Isolation and Functional Analysis of MbCBF2, a Malus baccata (L.) Borkh CBF Transcription Factor Gene, with Functions in Tolerance to Cold and Salt Stress in Transgenic Arabidopsis thaliana

CBF transcription factors (TFs) are key regulators of plant stress tolerance and play an integral role in plant tolerance to adverse growth environments. However, in the current research situation, there are few reports on the response of the CBF gene to Begonia stress. Therefore, this experiment investigated a novel CBF TF gene, named MbCBF2, which was isolated from M. baccata seedlings. According to the subcellular localization results, the MbCBF2 protein was located in the nucleus. In addition, the expression level of MbCBF2 was higher in new leaves and roots under low-temperature and high-salt induction. After the introduction of MbCBF2 into Arabidopsis thaliana, the adaptability of transgenic A. thaliana to cold and high-salt environments was significantly enhanced. In addition, the high expression of MbCBF2 can also change many physiological indicators in transgenic A. thaliana, such as increased chlorophyll and proline content, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity, and reduced malondialdehyde (MDA) content. Therefore, it can be seen from the above results that MbCBF2 can positively regulate the response of A. thaliana to low-temperature and osmotic stress. In addition, MbCBF2 can also regulate the expression of its downstream genes in transgenic lines. It can not only positively regulate the expression of the downstream key genes AtCOR15a, AtERD10, AtRD29a/b and AtCOR6.6/47, related to cold stress at low temperatures, but can also positively regulate the expression of the downstream key genes AtNCED3, AtCAT1, AtP5CS, AtPIF1/4 and AtSnRK2.4, related to salt stress. That is, the overexpression of the MbCBF2 gene further improved the adaptability and tolerance of transgenic plants to low-temperature and high-salt environments.


Introduction
Plants are constantly affected by various environmental pressures throughout their lives. These environmental factors include biological and abiotic factors, which bring great challenges to the growth and survival of plants [1]. Soil salinization, high-temperature, low-temperature, drought and other abiotic stresses often cause certain oxidative damage to plants, and seriously affect the yield and quality of crops [2][3][4]. Especially under cold conditions, the synthesis and accumulation of various substances in plants are affected, and various life activities and reactions will also undergo dramatic changes. For example, they lead to damage to cell membranes, causing the increased extravasation of soluble substances in cells, and disrupting the balance of substances inside and outside plant cells. The normal life activities of plants are even more seriously threatened. Therefore, the harm of low-temperature stress on plants is in need of attention [5,6]. In order to adapt to these et al. [33] found that the transcript levels of CBF2 and CBF3 in WT plants were significantly lower than those in HOS2 mutant plants under cold treatment, suggesting that HOS2 may act upstream of CBFs. In addition, CBF TFs can further regulate the transcription and expression of downstream genes, and can further enhance the tolerance of plants to low-temperature and salt stress. So far, it has been proven that many downstream genes responding to low-temperature stress and salt stress are regulated by CBF TFs, such as RD29a, COR15a, SnRK2.4, and so on. Studies have shown that COR15a and COR15b promoters were induced by cold stress in potato and tobacco [34]. A great deal of the downstream functional genes KIN1, COR6.6, COR15a, RD17, RD29a and ERD10 located in A. thaliana can also be activated by the overexpression of the CBF/DREB gene under cold stress [35]. Du et al. [36] found that the expression level of jasmonic acid (JA) biosynthesis genes such as OsAOS, OsOPR1, OsAOC, and OsLOX2 in rice increased under the induction of a low temperature, which led to the rapid increase of the endogenous JA level. SFR6 protein is a regulator located downstream of CBF. Wathugala et al. [37] found that SFR6 protein was not only related to low-temperature stress in plants but also regulated the expression of defense genes in salicylic acid (SA) and JA-dependent signal transduction pathways. SA and JA are plant hormones that mainly regulate plant development and improve plant stress tolerance [38,39].
Changes in stress-related physiological indicators can intuitively reflect the degree of damage to plants caused by adverse growth conditions. For example, MDA, one of the products of plant plasma membrane peroxidation, can cause serious damage to enzyme proteins and cell membrane systems in plant cells [40]. Therefore, the degree of peroxidation of the plant cell membrane can be reflected by the content of MDA. The higher the MDA content, the higher the degree of oxidation and the greater the damage to the plant. Chlorophyll is one of the crucial factors for the photosynthesis of plants. However, chlorophyll biosynthesis was clearly inhibited under cold stress, and the content of it will be greatly reduced [41,42], which affects the growth and development of plants. In addition, antioxidant enzymes such as SOD, POD, CAT and other enzymes in plants can effectively remove or inhibit peroxide ions in plant cells in a timely manner, thereby reducing the damage caused by stress to plants [43][44][45]. In addition, when the plant is stressed, proline will accumulate in a large amount. Therefore, its content can also be used as an index of plant tolerance to adverse environments [46].
However, there are few CBF TFs studies targeting Malus plants. Therefore, the signaling/regulatory pathway of CBF in the stress response of Malus plants is still unclear. In this study, the CBF TF gene MbCBF2 induced by low-temperature and salt stress was found through gene cloning. The main function of this CBF TF gene under low-temperature and high-salt conditions was verified through stable genetic transformation, and the expression of several key downstream genes under relevant stress conditions was identified. The deep-seated causes of cold tolerance and salt tolerance in mountain stator were preliminarily clarified, and were also prepared for the screening of the cold and salt tolerance of new Malus materials.

Cloning and Sequence Analysis of MbCBF2
Prot Param analysis showed that MbCBF2 encoded 220 amino acids, and its ORF was 663 bp ( Figure S1). The theoretical isoelectric point (pI) of the MbCBF2 protein was predicted to be 5.18, the theoretical molecular mass (MW) was 24.206 kDa, and the average hydrophilicity coefficient was −0.456. Among them, Ala (14.5%), Ser (10.0%), Glu (7.7%), and Pro (7.3%) account for a large proportion. Sequence analysis indicated that the sequence included a DNA binding site and a conserved sequence of AP2 consisting of 58 amino acids, which showed that MbCBF2 belonged to the AP2 family.

Phylogenetic Relationship of MbCBF2
In order to probe the evolutionary relationship between plant CBF proteins, the CBF proteins of MbCBF2 and 11 other different species were compared by DNAMAN. It can be seen from Figure 1A that the conserved amino acid sequences of the MbCBF2 gene are framed in red. The conserved fragments of the CBF TFs of different species had slight differences, but the sequence was roughly the same; in other words, the CBF amino acid sequence of different species had a higher homology in its conserved regions, while the homology in other regions was relatively low. According to the homologous phylogenetic tree ( Figure 1B), MbCBF2 (Malus baccata, TQE10660.1) and PdCBF4 (Populus davidiana, KF880602.1) belong to the same evolutionary branch, indicating that they have the highest homology and the closest evolutionary relationship, followed by FaCBF4 (Fragaria ananassa, AEK94313.1) homogenized in the According to the homologous phylogenetic tree ( Figure 1B), MbCBF2 (Malus baccata, TQE10660.1) and PdCBF4 (Populus davidiana, KF880602.1) belong to the same evolutionary branch, indicating that they have the highest homology and the closest evolutionary relationship, followed by FaCBF4 (Fragaria ananassa, AEK94313.1) homogenized in the first cluster of the phylogenetic tree. MdCBF4 (Malus domestica, ART85561.1), MdCBF2 (Malus domestica, AGL07698.1), MsCBF3 (Malus sieversii, ARO50175.1), and MsCBF4 (Malus sieversii, AFU52632.1) were all clustered in the second cluster of the phylogenetic tree, indicating that their kinship is also relatively close. AtCBF3 (A. thaliana, ABV27154.1), AtCBF2 (A. thaliana, ABV27118.1), BnCBF7 (Brassicanapus, AAM18959.1), LpCBF3 (Lepidium perfoliatum, AGY36892.1), AaCBF3 (Ageratina adenophora, AIT39763.1), and MbCBF2 (Malus baccata) had a low homology, and were located in the third cluster of the evolutionary tree.

Subcellular Localization of MbCBF2 Protein
The specific location of MbCBF2 protein was determined by constructing the fusion expression vector of green fluorescent protein (GFP) and the MbCBF2 gene. It can be seen from Figure 2 that the MbCBF2-GFP fusion protein can only be detected in the nucleus ( Figure 2E), while the GFP-as the control-can be observed in the whole cytoplasm ( Figure 2B). At the same time, the location of the nucleus was confirmed by 4 , DAPI staining ( Figure 2F). Therefore, it can be concluded that MbCBF2 is a nucleus-located protein.

Subcellular Localization of MbCBF2 Protein
The specific location of MbCBF2 protein was determined by constructing the fusion expression vector of green fluorescent protein (GFP) and the MbCBF2 gene. It can be seen from Figure 2 that the MbCBF2-GFP fusion protein can only be detected in the nucleus ( Figure 2E), while the GFP-as the control-can be observed in the whole cytoplasm ( Figure 2B). At the same time, the location of the nucleus was confirmed by 4′, DAPI staining (Figure2F). Therefore, it can be concluded that MbCBF2 is a nucleus-located protein.

Expression Analysis of MbCBF2 in M. baccata
When the plants were in the control condition (CK), the MbCBF2 gene had the highest expression in new leaves, followed by roots, while the expression was lower in stems and mature leaves. This indicated that the organs in which the MbCBF2 gene produces an effect are mainly related to nutrient transport, such as roots and new leaves ( Figure 3A).
Under the treatment with high salt, cold, drought and high temperature, the expression of the MbCBF2 gene in the new leaves of M. baccata showed a tendency to increase and then decrease. After treatment with cold, high salt, drought and high temperature for 9 h, 12 h, 6 h and 6 h, respectively, the expression level of the MBCBF2 gene reached its highest ( Figure 3B). The expression trend of MBCBF2 in the roots was generally consistent with that in new leaves, and reached the highest expression at 12 h, 9 h, 2 h and 6 h, respectively, and then decreased slightly ( Figure 3C). The results showed that in the new leaves and roots of the M. baccata, these four stresses can cause the up-regulation of MbCBF2 expression.

Expression Analysis of MbCBF2 in M. baccata
When the plants were in the control condition (CK), the MbCBF2 gene had the highest expression in new leaves, followed by roots, while the expression was lower in stems and mature leaves. This indicated that the organs in which the MbCBF2 gene produces an effect are mainly related to nutrient transport, such as roots and new leaves ( Figure 3A).
Under the treatment with high salt, cold, drought and high temperature, the expression of the MbCBF2 gene in the new leaves of M. baccata showed a tendency to increase and then decrease. After treatment with cold, high salt, drought and high temperature for 9 h, 12 h, 6 h and 6 h, respectively, the expression level of the MBCBF2 gene reached its highest ( Figure 3B). The expression trend of MBCBF2 in the roots was generally consistent with that in new leaves, and reached the highest expression at 12 h, 9 h, 2 h and 6 h, respectively, and and four stress conditions. Each datum in the figure is taken as the average value obtained by repeating three times. Compared with the control group, the asterisks above the column indicate significant differences and extremely significant differences (*, p ≤ 0.05; **, p ≤ 0.01).

Overexpression of MbCBF2 in A. thaliana Enhances Low-Temperature Tolerance
It can be seen from the results of qPCR that MbCBF2 was very sensitive to low-temperature and high-salt stresses. Therefore, controlled by the CaMV 35S promoter, the MbCBF2 gene was transformed into A. thaliana for verification. Using WT and unload line (UL, A. thaliana plants with an 'empty vector' after genetic transformation) as controls, the results of RT-PCR in all T2 generation transformation lines showed that there were target fragments in 6 transformation lines (S1, S2, S3, S4, S5 and S6) ( Figure 4A).
It can be seen from Figure 4B that under the control conditions (low-temperature for 0 h), the phenotypes of all of the A. thaliana strains (WT, UL, S1, S4 and S5) were basically the same. However, after 10 h of treatment at −4 °C, the WT, UL, and transgenic (S1, S4 and S5) lines were injured to varying degrees, among which UL and WT had more obvious leaf wilt, and the leaves were seriously damaged. Compared with UL and WT, although some of the leaves of transgenic plants were injured, the overall wilting situation was not particularly obvious. and four stress conditions. Each datum in the figure is taken as the average value obtained by repeating three times. Compared with the control group, the asterisks above the column indicate significant differences and extremely significant differences (*, p ≤ 0.05; **, p ≤ 0.01).

Overexpression of MbCBF2 in A. thaliana Enhances Low-Temperature Tolerance
It can be seen from the results of qPCR that MbCBF2 was very sensitive to lowtemperature and high-salt stresses. Therefore, controlled by the CaMV 35S promoter, the MbCBF2 gene was transformed into A. thaliana for verification. Using WT and unload line (UL, A. thaliana plants with an 'empty vector' after genetic transformation) as controls, the results of RT-PCR in all T 2 generation transformation lines showed that there were target fragments in 6 transformation lines (S1, S2, S3, S4, S5 and S6) ( Figure 4A).
It can be seen from Figure 4B that under the control conditions (low-temperature for 0 h), the phenotypes of all of the A. thaliana strains (WT, UL, S1, S4 and S5) were basically the same. However, after 10 h of treatment at −4 • C, the WT, UL, and transgenic (S1, S4 and S5) lines were injured to varying degrees, among which UL and WT had more obvious leaf wilt, and the leaves were seriously damaged. Compared with UL and WT, although some of the leaves of transgenic plants were injured, the overall wilting situation was not particularly obvious. After 3 days of recovery, the survival rate was calculated. Under control conditions, the survival rates of all A. thaliana lines (WT, UL, S1, S4 and S5) were basically the same. However, when treated at −4 °C for 10 h, the survival rates of WT and UL were only 20.53% and 19.62%, while the S1, S4 and S5 transgenic plants survived better; their survival rates were 60.63%, 60.75% and 57.33%, respectively. After growing at low-temperatures for a period of time, more transgenic plants survived than WT and UL ( Figure 4C), indicating that the transgenic plants have better low-temperature tolerance.
In the control conditions, the UL, WT and transgenic A. thaliana lines showed no significant differences among all of the indicators. After low-temperature treatment, the contents of proline, chlorophyll and MDA, and the activities of SOD, CAT and POD changed, compared with the transgenic lines, the MDA contents of WT and UL were higher, but other indicators were lower than transgenic A. thaliana ( Figure 5). These datas indicated that transgenic A. thaliana plants had stronger antioxidant capacity than UL and WT, and that they can scavenge the intracellular reactive oxygen species (ROS) thaliana lines (S1, S4, S5), WT and UL under low-temperature stress and recovery. (C) A t-test showed that there were significant differences in the survival rates between transgenic A. thaliana (S1, S4 and S5) and WT lines under low-temperature conditions (**, p ≤ 0.01). The scale bar corresponds to 3 cm.
After 3 days of recovery, the survival rate was calculated. Under control conditions, the survival rates of all A. thaliana lines (WT, UL, S1, S4 and S5) were basically the same. However, when treated at −4 • C for 10 h, the survival rates of WT and UL were only 20.53% and 19.62%, while the S1, S4 and S5 transgenic plants survived better; their survival rates were 60.63%, 60.75% and 57.33%, respectively. After growing at low-temperatures for a period of time, more transgenic plants survived than WT and UL ( Figure 4C), indicating that the transgenic plants have better low-temperature tolerance.
In the control conditions, the UL, WT and transgenic A. thaliana lines showed no significant differences among all of the indicators. After low-temperature treatment, the contents of proline, chlorophyll and MDA, and the activities of SOD, CAT and POD changed, compared with the transgenic lines, the MDA contents of WT and UL were higher, but other indicators were lower than transgenic A. thaliana ( Figure 5). These datas indicated that transgenic A. thaliana plants had stronger antioxidant capacity than UL and WT, and that they can scavenge the intracellular reactive oxygen species (ROS) whilst better preventing plasma membrane peroxidation; thereby, the resilience of transgenic A. thaliana to cold stress was improved. whilst better preventing plasma membrane peroxidation; thereby, the resilience of transgenic A. thaliana to cold stress was improved. There was a significant difference between transgenic and WT A. thaliana in the treatment group, which was indicated by asterisks above the error bars (**, p ≤ 0.01).

Expression Analysis of Cold-Resistant Downstream Genes in MbCBF2-OE A. thaliana
A. thaliana triggers cold stress tolerance through the translation of CBF as an early cold response TF. Therefore, under cold treatment the expression changes of six key genes located downstream of the CBF TFs were analyzed (  [49]. After the plants were treated at a low temperature of −4 °C for 10 h, the expression levels of six genes in the WT and UL lines were significantly lower than those in the MbCBF2 transgenic lines, suggesting that the MbCBF2 can promote the up-regulation of the expression of AtRD29a, AtCOR6.6, AtCOR15a, AtERD10, AtCOR47 and AtRD29b, so as to enhance the tolerance of the plants to low-temperature stress.   [49]. After the plants were treated at a low temperature of −4 • C for 10 h, the expression levels of six genes in the WT and UL lines were significantly lower than those in the MbCBF2 transgenic lines, suggesting that the MbCBF2 can promote the up-regulation of the expression of AtRD29a, AtCOR6.6, AtCOR15a, AtERD10, AtCOR47 and AtRD29b, so as to enhance the tolerance of the plants to low-temperature stress.

Overexpression of MbCBF2 in A. thaliana Improved High-Salt Tolerance
In order to explore the role of MbCBF2 when the plants are in a high-salt environment, WT, UL and transgenic A. thaliana strains (S1, S4 and S5) were transplanted into plastic square pots with a substrate (nutrient soil and vermiculite: 1:1); then, the square pots were placed into a tray, and the plants were irrigated with 200 mM NaCl through the holes at the bottom of the pot. After 7 d, the phenotype of each strain was observed ( Figure 7A). Under the salt stress treatment, the leaves of the WT, UL and transgenic plants were all yellowed. Compared with the transgenic plants, the UL and WT leaves' yellowing was more serious. The survival rate of each A. thaliana was counted after 3 days of recovery, as shown in Figure 7B. Under the control conditions, all of the A. thaliana strains (WT, UL, S1, S4 and S5) have basically the same survival rate. However, under salt stress, the survival rates of the transgenic plants were 54.87%, 52.78% and 51.99%, respectively, while UL was 17.98% and WT was 18.04%. It is obvious that transgenic A. thaliana has a higher survival rate than UL and WT in a high-salt environment.

Overexpression of MbCBF2 in A. thaliana Improved High-Salt Tolerance
In order to explore the role of MbCBF2 when the plants are in a high-salt environment, WT, UL and transgenic A. thaliana strains (S1, S4 and S5) were transplanted into plastic square pots with a substrate (nutrient soil and vermiculite: 1:1); then, the square pots were placed into a tray, and the plants were irrigated with 200 mM NaCl through the holes at the bottom of the pot. After 7 d, the phenotype of each strain was observed ( Figure 7A). Under the salt stress treatment, the leaves of the WT, UL and transgenic plants were all yellowed. Compared with the transgenic plants, the UL and WT leaves' yellowing was more serious. The survival rate of each A. thaliana was counted after 3 days of recovery, as shown in Figure 7B. Under the control conditions, all of the A. thaliana strains (WT, UL, S1, S4 and S5) have basically the same survival rate. However, under salt stress, the survival rates of the transgenic plants were 54.87%, 52.78% and 51.99%, respectively, while UL was 17.98% and WT was 18.04%. It is obvious that transgenic A. thaliana has a higher survival rate than UL and WT in a high-salt environment.  In addition, the physiological indexes of all of the A. thaliana under normal and different concentrations of salt stress were measured. When growing under normal conditions, there were almost no differences in the physiological indexes of any of the A. thaliana, but after salt stress treatment, the contents of MDA and proline, and the activities of CAT, POD and SOD of transgenic A. thaliana were significantly higher than those of WT and UL, while the content of chlorophyll was significantly lower than that of WT and UL (Figure 8). The results showed that due to the expression of the MbCBF2 gene, transgenic A. thaliana can better adapt to a high-salt environment.   [53], it can be seen that in a normal growth environment, the expression of these genes in all A. thaliana strains was almost the same. (Figure 9). However, after 7 days of treatment with 200 mM NaCl, compared with the WT and UL strains, the expressions of AtCAT1, AtP5CS, AtNCED3, AtPIF1, AtPIF4 and AtSnRK2.4 were significantly higher in MbCBF2-overexpression strains. The results made clear that MbCBF2 could enhance the tolerance of plants to salt stress in two ways. There was a significant difference between transgenic and WT A. thaliana in the treatment group, which is indicated by asterisks above the error bars (**, p ≤ 0.01).  [53], it can be seen that in a normal growth environment, the expression of these genes in all A. thaliana strains was almost the same. (Figure 9). However, after 7 days of treatment with 200 mM NaCl, compared with the WT and UL strains, the expressions of AtCAT1, AtP5CS, AtNCED3, AtPIF1, AtPIF4 and AtSnRK2.4 were significantly higher in MbCBF2-overexpression strains. The results made clear that MbCBF2 could enhance the tolerance of plants to salt stress in two ways.

Discussion
In recent years, there has been more and more evidence that CBF TF is an indispensable factor in the plant stress response. CBF TFs can regulate the response of plants to drought, low-temperature and high-salt stress. The cold resistance of Forage Maize SAUMZ1 was enhanced by CBF1 gene [54], while LeCBF1 [55] helps to improve the ability of A. thaliana to adapt to drought/freezing injury. For MbDREB1 [56], its expression affects the growth state of A. thaliana under low temperature, high-salt and drought stress environments, and its overexpression can improve the tolerance of A. thaliana to these stresses. In addition, hormones also participate in the regulation of CBFs. Low temperatures can induce JA to remove ICE1/2 inhibition by JAZ1/4, a repressor of the JA signaling pathway, and to positively regulate the transcription of ICE and the expression of CBF1-3 [57]. The mutation of BIN2, a brassinolide-insensitive factor, enhanced the antifreeze performance of plants [58], while BZR1, BES1 and CES-the downstream TFs of BIN2-positively regulated the expression of A. thaliana CBFs and the antifreeze performance of plants [58,59].
Sequence homology analysis showed that the ORF of MbCBF2 was 663 bp, and that it encoded 220 amino acids. MbCBF2 protein had a conserved AP2 domain, in which the N-terminal region had nuclear localization signals, and the C-terminal region contains transcriptional activation domains belonging to the CBF family, with the typical structure of the CBF family [60][61][62]. MbCBF2 protein was different from other CBF family proteins in length, but had highly similar conserved sequences, such that it had similar functions, indicating that CBF family genes were highly conserved in the process of evolution. In addition, MbCBF2 protein was hydrophilic, because its average hydrophilic coefficient is −0.456. Through phylogenetic tree analysis, it can be concluded that MbCBF2 protein had the highest homology with PdCBF4 (Populus davidiana, KF880602.1) (Figure 1). From the results of subcellular localization, it can be concluded that MbCBF2 is a nuclear localization protein (Figure 2). Gene expression is specifical in different parts of the plant. Under normal culture conditions, MbCBF2 was expressed in the new leaves, roots, stems, and mature leaves.

Discussion
In recent years, there has been more and more evidence that CBF TF is an indispensable factor in the plant stress response. CBF TFs can regulate the response of plants to drought, low-temperature and high-salt stress. The cold resistance of Forage Maize SAUMZ1 was enhanced by CBF1 gene [54], while LeCBF1 [55] helps to improve the ability of A. thaliana to adapt to drought/freezing injury. For MbDREB1 [56], its expression affects the growth state of A. thaliana under low temperature, high-salt and drought stress environments, and its overexpression can improve the tolerance of A. thaliana to these stresses. In addition, hormones also participate in the regulation of CBFs. Low temperatures can induce JA to remove ICE1/2 inhibition by JAZ1/4, a repressor of the JA signaling pathway, and to positively regulate the transcription of ICE and the expression of CBF1-3 [57]. The mutation of BIN2, a brassinolide-insensitive factor, enhanced the antifreeze performance of plants [58], while BZR1, BES1 and CES-the downstream TFs of BIN2-positively regulated the expression of A. thaliana CBFs and the antifreeze performance of plants [58,59].
Sequence homology analysis showed that the ORF of MbCBF2 was 663 bp, and that it encoded 220 amino acids. MbCBF2 protein had a conserved AP2 domain, in which the N-terminal region had nuclear localization signals, and the C-terminal region contains transcriptional activation domains belonging to the CBF family, with the typical structure of the CBF family [60][61][62]. MbCBF2 protein was different from other CBF family proteins in length, but had highly similar conserved sequences, such that it had similar functions, indicating that CBF family genes were highly conserved in the process of evolution. In addition, MbCBF2 protein was hydrophilic, because its average hydrophilic coefficient is −0.456. Through phylogenetic tree analysis, it can be concluded that MbCBF2 protein had the highest homology with PdCBF4 (Populus davidiana, KF880602.1) (Figure 1). From the results of subcellular localization, it can be concluded that MbCBF2 is a nuclear localization protein (Figure 2). Gene expression is specifical in different parts of the plant. Under normal culture conditions, MbCBF2 was expressed in the new leaves, roots, stems, and mature leaves. Among them, MbCBF2 had the highest expression in new leaves, followed by roots and stems, and the lowest expression in mature leaves, as shown in Figure 3A. According to the results of real-time quantitative PCR, it can be known that when the new leaves were under the conditions of low temperatures, high salt, drought and high temperatures, the expression level of the MbCBF2 gene reached its highest at 9 h, 12 h, 6 h and 6 h, respectively ( Figure 3B); meanwhile, when the roots were also under such conditions, the expression level of the MbCBF2 gene reached its highest at 12 h, 9 h, 2 h and 6 h, respectively ( Figure 3C). This situation may be due to the fact that new leaves are less sensitive to salt and drought stress than the roots, because the root always suffers from these two stresses first. Compared with the roots, the response of new leaves to low-and high-temperature stress was faster, indicating that new leaves were the first to be stressed in low-temperature and high-salt environments. Therefore, the MbCBF2 gene can be induced and expressed by cold, high-salt, drought and high-temperature stress. At the same time, it can be seen that the sensitivity of MbCBF2 to low-temperature and high-salt stress is higher than that for drought and high-temperature stress.
In general, the production and elimination of free radicals maintain a dynamic balance, such that plant cells will not be damaged [63]. However, when plants are under abiotic stress, they will accumulate a large amount of reactive oxygen species in cells. In the short-term period of stress, plants can rely on their own protective enzymes to remove reactive oxygen species. However, over a certain period of time, the amount of reactive oxygen species exceeds the ability of plants to remove reactive oxygen species, which will damage plants [64,65]. The enzyme removal system in plants mainly includes SOD, POD and CAT. When plants are under environmental stress, H 2 O 2 is converted into reactive oxygen species in order to avoid damage [66]. The amount of MDA can determine the damage degree of the plant cell membrane, and its content is positively correlated with membrane permeability [67]. Compared with Chinese kale plants grown under normal conditions, kale plants under salt stress showed higher levels of MDA [68]. Overexpressed PeCBF4a enhanced the antioxidant capacity of transgenic plants and reduced the MDA content, but raised the activities of SOD, CAT and POD so as to reduce the harm to plants brought about by various adverse growth conditions [69]. This experiment studied the effects of MbCBF2 on plant growth status and growth under low-temperature and high-salt stress. Figures 4B and 7A showed the results of low-temperature and high-salt stress treatment on WT, UL and transgenic plants. UL, that is, A. thaliana without the vector inserted with the MbCBF2, was used as a negative control group to evaluate the function and effect of the foreign gene. From the morphological results, compared with the WT, the transgenic plants had stronger adaptability in low-temperature and high-salt environments. The survival rate of A. thaliana can be seen in Figures 4C and 7B. The transgenic plants had a higher survival rate, while the survival rates of UL and WT were basically the same, and both were lower than those of the transgenic plants. This result showed that transgenic plants have stronger cold tolerance and salt tolerance than WT and UL plants.
Under low-temperature and high-salt stress, the measured physiological index changes showed similar change trends, as shown in Figures 5 and 8. Compared with the WT and UL, CAT, SOD and POD activities, and chlorophyll, proline and MDA contents, the transgenic plants had significant changes. In addition to the decrease of MDA content, other indicators increased significantly, indicating that transgenic plants had strong vitality under lowtemperature and high-salt stress. In addition, the enzyme activity in transgenic plants also increased significantly, which improved the ability of transgenic plants to remove peroxides, thereby reducing the peroxidation of the cell plasma membrane and improving the survival rate of the plants under low-temperature and high-salt conditions. Although the enzyme activity of UL and WT also increased, it was still lower than that of the transgenic plants. At the same time, under low-temperature and high-salt stress, the stability of the chlorophyll content and the increase of the proline content provide a guarantee for the normal life activities of plants.
The expression of stress tolerance genes is affected by a variety of TFs, which play an extremely important role in the response of plants to stress (including biological and abiotic stress). CBF binds to the CRT/DRE cis-acting elements of downstream genes to stimulate the up-regulation of the expression of these genes, thereby enhancing the stress tolerance of the plants. The expression results of cold-responsive genes (AtCOR15a, AtRD29a, AtCOR6.6, AtERD10, AtCOR47 and AtRD29b) downstream of CBF were analyzed under control and low-temperature treatment conditions. As shown in Figure 6, the results showed that MbCBF2 TF promoted the expression of AtCOR15a, AtRD29a, AtCOR6.6, AtERD10, AtCOR47 and AtRD29b more obviously under low-temperature conditions. ABA is the main substance for plants to deal with salt stress. ABA can alleviate the ion stress and osmotic stress caused by excessive salt. Therefore, the water balance and the integrity of the cell membrane structure are maintained. Under high-salt conditions, the expression of the ABA synthesis-related gene AtNCED3 and the ABA signal transduction-related gene AtSnRK2.4 were significantly up regulated in transgenic A. thaliana. According to this phenomenon, it can be speculated that MbCBF2 may participate in the response of plants to salt stress by regulating ABA synthesis and signal transduction. As shown in Figure 9, due to the induction of MbCBF2 expression in transgenic A. thaliana, the other salt stress-responsive genes AtCAT1, AtP5CS, AtPIF1 and AtPIF4 were also significantly up-regulated, thereby enhancing the plant adaptation to salt stress.
To sum up, combined with previous studies and the above results, a potential model was derived to describe the mode of action of MbCBF2 under salt and cold stress ( Figure 10). First, salt stress induced the expression of MbCBF2, thereby evidently in creasing the expression level of AtNCED3, promoting ABA biosynthesis and signal transduction, causing a significant up-regulation of AtSnRK2.4 expression and enhancing the adaptability of transgenic plants to salt stress. In addition, the expression levels of the key genes AtCAT1, AtP5CS, AtPIF1 and AtPIF4 in response to salt stress were also significantly increased when plants were stressed, indicating that key genes under salt stress could be regulated by MbCBF2, thereby enhancing the salt tolerance of transgenic plants. When a cold shock signal is felt, MbCBF2 is induced by cold stress, and its encoded product specifically binds to the CRT/DRE cis-elements on the promoters of multiple anti-stress genes, causing the high expression of downstream cold-regulated genes (AtCOR15a, AtRD29a, AtCOR6, AtERD10, AtCOR47 and AtRD29b), thereby enhancing the cold tolerance of plants.
The expression of stress tolerance genes is affected by a variety of TFs, which play an extremely important role in the response of plants to stress (including biological and abiotic stress). CBF binds to the CRT/DRE cis-acting elements of downstream genes to stimulate the up-regulation of the expression of these genes, thereby enhancing the stress tolerance of the plants. The expression results of cold-responsive genes (AtCOR15a, AtRD29a, AtCOR6.6, AtERD10, AtCOR47 and AtRD29b) downstream of CBF were analyzed under control and low-temperature treatment conditions. As shown in Figure 6, the results showed that MbCBF2 TF promoted the expression of AtCOR15a, AtRD29a, AtCOR6.6, AtERD10, AtCOR47 and AtRD29b more obviously under low-temperature conditions. ABA is the main substance for plants to deal with salt stress. ABA can alleviate the ion stress and osmotic stress caused by excessive salt. Therefore, the water balance and the integrity of the cell membrane structure are maintained. Under high-salt conditions, the expression of the ABA synthesis-related gene AtNCED3 and the ABA signal transduction-related gene AtSnRK2.4 were significantly up regulated in transgenic A. thaliana. According to this phenomenon, it can be speculated that MbCBF2 may participate in the response of plants to salt stress by regulating ABA synthesis and signal transduction. As shown in Figure 9, due to the induction of MbCBF2 expression in transgenic A. thaliana, the other salt stress-responsive genes AtCAT1, AtP5CS, AtPIF1 and AtPIF4 were also significantly up-regulated, thereby enhancing the plant adaptation to salt stress.
To sum up, combined with previous studies and the above results, a potential model was derived to describe the mode of action of MbCBF2 under salt and cold stress ( Figure 10). First, salt stress induced the expression of MbCBF2, thereby evidently in creasing the expression level of AtNCED3, promoting ABA biosynthesis and signal transduction, causing a significant up-regulation of AtSnRK2.4 expression and enhancing the adaptability of transgenic plants to salt stress. In addition, the expression levels of the key genes AtCAT1, AtP5CS, AtPIF1 and AtPIF4 in response to salt stress were also significantly increased when plants were stressed, indicating that key genes under salt stress could be regulated by MbCBF2, thereby enhancing the salt tolerance of transgenic plants. When a cold shock signal is felt, MbCBF2 is induced by cold stress, and its encoded product specifically binds to the CRT/DRE cis-elements on the promoters of multiple anti-stress genes, causing the high expression of downstream cold-regulated genes (AtCOR15a, AtRD29a, AtCOR6, AtERD10, AtCOR47 and AtRD29b), thereby enhancing the cold tolerance of plants.

Plant Material and Growth Conditions
The tissue culture seedlings of M. baccata (Malus baccata (L.) Borkh) came from the National Apple Germplasm Resource Garden, and were a cold-resistant genotype with ID DGB0367 grown in propagation medium containing Murashige and Skoog (MS), Agar, and 1 mg/L 6-benzylaminopurine (6-BAP) + 0.5 mg/L indole-3-butyric acid (IBA). After growing for 30 days, the robust tissue culture seedlings were transferred to the rooting medium. The medium used was MS solid medium of 1.2 mg/L IBA. The tissue culture seedlings were grown in the medium for 45 days. The seedlings with thick roots were moved into Hogland nutrient solution to continue to grow, and the nutrient solution was changed every 3 days. Clean and sterilized glass bottles with a height of 20 cm were used for the hydroponics. At the same time, a floating plate was placed at the mouth of the bottle to carry the seedlings for colonization. In total, 2/3 of them were immersed in the nutrient solution, and the environment was kept ventilated during the root seedling raising process. The incubator always maintained a temperature range of 24-26 • C and a relative humidity of about 80% [66]. The stress treatment was carried out according to the method of Han et al. [70]. When 8 to 9 mature leaves were grown (the leaves were fully expanded), the hydroponic seedlings were placed under the conditions of high salt (the small plants were irrigated with Hogland nutrient solution containing 200 mM NaCl), low temperatures (the incubator temperature was set to −4 • C), dehydration (the small plants were irrigated with Hogland nutrient solution containing 20% PEG6000 to simulate drought conditions) and high temperatures (the incubator temperature was 37 • C). The control group consisted of hydroponic seedlings cultured in normal Hoagland nutrient solution. After 0, 2, 6, 9, 12 and 16 h of treatment, root, stem, new leaf and mature leaf samples (100 mg) were randomly collected and sealed, including control and treated plant samples (3 plants were selected, respectively), and were immediately put into liquid nitrogen. Then, for the later extraction of RNA, they were stored at −80 • C [71]. All of the results were obtained from 3 independent experiments on each of the plants at each time point.

Cloning and Quantitative Expression Analysis of MbCBF2
The EasyPure Plant RNA Kit (TransGen Biotech, Beijing, China) was used to extract the total RNA from the new leaves, mature leaves, roots and stems, and the DNA was purified by RNase-Free DNase I purification. We used the HiFiScript gDNA Removal cDNA Synthesis Kit (Kangweishiji, Beijing, China) and Oligo (dT) 18 as the primer for reverse transcription to complete the synthesis of the cDNA First Strands. A pair of specific primers (MbCBF2-F and MbCBF2-R, Table S1) were designed and synthesized according to the homologous region of MbCBF2, such that the full-length sequence of cDNA could be amplified. The PCR amplification used cDNA as a template to obtain the target fragment; it was ligated into the pEASY-T1 Cloning Kit (TransGen Biotech, Beijing, China), and positive colonies were transformed, screened and sent for sequencing. According to the viewpoints of Jiang and Zhou, the MbCBF2 method was analyzed by real-time PCR [72].
The real-time PCR primers q-PCR-F and q-PCR-R were designed based on the conservative part of the MbCBF2 sequence. The control group was the Actin gene, which can be stably expressed under different conditions. Actin primers were designed according to the sequences in the GenBank database (Actin-F and Actin-R). Table S1 showed the primer sequences. The expression level of the MbCBF2 gene was detected using TransStart ® Green qPCR Super Mix (TransGen Biotech, Beijing, China) according to the manufacturer's protocol. The following was the PCR reaction system: 2xMix 12.5 µL, ddH 2 O 9 µL, cDNA 1.5 µL, primer F 1 µL, and primer R 1 µL. The PCR reaction procedure was as follows: 95 • C for 5 min, 95 • C for 45 s, 56 • C for 1 min, 72 • C for 1 min, 35 cycles, and 72 • C for 5 min. We used the 2 −∆∆CT method to analyze the relative transcript level [73,74].

Subcellular Localization Analysis of the MbCBF2 Protein
SalI and BamHI are two sites of the pSAT6-GFP-N1 vector. MBCBF2 ORF was cloned between the two sites. The transformation of the MBCBF2 GFP construct containing modified red-shift GFP at the SalI-BamHI site into onion epidermal cells was achieved using particle bombardment technology [75,76]. The nuclear marker in the nuclear detection was DAPI staining. Confocal microscopy observed the transient expression of MbCBF2-GFP fusion protein (LSM 510 Meta, Zeiss, Germany).

Construction of Transgenic A. thaliana
Firstly, the restriction sites of SalI and BamHI were added at the 5 and 3 ends of MbCBF2 cDNA by PCR to build an expression vector of transformed columbia-0 ecotype A. thaliana. In order to construct the pCAMBIA2300-MbCBF2 overexpression vector, a homologous recombinase (Novizan C115-01/02) was used to join the PCR product with the restriction site to the pCAMBIA2300 vector. The Agrobacterium-mediated method was used in the GV3101 transformation, and the MbCBF2 gene was introduced into columbia-0 ecotype A. thaliana by the inflorescence infection method; the transformation of the empty plasmids pCAMBIA2300-MbCBF2 and pCAMBIA2300 into WT A. thaliana was realized [77]. Plants successfully transfected with the MbCBF2 gene on 1/2 MS medium containing 50 mg/L Kanamicin were selected. Through further screening, T 3 generation plants were obtained and further analyzed.

Determination of the Related Physiological Indicators
The selected WT, UL and transgenic lines (S1, S4, S5) were sown in the medium, respectively, until the A. thaliana seedlings grew four leaves; they were then moved to a nutrition bowl and marked. The UL, WT and transgenic plants were divided into three groups: one group was cultured normally at 23 • C, while the other two groups were placed under −4 • C for cold treatment, and were treated with 200 mM NaCl, respectively. After continuous watering for 7 days, we observed the phenotype of A. thaliana. Each A. thaliana leaf was sampled, and the content of chlorophyll, proline and MDA, and the activities of SOD, POD and CAT were measured [78][79][80][81]. The treated A. thaliana were placed in a lighted incubator for normal watering in order to recover; we then observed the phenotype of each A. thaliana, and ascertained its survival rate.

Analysis of the Downstream Gene Expression of MbCBF2
We extracted the mRNA of WT, UL and MbCBF2 transgenic A. thaliana grown under normal conditions, low-temperature stress, and salt stress, respectively, and reverse transcribed them into the first-strand cDNA, which was used as a template. With AtActin as the internal reference, qPCR tests were conducted on several significant regulatory genes downstream of CBF TF: key genes in response to low-temperature stress (AtCOR15a, AtRD29a, AtCOR6.6, AtERD10, AtCOR47 and AtRD29) and salt-related genes (AtNCED3, AtCAT1, AtP5CS, AtPIF1, AtPIF4 and AtSnRK2.4). The specific primers used are shown in Table S1. The reaction system is the same as that in Section 2.2.

Statistical Analysis
One-way analysis of variance was performed using SPASS data processing system software. We repeated all of the experiments 3 times, and measured the standard error (±SE), respectively. The statistical difference is called significant at * p ≤ 0.05 and ** p ≤ 0.01.

Conclusions
In this experiment, a new CBF-TF named MbCBF2 was isolated from Malus plants. The MbCBF2 protein had a conserved AP2 domain and a typical structure for the CBF family, as it belonged to the CBF family. According to the subcellular localization results, the MbCBF2 protein was located in the nucleus. According to the phylogenetic tree results, the MbCBF2 protein had the highest homology with PdCBF4. Low-temperature, high-salt, hightemperature and drought stress can all induce the up-regulated expression of the MbCBF2 gene in M. baccata. In addition, under the stimulation of low-temperature and high-salt conditions, the expression of MbCBF2 in new leaves and roots was higher. In A. thaliana, MbCBF2 responds positively to low-temperature and osmotic stress. After the introduction of MbCBF2 into A. thaliana, the adaptability of transgenic A. thaliana to cold and high-salt environments was significantly enhanced. In addition, the high expression of MbCBF2 also significantly changed many physiological indexes of transgenic A. thaliana. Except for the content of MDA, the contents of proline and chlorophyll, and the activities of CAT, POD, and SOD of the WT and UL lines were significantly lower than those of transgenic A. thaliana. MbCBF2 can also regulate the expression of its downstream genes in transgenic lines. After CBF2 binds with cis-acting elements at low temperature, it positively regulates the expression of the key genes AtCOR15a, AtRD29A/B and AtCOR6.6/47, and positively regulates the expression of AtCAT1, AtP5CS and AtPIF1/4 under salt stress. It was also found that the expression of the ABA synthesis-related gene AtNCED3 and the ABA signal transduction-related gene AtSNRK2.4 were also upregulated in transgenic A. thaliana under high-salt conditions. Therefore, the overexpression of the MbCBF2 gene further improved the adaptability and tolerance of transgenic plants to low-temperature and high-salt environments. Therefore, the overexpression of the MbCBF2 gene further improved the adaptability and tolerance of transgenic plants to low-temperature and high-salt environments.