Overexpression of a Malus baccata MYB Transcription Factor Gene MbMYB4 Increases Cold and Drought Tolerance in Arabidopsis thaliana

In the natural environment, plants often face unfavorable factors such as drought, cold, and freezing, which affect their growth and yield. The MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor family is widely involved in plant responses to biotic and abiotic stresses. In this study, Malus baccata (L.) Borkh was used as the research material, and a gene MbMYB4 of the MYB family was cloned from it. The open reading frame (ORF) of MbMYB4 was found to be 762 bp, encoding 253 amino acids; sequence alignment results and predictions of the protein structure indicated that the MbMYB4 protein contained the conserved MYB domain. Subcellular localization showed that MbMYB4 was localized in the nucleus. In addition, the use of quantitative real-time PCR (qPCR) technology found that the expression of MbMYB4 was enriched in the young leaf and root, and it was highly affected by cold and drought treatments in M. baccata seedlings. When MbMYB4 was introduced into Arabidopsis thaliana, it greatly increased the cold and drought tolerance in the transgenic plant. Under cold and drought stresses, the proline and chlorophyll content, and peroxidase (POD) and catalase (CAT) activities of transgenic A. thaliana increased significantly, and the content of malondialdehyde (MDA) and the relative conductivity decreased significantly, indicating that the plasma membrane damage of transgenic A. thaliana was lesser. Therefore, the overexpression of the MbMYB4 gene in A. thaliana can enhance the tolerance of transgenic plants to cold and drought stresses.


Introduction
In nature, the growth and development of plants are often affected by the environment, where the harsh natural environment may even cause irreversible damage to plants, leading to plants' death [1]. In recent years, the development of molecular biology has allowed people to deeply understand the resistance mechanism of plants to adversity stress in terms of gene expression, transcriptional regulation, and signal transduction [2]. Among them, transcriptional regulation plays a connecting role in plant stress response. Transcriptional regulation means that transcription factors regulate the expression of a series of genes by binding to the cis-acting elements in the promoter region, and it is an important part of the plant stress response mechanism [3,4].
The MYB (v-myb avian myeloblastosis viral oncogene homolog) transcription factor family is one of the largest transcription families in plants [5,6], and it plays an important role in plant growth, development, and metabolic regulation [7]. The R2R3-MYB transcription factor is the most abundant MYB protein in plants. Its DNA binding region is composed of two homologous MYB domains (R2, R3) and binds to the target sequence through the synergistic effect of R2 and R3 specifically [8,9]. Most R2R3-MYB proteins have transcriptional activation domains at the C-terminus, which play a key role in the process of plant cell differentiation, hormone response, secondary metabolism, and resistance to biotic and abiotic stresses [10][11][12].
Cold is a common abiotic stress during plant growth and development. Cold stress will increase the permeability of plant cell membranes, leading to the extravasation of solutes in the membrane, imbalance of water metabolism, and abnormal photosynthesis and respiration rates [13]. It further causes plant leaves to wither, which seriously affects plant growth and yield [14,15]. MYB transcription factor genes can play a key role in plant response to cold stress. Studies have shown that OsMYB2 encodes a type of R2R3 transcription factor in rice, and the overexpression of this gene in rice can enhance the resistance of transgenic plants to cold [16]; MdMYB108L can regulate the expression of downstream CBF genes, thereby improving the cold resistance of Malus domestica [17]; MdMYB23 can enhance the cold resistance of M. domestica mainly by the accumulation of proanthocyanidins [18], and similar findings have also been found in Arabidopsis thaliana and maize [19,20].
During the growth of plants, drought and salt stress usually occur concurrently. At present, many MYB genes have been isolated and cloned in A. thaliana, tobacco, and some food and commercial crops [5,21], where the salt and drought resistance of transgenic plants are significantly improved due to the expression of these genes. Studies have shown that the expression of AtMYB20 is induced by high salt and drought stress; under stress conditions, the chlorophyll content of AtMYB20 transgenic plants is significantly higher than that of wild-type (WT); and the water loss rate is lower than that of WT, indicating that overexpression of AtMYB20 can improve the salt and drought tolerance of transgenic plants [22]. Overexpression of the OsMYB48-1 gene in rice increased the expression of abscisic acid (ABA) synthesis genes (OsNCED4, OsNCED5) in transgenic plants, and the survival rate of transgenic plants was significantly higher than that of WT, indicating that the OsMYB48-1 gene regulates ABA accumulation to improve the drought resistance of transgenic plants [23].

Cloning and Bioinformatics Analysis of MbMYB4
ExPASy ProtParam analysis results showed that the full length of the MbMYB4 gene was 762 bp and encoded 253 amino acids (Supplementary Figure S1A). The predicted theoretical molecular weight (MW) of the protein was 28.843 kDa, the theoretical isoelectric point (pI) was 6.84, and the average hydrophilic coefficient was −0.839, which indicated that the protein was hydrophilic. Among the amino acids contained in the MbMYB4 protein, Ser (12.0%), Asn (8.0%), Lys (7.2%), and Glu (6.8%) accounted for a large proportion.
MYB amino acid sequences of other species were compared with the MbMYB4 protein sequence. The results showed that the MbMYB4 protein and other MYB proteins had roughly the same R2 and R3 conserved domains ( Figure 1A). Furthermore, the phylogenetic tree showed that the MbMYB4 protein had the highest homology with MdMYB4 (M. domestica) ( Figure 1B). As shown in Figure 2A, the secondary structure of the MbMYB4 protein contained 25.90% of α helix, 4.38% of β turn, 66.14% of random coils, and 3.59% of extended chain. The amino acid sequence of MbMYB4 had two SANT conserved domains ( Figure 2B), and it was speculated that MbMYB4 belongs to the R2R3-MYB family. Using the SWISS-MODEL online analysis tool to predict the tertiary structure of MbMYB4, it was found that the protein had a HTH structure, which was consistent with the predicted result of its secondary structure ( Figure 2C).

Subcellular Localization of MbMYB4 Protein
Based on the subcellular localization, the specific location of MbMYB4 protein functioning in the cell can be determined. Under the fluorescence confocal microscope (LSM 900, Precise, Beijing, China), the green fluorescent protein (GFP) as a control was distributed throughout the cell ( Figure 3B), while the fluorescence of the MbMYB4-GFP fusion protein was only distributed in the nucleus ( Figure 3E). In addition, observing the condition of the cell nucleus after DAPI staining can further prove that the MbMYB4 protein was located in the cell nucleus ( Figure 3C,F).

Expression Analysis of MbMYB4 in Malus baccata
Analyzing the expression pattern of the MbMYB4 gene found that MbMYB4 can be expressed in the young leaf, stem, root, and mature leaf, but the expression levels in these parts were different. Under control conditions, the expression of MbMYB4 was higher in the root and young leaf than the mature leaf and stem of M. baccata seedlings ( Figure 4A). Under the five stresses of low-temperature, high-salt, dehydration, high-temperature, and ABA, the expression level of MbMYB4 in the young leaf increased first and then decreased within 12 h after treatment, and the expression level peaked at 3 h, 7 h, 7 h, 3 h, and 5 h of treatment, respectively ( Figure 4B). Similarly, the expression level in the root also showed the same trend, reaching peaks at 5 h, 5 h, 3 h, 5 h, and 3 h of treatment, respectively ( Figure 4C), and under cold and drought stresses, the expression levels of MbMYB4 in the young leaf and root were higher than those under other stress treatments.

Overexpression of MbMYB4 in A. thaliana Improved Cold Tolerance
To investigate the role of MbMYB4 in response to cold stress, MbMYB4-overexpressing transgenic A. thaliana was generated. Using wild-type (WT) and empty vector (UL) lines as controls, quantitative real-time PCR (qPCR) analysis was performed on six T 2 transformed lines (S1, S2, S3, S4, S5, and S6 in Figure 5A). The results showed that the target fragment did not appear in WT and UL, while the six T 2 transgenic plants amplified bands consistent with the target gene fragment, indicating that the target gene had been integrated into T 2 generation transgenic lines (S1, S2, S3, S4, S5, and S6 in Figure 5A). As, among the six transgenic lines, S1, S3, and S5 have higher DNA relative expression levels, these three lines were cultured to the T 3 generation of transgenic A. thaliana.
WT, UL, and T 3 transgenic A. thaliana were cultured in the light incubator for about 3 weeks, moved to −6 • C for cold treatment for 14 h, and finally cultivated under normal conditions for 7 days. The results showed that cold treatment caused different levels of damage to transgenic A. thaliana (S1, S3, and S5), UL, and WT ( Figure 5B), but WT and UL A. thaliana were more severely damaged, and their leaves wilted more deeply. Seven days after the stress was removed, most of WT and UL died, the survival rates were only 36% and 26%, while the average survival rate of transgenic A. thaliana was 77% ( Figure 5C).
In addition, we measured the content of MDA, chlorophyll, and proline, the POD and CAT activity, and relative conductivity in WT, UL, and transgenic A. thaliana (S1, S3, and S5) under normal conditions (25 • C) and cold treatments ( Figure 6). There was almost no difference in the physiological indicators of each plant before treatment. After cold treatment, the activity of POD and CAT and the content of proline increased in all plants, but in contrast, the increases in these indexes were greater in transgenic A. thaliana; the content of MDA and relative conductivity also increased due to cold stress, while the increase in transgenic A. thaliana was lower than those in WT and UL; the chlorophyll content of all A. thaliana lines decreased under cold conditions, but the chlorophyll content of transgenic A. thaliana decreased slighter compared with WT and UL. These indicate that the expression of MbMYB4 improves the cold resistance of transgenic A. thaliana.

Expression Analysis of Cold Tolerance-Related Genes in A. thaliana Overexpressing MbMYB4
The molecular regulation pathway of A. thaliana responding to cold stress mainly relies on the CBF (CRT/DRE-binding factor) transcription factor [24]. Therefore, we analyzed the expression changes of several key genes AtCBF1, AtCBF3, AtRD29a, and AtCOR15a located downstream of the MYB transcription factor under cold treatment ( Figure 7). After 14 h of cold treatment at −6 • C, the expression levels of AtCBF1, AtCBF3, AtRD29a, and AtCOR15a in MbMYB4-overexpression transgenic lines were significantly higher than those of WT and UL lines, indicating that the MbMYB4 transcription factor positively regulates AtCBF1 and AtCBF3. Then, we further promoted the expression of AtRD29a and AtCOR15a to improve the resistance of plants to cold stress.

Overexpression of MbMYB4 in A. thaliana Improved Drought Tolerance
In order to understand the role of MbMYB4 in response to drought stress, we stopped watering transgenic A. thaliana (S1, S3, and S5 in Figure 8A), UL, and WT for 10 days to simulate drought stress. Under normal growth conditions, the transgenic lines have the same growth state as WT and UL plants. After stopping watering for 10 days, the leaves of all plants turned slightly yellow. Then, after normal watering (stress removed) for 7 days, the state of transgenic lines was significantly better than that of other lines ( Figure 8A). The survival rates of S1, S3, and S5 reached 76%, 74%, and 85%, respectively, while WT and UL survival rates were only 27% and 49%, respectively ( Figure 8B).    In addition, the measurement of the physiological indicators of all A. thaliana under normal and drought conditions found that when plants were under sufficient water conditions, there were no significant differences in the physiological indexes of WT, UL, and transgenic A. thaliana, but when plants were under drought stress, the activity of POD, CAT, and the content of proline in transgenic A. thaliana were higher than those in WT and UL plants. The increase in MDA content and relative conductivity and the decrease in chlorophyll content in WT and UL A. thaliana were greater than those of transgenic A. thaliana ( Figure 9). The results showed that the expression of the MbMYB4 gene increased the resistance of transgenic A. thaliana to drought stress.

Expression Analysis of Drought Tolerance-Related Genes in A. thaliana Overexpressing MbMYB4
The results of the analysis of MbMYB4 expression showed that ABA can induce the expression of MbMYB4. The expression of the ABA synthesis gene AtNCED3 and the ABA signal transduction-related gene AtSnRK2.4 in MbMYB4 transgenic A. thaliana was further explored (Figure 10), and it was found that under normal conditions, the expression levels of AtNCED3 and AtSnRK2.4 were not significantly different among all A. thaliana lines, but under drought stress, the expression levels of AtNCED3 and AtSnRK2.4 in MbMYB4overexpression lines were significantly higher than those of WT and UL lines, indicating that MbMYB4 can participate in the response of plants to drought stress through two pathways: regulating ABA synthesis and ABA signal transduction. In addition, the expression of AtCAT1 and AtP5CS genes increased significantly under drought stress. This indicates that MbMYB4 can regulate the key genes of drought stress to improve the scavenging ability of reactive oxygen species (ROS), thereby enhancing the drought resistance of plants.

Discussion
MYB transcription factors in plants contain a conserved MYB domain, and its DNA binding domain contains 1 to 4 incompletely repeated amino acid sequence structures, called R structures (R1, R2, and R3). The R structure is a folded protein composed of about 50 amino acids, including a series of highly conserved amino acid residues and spacer sequences, among which the amino acid residues participate in the DNA binding process in the form of helix-turn-helix (HTH) [25][26][27]. The R2R3-MYB protein is the largest MYB protein subclass in plants, and it contains two conserved domains, R2 and R3. This experiment used M. baccata as the test material, and its genome has not been sequenced. Therefore, using MdMYB25 (NM_001293983.1, M. domestica) as the reference sequence, specific primers were designed to amplify the target gene MbMYB4. Sequence analysis revealed that the ORF of MbMYB4 is 762 bp, encoding 253 amino acids; the average hydrophilic coefficient of the protein is -0.839, which is a hydrophilic protein (Supplementary Figure S1A). Analysis of the conserved domains of MbMYB4 protein revealed that there are two SANT-MYB DNA binding domains, which is speculated to belong to the R2R3-MYB family ( Figure 1A). Compared with the highly conserved MYB domain, other regions of the R2R3-MYB protein have a high degree of variability [28]. The MbMYB4 protein sequence was compared with the MYB protein sequences of other species. The results showed that the MbMYB4 protein is highly similar to the conserved sequences of other MYB family proteins, but the nonconserved region is quite different. This feature is consistent with characteristics of transcription factors. The evolutionary tree showed that MbMYB4 is most related to M. domestica MdMYB4 ( Figure 1B).
Studies have shown that the functional area of transcription factors in plants is mainly located in the nucleus. Han et al. found that MbNAC transcription factor proteins are located in the nucleus [29,30]. After constructing the MbMYB4-GFP fusion transient expression vector, it was introduced into onion epidermal cells by the particle bombardment method, the distribution of the tag protein in the cells was observed under a confocal microscope, and the results showed that the MbMYB4 protein was localized in the nucleus ( Figure 3). This is also consistent with the distribution of the MYB protein in the M. domestica by previous studies [31,32].
MYB transcription factors are involved in the growth and development of plants. For example, AtMYB33 and AtMYB65 are expressed differently in various tissues and organs [33]; AtMYB118 is highly expressed in the leaf and participates in leaf morphogenesis [34]. In this study, the expression levels of MbMYB4 in the young leaf and root of M. baccata were higher ( Figure 4A), indicating that it may play an important role in the growth and development of the root and young leaf. The results of this study showed that abiotic stresses such as low-temperature, high-salt, dehydration, high-temperature, and ABA all induce the expression of MbMYB4, and the expression of the gene under different stresses changes with the treatment time. Under low-temperature stress and dehydration stress, the expressions of MbMYB4 in the young leaf and root were upregulated most significantly ( Figure 4B,C). Under low-temperature stress, the expression levels of MbMYB4 in the young leaf and root reached their peaks at 3 h and 5 h of treatment, and they were 7.8 times and 7.9 times higher than those in untreated groups, respectively. Under dehydration stress, the expression levels of MbMYB4 in the young leaf and root reached their peaks at 7 h and 3 h of treatment, and the expression level was 8.7 times and 7.5 times those of the untreated group, respectively. Most MYB transcription factors involved in the response to drought stress require ABA as a medium [35]. Under ABA stress, the expression levels of MbMYB4 in the young leaf and root were significantly different from the control at 5 h and 3 h, and the expression levels were significantly higher than those of the untreated group. In high-salt and high-temperature stress, MbMYB4 in the root was more sensitive to high-salt stress ( Figure 4C). This showed that when M. baccata are in a growth environment with a high salt concentration, the root receives the salt stress signal first. Therefore, the root can give priority to responding to adverse conditions. After transforming the model plant A. thaliana with MbMYB4, WT, UL, and transgenic lines were treated with cold and drought. After cold and drought stresses, all plants showed damage. However, the degree of yellowing and wilting of transgenic A. thaliana was lighter. Statistics of their survival rate showed that the survival rate of transgenic A. thaliana is higher than those of WT and UL A. thaliana. The results showed that MbMYB4 improves the cold and drought tolerance of transgenic plants.
In order to explore the mechanism by which this gene enhances plant cold and drought tolerance, it is necessary to study its physiological, biochemical, and molecular regulation levels. When plants are under cold stress, their cell plasma membranes become more permeable, electrolytes and soluble substances leak out, and electrical conductivity increases. Therefore, the measurement of relative electrical conductivity is a method used to determine the resistance of plants to stress [36]. Under cold and drought stresses, compared with WT and UL, the relative conductivity of overexpression transgenic lines has a smaller increase, indicating that the cell membrane has less damage and plants have less damage from stress. According to the phenotypic analysis of plants under cold and drought stresses, cold stress can decompose the chlorophyll in plants and turn leaves yellow, make plants unable to photosynthesize, and die in severe cases. Therefore, the chlorophyll content in plants can also be used as a measure of plant resistance [37]. Under stress conditions, the chlorophyll content of transgenic plants was indeed significantly higher than that of wild type. In addition, plants subjected to cold and drought stresses can also enhance the water holding capacity by accumulating osmotic adjustment substances [38]. Proline is an important type of osmotic adjustment substance. Proline in plants can promote protein hydration and increase the content of soluble protein [39], thereby maintaining enzyme activity under cold and drought conditions. According to the changes in the proline content of each line, it can be seen that the cytoplasm of transgenic plants suffers less damage under cold and drought conditions. Due to the high level of ROS in the body when plants are dehydrated, which causes membrane damage, protective enzymes such as SOD, POD, and CAT begin to work to remove excessive reactive oxygen species to maintain intracellular balance. At the same time, studies have found that the activities of POD and CAT in the root of cold-acclimated rice are significantly enhanced, indicating that these antioxidant enzymes also play the most important role in cold resistance [40]. The results of the study showed that, compared with WT and UL A. thaliana, the activity of CAT and POD in transgenic A. thaliana with MbMYB4 was significantly increased, and the content of MDA was significantly reduced. This shows that transgenic A. thaliana has a stronger ability to scavenge superoxide ions and a stronger tolerance to cold and drought.
Studies have shown that MYB transcription factors can bind to the CBF promoter region [41,42]. CBF induces the expression of downstream genes such as CORs, RDs, and LTIs by combining CRT/DRE cis-acting elements to improve the cold resistance of plants [43][44][45][46][47][48]. The expressions of two key genes CBF1 and CBF3 [44] in the CBF-dependent pathway and their downstream cold-responsive genes COR15a [14] and RD29a [49] were analyzed under normal conditions and cold treatment. The results showed that MbMYB4 can promote the expression of AtCBF1, AtCBF3, AtCOR15a, and AtRD29a in transgenic A. thaliana. Therefore, MbMYB4 can participate in the response to cold stress through a CBF-dependent pathway.
The ABA signal transduction pathway is an important pathway in plant drought stress response. Under drought stress, plants can induce stomatal closure by synthesizing ABA, thereby reducing water loss and improving drought tolerance. In A. thaliana, a heterologous expression of MYB10 has been shown to increase ABA hypersensitivity and enhance drought tolerance [22,50]. The expressions of ABA synthesis-related gene AtNCED3 and ABA signal transduction-related gene AtSnRK2.4 in transgenic A. thaliana were analyzed, and it was found that their expressions were upregulated under drought conditions. This indicated that MbMYB4 could participate in the process of response to drought by regulating ABA synthesis and ABA signal transduction. In addition, the overexpression of GaMYB85 can promote the accumulation of free proline in transgenic A. thaliana, and the drought stress-responsive genes RD22, ADH1, RD29A, P5CS, and ABI5 are also induced to express, thereby enhancing the resistance of plants to drought and salt stress [51]. Drought stress can induce the expression of CAT1 and significantly increase the activity of catalase. CAT1 acts as a major scavenger of H 2 O 2 and plays an important role in adaptation to drought stress [52]. The expression of MbMYB4 in transgenic A. thaliana induces the expression of the drought stress-responsive genes AtCAT1 and AtP5CS, thereby enhancing the resistance of plants to drought stress.
In summary, we can derive a potential model to describe the role of MbMYB4 in cold and drought stresses based on the above results and previous studies ( Figure 11). First, cold stress induces the expression of MbMYB4 so that it can bind to the CBF promoter region. Two key genes in the CBF-dependent pathway, CBF1 and CBF3, directly activate their expression by binding to the CRT/DRE cis-acting elements of the downstream coldresponsive genes COR15a and RD29a, enhancing the cold resistance of plants. Secondly, drought stress induces the expression of MbMYB4, regulates the up-regulated expression of NCED3 and SnRK2.4, and promotes ABA biosynthesis and signal transduction, thereby enhancing the drought resistance of transgenic plants. At the same time, the production of ABA can also induce the expression of MbMYB4. In addition, under stress conditions, the expressions of drought-responsive key genes CAT1 and P5CS increase, indicating that MbMYB4 can regulate key genes under drought stress to reduce the accumulation of reactive oxygen species, and can also increase the level of proline to improve the waterholding capacity of plant cells under drought conditions, thereby enhancing the drought resistance of transgenic plants.

Plant Material and Growth Conditions
The test-tube plantlets of M. baccata (Supplementary Figure S2) were propagated on MS growth medium (MS + 0.6 mg/L 6-Benzylaminopurine (6-BA) + 0.6 mg/L Indole-Butytric acid (IBA)) for 30 days, and then transferred to MS rooting medium (MS + 1.2 mg/L IBA) to continue culturing until white roots grew. Finally, the seedlings were transferred to Hoagland solution for 40 days for growth. The solution was changed every 2-3 days. The temperature of the culture chamber was maintained at 20 • C and the relative humidity was maintained at about 85%. When the seedlings had 7-9 fully expanded leaves and the roots were well developed, we selected 60 seedlings with better morphology and basically the same growth. First, we sampled the four different parts of the young leaf, root, mature leaf, and stem of 10 seedlings, then the remaining 50 seedlings were divided into five groups for different stress treatments: low-temperature stress (hydroponic seedlings (Supplementary Figure S2C) were cultured at 4 • C), salt stress (hydroponic seedlings were cultured in Hoagland solution containing 200 mM of NaCl), dehydration stress (hydroponic seedlings were cultured in Hoagland solution with a concentration of 20% PEG6000), hightemperature stress (hydroponic seedlings were cultured in a lighted incubator at 37 • C), and ABA stress (hydroponic seedlings were cultured in Hoagland solution with ABA concentration of 50 µM). After 0, 1, 3, 5, 7, 9, and 12 h of stress treatment, we took samples of the young leaf and root of these seedlings, and the obtained plant materials were frozen with liquid nitrogen and stored at −80 • C for RNA extraction.

Isolation and Cloning of MbMYB4
The OminiPlant RNA Kit (Kangweishiji, Beijing, China) was used for RNA extraction. Then, RNA was used as the template to synthesize the first-strand cDNA with TransScript ® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) [53]. RNA and cDNA were assessed by 1.0% agarose gel electrophoresis [54]. The CDS region of MdMYB25 (NM_001293983.1, M. domestica) was used as the reference sequence, and primers (MbMYB4-F and MbMYB4-R, Supplementary Table S1) were designed with Primer 5.0 software. After the primers were synthesized, they were used for the amplification of MbMYB4. The purified DNA was linked to the pEASY ® -T1 Cloning Kit (TransGen Biotech, Beijing, China) and sequenced [55,56].

Bioinformatics Analysis of MbMYB4
DNAMAN5.2 was used to translate the MbMYB4 gene sequence, the amino acid sequence of the MbMYB4 protein was compared with MYB sequences with higher homology in other species, and ExPASy (https://web.expasy.org/protparam/, accessed on 31 January 2022) was used to predict the primary structure of the MbMYB4 protein. The domain of the MbMYB4 protein was predicted on the SMART website (http://smart.embl-heidelberg.de/, accessed on 31 January 2022), and the SWISS-MODEL (https://swissmodel.expasy.org/, accessed on 31 January 2022) was used to predict the tertiary structure of the MbMYB4 protein. MEGA7.0 (http://www.megasoftware.net, accessed on 5 April 2021) was used to construct a homologous phylogenetic tree of MbMYB4.

Subcellular Localization Analysis of MbMYB4 Protein
The upstream and downstream primers with BamHI and SalI restriction sites were designed (site-F and site-R, Supplementary Table S1), and the DNA fragments of MbMYB4 were cloned into BamHI and SalI sites of the pSAT6-GFP-N1 vector. The recombinant plasmid containing the MbMYB4 gene was injected into onion epidermal cells by particle bombardment [57]. After overnight culture, it was placed under a confocal microscope to observe the fluorescent signals of the control protein and the MbMYB4-GFP fusion protein, to observe the location of the protein encoded by the gene in the cells. DAPI staining was used as a nucleus marker for nucleus detection.

Quantitative Real-Time PCR (qPCR) Analysis of MbMYB4
The expressions of the MbMYB4 gene under different treatments were determined by qPCR with a pair of specific primers (MbMYB4-qF and MbMYB4-qR, Supplementary Table S1). The PCR condition was as follows: 30 s at 94 • C; 40 cycles of 5 s at 95 • C, 40 s at 54 • C, and 30 s at 72 • C; and then 10 min at 72 • C. The expression of the MbMYB4 gene was detected by the TB Green ™ Premix Ex Taq ™ II (Tli RNaseH Plus kit) (TaKaRa, Beijing, China) according to the manufacturer's protocol, and the Actin gene (NC_024251.1, M. domestica) was used as the internal reference gene. MbMYB4 expression level was analyzed using the 2 −∆∆CT method.

Vector Construction and Agrobacterium-Mediated A. thaliana Transformation
According to the principle of homologous recombination to connect the expression vector, the primers including the target fragment-specific primer sequence, restriction sequence (BamHI and SalI), and overlapping sequence were designed (HR-F and HR-R, Supplementary Table S1). The target fragment was ligated with the digested pCAMBIA2300 plasmid and transformed into Agrobacterium tumefaciens GV3101 [58].
The right amount of Colombian WT A. thaliana seeds was prepared and fully disinfected with 75% alcohol and 10% NaClO and then dried. The seeds were sown evenly on the sowing medium (1/2MS + 20% sucrose + 0.7% agar) until 2-4 leaves grew. We selected seedlings with well-developed roots and transferred them to nutrient bowls (ratio of turfy soil to vermiculite is 2:1), and then cultivated them in a light incubator (temperature setting: 25 • C daily/22 • C night, humidity 60%, 16 h light/8 h darkness). After WT A. thaliana entered the blooming period, we infected it by the inflorescence dip method and harvested the seeds [59]. The harvested seeds were evenly sown on the screening medium (4.3 g/L MS + 30 g/L sucrose + 7.5 g/L agar + 50 mg/L kanamycin) [60] until T 3 generation. We continued to sow T 3 generation seeds on MS medium containing antibiotics (50 mg/L kanamycin). After a period of time, the selection medium was full of positive seedlings. At this time, the transgenic A. thaliana obtained was the homozygous transgenic line. Transgenic A. thaliana, UL, and WT were transplanted into nutrient bowls and grown in a light incubator.

Determination of Related Physiological Indicators
WT, UL, and transgenic A. thaliana were divided into two groups after three weeks of growth. One group was treated at −6 • C for 14 h to cause cold stress; the other group was not watered for 10 d, thereby causing drought stress to them. After the stress treatment, the A. thaliana of each group was cultivated under normal conditions for one week, and the morphological changes of each group under stress and normal environments were observed. Fresh leaves were immersed in an ethanol-acetone mixture solution for 24 h and the chlorophyll content was calculated by measuring the absorbance value at 645 and 663 nm of the mixed solution using a spectrophotometer [61]. Proline was extracted with sulfosalicylic acid, and the content was calculated using the method described by Huang et al. [62]. The A. thaliana leaves were put into deionized water and were repeatedly pumped and deflated until the leaves were completely submerged in deionized water. We measured the conductance of the extract solution with a conductivity meter, heated it in a boiling water bath for 30 min, cooled it down sufficiently, measured the conductance again, and used the formula to calculate the relative conductivity [63]. The determination of POD activity was based on the guaiacol method [64]. CAT activity was measured following the ultraviolet (UV) absorption method [65]. MDA activity was measured by the colorimetric method using a spectrophotometer [66]. Calculation formulas for all indicators are listed in Supplementary Table S2.

Analysis of Downstream Gene Expression of MbMYB4
The mRNAs of WT, UL, and MbMYB4 transgenic A. thaliana under normal conditions and after cold and drought stresses were extracted and reverse-transcribed into the firststrand cDNA as the template, and AtActin was used as an internal reference. Specific primers were designed (Supplementary Table S1) to perform qPCR experiments on several important regulatory genes downstream of MYB transcription factors: cold-responsive key genes (AtCBF1, AtCBF3, AtCOR15a, AtRD29a) and drought-responsive related genes (AtNCED3, AtSnRK2. 4, AtCAT1, AtP5CS).

Statistical Analysis
SPSS software was used to analyze the differences with Duncan's multiple range tests. Statistical differences were referred to as significant when *p ≤ 0.05, ** p ≤ 0.01.