as a control, it localized to the cytoplasm in tobacco leaf epidermis and Arabidopsis root hair cells ( Figure 3A,B). These results suggest that PsCor413pm2-GFP localized to the plasma membrane.

Overexpression of PsCor413pm2 in Arabidopsis Enhances Tolerance to Low Temperature
Transgenic Arabidopsis plants overexpressing PsCor413pm2 driven by a CaMV35S promoter were generated ( Figure 4A) to evaluate its possible role in the plant response to multiple stresses. First, we compared the germination rate of seeds from the transgenic lines and wild type (WT) plants on 1/2 strength Murashige and Skoog (MS) medium under cold (4 °C) stress. At normal temperatures (22 °C), germination rates in the transgenic seeds were similar to those of WT seeds. However, under cold (4 °C) stress conditions, germination rates of the transgenic seeds were significantly higher than those of WT seeds after 18 and 22 days of cultivation ( Figure 4B,C). Moreover, we compared the maximum photochemical efficiency (Fv/Fm) of leaves of transgenic lines and WT plants growing in soil under normal and cold (4 °C) stress ( Figure 4D). As shown in Figure 4E, cold (4 °C, 48 h) treatment resulted in marked decreases in Fv/Fm in both the WT and transgenic plants, but the Fv/Fm of the transgenic plants (#10 and #16) upon stress treatment was higher than that of the WT plants. Further, we compared electrical conductivity (EC) and survival rate of transgenic lines and WT plants growing in soil under normal and freezing (−10 °C) stress. The EC of transgenic plants was significantly lower than that of WT plants after freezing (−10 °C) treatment for 2 h ( Figure 5A,B). The EC results indicated less damage from the freezing treatment in transgenic plant leaves than in WT leaves. Furthermore, the survival rate of transgenic plants was higher than that of WT after freezing (−10 °C) treatment ( Figure 5A,C). These results suggest that overexpression of PsCor413pm2 improves tolerance to low temperature stress in Arabidopsis plants.
In addition, we compared seedling growth in the transgenic lines and WT plants under osmotic stress. On 1/2 strength MS supplemented with mannitol, germination rate (with 250 mM mannitol),

Overexpression of PsCor413pm2 in Arabidopsis Enhances Tolerance to Low Temperature
Transgenic Arabidopsis plants overexpressing PsCor413pm2 driven by a CaMV35S promoter were generated ( Figure 4A) to evaluate its possible role in the plant response to multiple stresses. First, we compared the germination rate of seeds from the transgenic lines and wild type (WT) plants on 1/2 strength Murashige and Skoog (MS) medium under cold (4 • C) stress. At normal temperatures (22 • C), germination rates in the transgenic seeds were similar to those of WT seeds. However, under cold (4 • C) stress conditions, germination rates of the transgenic seeds were significantly higher than those of WT seeds after 18 and 22 days of cultivation ( Figure 4B,C). Moreover, we compared the maximum photochemical efficiency (Fv/Fm) of leaves of transgenic lines and WT plants growing in soil under normal and cold (4 • C) stress ( Figure 4D). As shown in Figure 4E, cold (4 • C, 48 h) treatment resulted in marked decreases in Fv/Fm in both the WT and transgenic plants, but the Fv/Fm of the transgenic plants (#10 and #16) upon stress treatment was higher than that of the WT plants. Further, we compared electrical conductivity (EC) and survival rate of transgenic lines and WT plants growing in soil under normal and freezing (−10 • C) stress. The EC of transgenic plants was significantly lower than that of WT plants after freezing (−10 • C) treatment for 2 h ( Figure 5A,B). The EC results indicated less damage from the freezing treatment in transgenic plant leaves than in WT leaves. Furthermore, the survival rate of transgenic plants was higher than that of WT after freezing (−10 • C) treatment ( Figure 5A,C). These results suggest that overexpression of PsCor413pm2 improves tolerance to low temperature stress in Arabidopsis plants.
In addition, we compared seedling growth in the transgenic lines and WT plants under osmotic stress. On 1/2 strength MS supplemented with mannitol, germination rate (with 250 mM mannitol), primary root length, and fresh weight (with 175 and 200 mM mannitol) of the transgenic seedlings were significantly greater than those of WT plants (Supplementary Figure S1).  Figure S1).

Overexpression of PsCor413pm2 in Arabidopsis Affects Ca 2+ Flux and Stress-Related Genes Expression
The initial plant response to cold was confirmed to be an influx of Ca 2+ as a signaling molecule into the cytosol through the plasma membrane [1]. Net Ca 2+ flux in response to cold shock in seedling roots from transgenic Arabidopsis and WT plants was monitored using NMT ( Figure 6A). At normal temperatures (22 °C), there was a significant influx of extracellular Ca 2+ , with a minus peak in transgenic Arabidopsis and WT roots. Upon cold (4 °C) shock, WT roots exhibited a slight Ca 2+ efflux, while transgenic Arabidopsis roots exhibited a greater influx of Ca 2+ ( Figure 6B). The mean Ca 2+ influx in untreated transgenic Arabidopsis roots was slightly higher than that in WT plants. However, there were significant differences in the mean Ca 2+ flux attributable to cold shock between transgenic Arabidopsis and WT plants ( Figure 6C). These results suggest that overexpression of PsCor413pm2 affects Ca 2+ flux in Arabidopsis roots under cold shock.

Overexpression of PsCor413pm2 in Arabidopsis Affects Ca 2+ Flux and Stress-Related Genes Expression
The initial plant response to cold was confirmed to be an influx of Ca 2+ as a signaling molecule into the cytosol through the plasma membrane [1]. Net Ca 2+ flux in response to cold shock in seedling roots from transgenic Arabidopsis and WT plants was monitored using NMT ( Figure 6A). At normal temperatures (22 • C), there was a significant influx of extracellular Ca 2+ , with a minus peak in transgenic Arabidopsis and WT roots. Upon cold (4 • C) shock, WT roots exhibited a slight Ca 2+ efflux, while transgenic Arabidopsis roots exhibited a greater influx of Ca 2+ ( Figure 6B). The mean Ca 2+ influx in untreated transgenic Arabidopsis roots was slightly higher than that in WT plants. However, there were significant differences in the mean Ca 2+ flux attributable to cold shock between transgenic Arabidopsis and WT plants ( Figure 6C). These results suggest that overexpression of PsCor413pm2 affects Ca 2+ flux in Arabidopsis roots under cold shock.

Overexpression of PsCor413pm2 in Arabidopsis Affects Ca 2+ Flux and Stress-Related Genes Expression
The initial plant response to cold was confirmed to be an influx of Ca 2+ as a signaling molecule into the cytosol through the plasma membrane [1]. Net Ca 2+ flux in response to cold shock in seedling roots from transgenic Arabidopsis and WT plants was monitored using NMT ( Figure 6A). At normal temperatures (22 °C), there was a significant influx of extracellular Ca 2+ , with a minus peak in transgenic Arabidopsis and WT roots. Upon cold (4 °C) shock, WT roots exhibited a slight Ca 2+ efflux, while transgenic Arabidopsis roots exhibited a greater influx of Ca 2+ ( Figure 6B). The mean Ca 2+ influx in untreated transgenic Arabidopsis roots was slightly higher than that in WT plants. However, there were significant differences in the mean Ca 2+ flux attributable to cold shock between transgenic Arabidopsis and WT plants ( Figure 6C). These results suggest that overexpression of PsCor413pm2 affects Ca 2+ flux in Arabidopsis roots under cold shock.  Further, the expression of five COR genes (AtCor6.6/AtKIN2, AtCor15A, AtCor15B, AtCor47, and AtCor78/AtRD29) in transgenic Arabidopsis and WT plants was investigated under conditions of cold stress. Under normal and cold (4 • C) conditions, the transcript abundance of these five COR genes in transgenic Arabidopsis plants was evidently higher than in the WT plants ( Figure 7A and Supplementary Figure S2A). The expression of many COR genes was regulated by CBF transcription factors. Further, we compared the expression of AtCBFs in transgenic Arabidopsis and WT plants under normal and cold (4 • C) stress. Under cold (4 • C) conditions, AtCBF2 and AtCBF3 expression from the AtCBF gene family was evidently higher than in the WT plants ( Figure 7B and Supplementary Figure S2B). These results suggest that overexpression of PsCor413pm2 affects expression of two CBF and five COR genes in Arabidopsis plants under cold stress. transgenic Arabidopsis plants was evidently higher than in the WT plants ( Figure 7A and Supplementary Figure S2A). The expression of many COR genes was regulated by CBF transcription factors. Further, we compared the expression of AtCBFs in transgenic Arabidopsis and WT plants under normal and cold (4 °C) stress. Under cold (4 °C) conditions, AtCBF2 and AtCBF3 expression from the AtCBF gene family was evidently higher than in the WT plants ( Figure 7B and Supplementary Figure S2B). These results suggest that overexpression of PsCor413pm2 affects expression of two CBF and five COR genes in Arabidopsis plants under cold stress.
(A) (B) Figure 7. Expression of stress-associated genes in transgenic Arabidopsis and wild type (WT) seedlings under cold stress. Two-week-old transgenic Arabidopsis line (#10) overexpressing PsCor413pm2 and WT seedlings were treated at 4 °C for the indicated time periods. Expression of the AtCor6.6/AtKIN2, AtCor15A, AtCor15B, AtCor47, AtCor78/AtRD29 (A) and AtCBF1 to AtCBF4 (B) genes was investigated using qPCR. The AtActin2 gene was used as an internal control, and the transcript level in the untreated WT seedlings was set as 1.0. Asterisks indicate significant differences between WT and transgenic lines (* p < 0.05; ** p < 0.01; student's t test). Error bars show the SD of the values from three replicates. Figure 7. Expression of stress-associated genes in transgenic Arabidopsis and wild type (WT) seedlings under cold stress. Two-week-old transgenic Arabidopsis line (#10) overexpressing PsCor413pm2 and WT seedlings were treated at 4 • C for the indicated time periods. Expression of the AtCor6.6/AtKIN2, AtCor15A, AtCor15B, AtCor47, AtCor78/AtRD29 (A) and AtCBF1 to AtCBF4 (B) genes was investigated using qPCR. The AtActin2 gene was used as an internal control, and the transcript level in the untreated WT seedlings was set as 1.0. Asterisks indicate significant differences between WT and transgenic lines (* p < 0.05; ** p < 0.01; student's t test). Error bars show the SD of the values from three replicates.

Discussion
The CBF pathway activation is an important plant response pathway to low temperature stress. Plants likely sense low temperatures through plasma membrane proteins such as chilling-tolerance-divergence 1 (COLD1), which might induce a cytosolic Ca 2+ signal and activate protein kinases [23]. Protein kinases are assumed to activate downstream transcription factors such as calmodulin-binding transcription activators (CAMTAs) and ICE1 through sumoylation and phosphorylation. ICE1-activated CBF regulates the expression of COR genes, which confer low temperature tolerance in plants [1,[4][5][6]. Cor413 is a member of the COR gene family, and it is divided into two groups, Cor413pm and Cor413im. In our study, the presence of a homolog of Cor413pm2 was revealed in the genome of P. subulata. The sequence analysis showed that it might be a conserved membrane protein with five transmembrane domains (Figure 1). qPCR analysis showed that PsCor413pm2 expression was obviously induced by cold and mannitol stress (Figure 2). Similarly, the AtCor413pm2 expression in Arabidopsis was also induced by cold and mannitol stress (data from Arabidopsis eFP Browser). Subcellular localization of GFP as a fusion marker showed that PsCor413pm2-GFP was localized to the plasma membrane in Arabidopsis (Figure 3), indicating that the fusion of GFP did not affect the normal localization of PsCor413pm2. These results suggest that PsCor413pm2 is a cold-regulated protein located on the plasma membrane. Furthermore, seed germination, leaf photochemical efficiency, electrical conductivity, and survival rate analysis showed that PsCor413pm2 transgenic Arabidopsis are more tolerant to low temperatures than WT (Figures 4 and 5). In addition, our study showed that the transcript abundance of five AtCOR (AtCor6.6/AtKIN2, AtCor15A, AtCor15B, AtCor47, and AtCor78/AtRD29) and two AtCBF (AtCBF2 and AtCBF3) genes from PsCor413pm2 transgenic Arabidopsis was significantly higher than that of WT plants under cold stress (Figure 7 and Supplementary Figure S2). These results suggest that overexpression of PsCor413pm2 in Arabidopsis may confer tolerance to low temperatures by influencing the expression of COR and CBF genes. NMT analysis revealed a greater Ca 2+ influx in transgenic Arabidopsis roots under cold shock ( Figure 6). Under cold stress, an increase in cytosolic Ca 2+ as a signaling molecule activates downstream COR gene expression [24]. Therefore, the influx of Ca 2+ may cause an increase in COR gene expression in transgenic Arabidopsis. However, how the overexpression of PsCor413pm2 affects the flux of Ca 2+ is unclear. We speculate that there are two possibilities. Subcellular localization showed that PsCor413pm2 is a plasma membrane protein (Figure 3), and it is known that the plasma membrane is the primary site of chilling and freezing injury. Therefore, the first proposed hypothesis is that PsCor413pm2 may be a plasma membrane protein associated with Ca 2+ signaling. Similarly, COLD1, a cold-related protein localized on the plasma membrane in Japonica rice, also affects the flux of Ca 2+ in the root under cold shock. Further, researchers have demonstrated that COLD1, as a regulator of G-protein signaling, confers cold tolerance by modulating Ca 2+ signaling in rice [25,26]. Early studies predicted that Cor413 proteins are G protein-coupled receptors [13]. The second hypothesis is that PsCor413pm2 is a membrane-stabilizing protein. As an integral membrane protein, PsCor413pm2 could play a structural role by stabilizing the plasma membrane lipid bilayer. The stability of the plasma membrane contributes to the function of plasma membrane proteins, such as Ca 2+ -flux regulating related proteins. The expression of AtCBF2 and AtCBF3 in transgenic Arabidopsis may be affected by the change of Ca 2+ flux caused by PsCor413pm2 overexpression.
In addition, PsCor413pm2 overexpression enhances tolerance to osmotic stress in Arabidopsis (Supplementary Figure S1). Under normal conditions, the expression of AtCor78 in transgenic plants was significantly higher than that in WT plants (Figure 7 and Supplementary Figure S2A). AtCor78 is also known as AtRD29A, which is involved in plant responses to dehydration stress [27]. Thus, overexpression of PsCor413pm2 enhances osmotic tolerance in Arabidopsis plants and may be related to the enhanced expression of AtCor78/AtRD29A under normal conditions. Taken together, our results suggest that overexpression of PsCor413pm2 enhances low temperature tolerance in Arabidopsis plants by affecting Ca 2+ flux and the expression of stress-related COR and CBF genes. Nevertheless, we will continue to study these mechanisms, as they should be further evaluated and verified.

Identification of PsCor413pm2 and Sequence Analysis
Based on sequence similarity, the PsCor413pm2 gene (GenBank accession number: KT337405.1) was identified using transcriptome sequencing data from cold-treated P. subulata. The amino acid sequences of PsCor413pm2 and its homologs were aligned using ClustalW. The transmembrane domains in PsCor413pm2 were predicted using the TMHMM algorithm [28], and the phylogenetic tree was constructed via the neighbor-joining method using the molecular evolutionary genetics analysis (MEGA) 4.1 software [29] with 1000 bootstrap replicates. The bootstrap scores <50% were deleted. For abiotic stress treatments, 2-week-old P. subulata or Arabidopsis seedlings were exposed to 4 • C, 100 µM ABA, 150 mM NaCl, or 300 mM mannitol. At least 10 seedlings from each treatment group were harvested and pooled at different time points (0, 3, 6, 12, or 24 h after treatment), frozen immediately in liquid nitrogen, and stored at −80 • C for RNA preparation.

RNA Extraction and Real-Time Quantitative PCR (qPCR) Analyses
Total RNA was extracted using a RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 µg total RNA with the M-MLV RTase cDNA synthesis kit (TaKaRa, Shiga, Japan). Real-time quantitative PCR analysis was performed using the SYBR green mix (Agilent Technologies, Palo Alto, CA, USA) in an optical 96-well plate on a Mx3000P system (Agilent). Three biological repeats and three technical repeats were performed for qPCR analysis. The primers used in this study are shown in Supplementary Table S1.

Vector Construction and Plant Transformation
To create the pBI121-PsCor413pm2 construct, the ORF of PsCor413pm2 was amplified via PCR and cloned at the XbaI and SacI sites of the pBI121 vector. To construct the GFP fusion genes, the ORF of PsCor413pm2, without the stop codon, was amplified using PCR and cloned at the XbaI and KpnI sites of the pBI121-GFP vector. The accuracy of the above constructs was confirmed by sequencing, and the specific primers used in this study are shown in Supplementary Table S1.
The constructs were transformed into the Agrobacterium tumefaciens strain EHA105 for plant transformation. Transient expression in tobacco leaves was performed according to a previously described procedure [22]. Arabidopsis plants were transformed using the floral dip method [30]; the transgenic plants were then selected on 1/2 strength MS medium containing 30 µg mL −1 kanamycin. Expression of PsCor413pm2 in the transgenic lines was assessed via semi-quantitative reverse transcription PCR analyses using the T3 generation.

Confocal Laser Scanning Microscopy
The tobacco leaf epidermis was peeled to make a temporary squash and then visualized using confocal laser scanning microscopy (CLSM; Nikon, A1, Tokyo, Japan). Four-day-old Arabidopsis seedlings grown on vertical 1/2 strength MS agar plates were incubated in 1 mL liquid 1/2 strength MS medium (0.5% sucrose, pH 5.8) containing 4 µM FM4-64 (Invitrogen, Carlsbad, CA, USA) for 1 min at room temperature (22 • C). Roots of transgenic Arabidopsis seedlings were washed twice with liquid 1/2 strength MS medium immediately before visualizing via CLSM. GFP signals were detected using a 500-530 nm emission filter. FM4-64 signals were detected using a 620-680 nm emission filter.

Low Temperature and Osmosis Tolerance Assay
Arabidopsis seeds were treated at 4 • C for 2 days and then grown on 1/2 strength MS medium at different temperatures (22 • C or 4 • C) or 1/2 strength MS medium supplemented with mannitol (0, 175, 200 mM, or 250 mM) before measuring seed germination rate, seedling root length, and fresh weight. The experiment was replicated three times.
Arabidopsis plants grown in soil for 20 days were treated with 4 • C for 48 h or −10 • C for 2 h, then the leaves of 2 cm diameter were used for maximal Fv/Fm and relative EC analysis. Fv/Fm was measured using an Imaging-PAM Chlorophyll Fluorometer (M-series, Heinz Walz GmbH, Frankfurt am Main, Germany) as described previously [31]. The percent relative EC was calculated as EC% = Post freezing EC/Post autoclaving EC × 100. These experiments were replicated three times with ten plants per treatment. After the −10 • C treatment, the plants were transferred to light (12 h light/12 h dark photoperiod) at 22 • C. The survival rates of the plants were scored visually after 2 weeks. The experiment was repeated three times and the average values were calculated. For each experiment, 5 pots from WT and three transgenic lines were used, with 4-5 seedlings per pot.

Net Ca 2+ Flux Measurements
Net Ca 2+ flux was measured using NMT (NMT100 Series, Younger USA LLC, Amherst, MA, USA) as described previously [25,32]. Seven-day-old seedling root segments were immobilized in measuring solution (0.1 mM KCl, 0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.5 mM NaCl, 0.3 mM 2-(N-morpholino) ethanesulfonic acid (MES), and 0.2 mM Na 2 SO 4 , pH 6.0) to measure Ca 2+ flux. For sudden cold-shock treatment, transient Ca 2+ flux kinetics in response to cold (4 • C) were measured in seedling root tips. After measuring for 5-6 min at room temperature, ice-cold test buffer (4 • C) was quickly added to the container around the roots; Ca 2+ flux was simultaneously measured for a further 10 min. Six biological repeats were performed for each analysis.

Statistical Analyses
The data were analyzed via one-way analysis of variance using SPSS software (Version 19.0, SPSS Inc., Chicago, IL, USA), and statistically significant differences were calculated based on the student's t-test, with p < 0.05 (*) and p < 0.01 (**) as the thresholds for significance.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/9/ 2579/s1. Figure S1: Phenotypes of transgenic Arabidopsis and wild type (WT) seedlings under osmotic Stress; Figure S2: Expression of stress-associated genes in transgenic Arabidopsis and wild type (WT) seedlings under cold stress; Table S1: List of primers used in this study.