Overexpression of ZmDHN15 Enhances Cold Tolerance in Yeast and Arabidopsis

Maize (Zea mays L.) originates from the subtropical region and is a warm-loving crop affected by low-temperature stress. Dehydrin (DHN) protein, a member of the Group 2 LEA (late embryogenesis abundant proteins) family, plays an important role in plant abiotic stress. In this study, five maize DHN genes were screened based on the previous transcriptome sequencing data in our laboratory, and we performed sequence analysis and promoter analysis on these five DHN genes. The results showed that the promoter region has many cis-acting elements related to cold stress. The significantly upregulated ZmDHN15 gene has been further screened by expression pattern analysis. The subcellular localization results show that ZmDHN15 fusion protein is localized in the cytoplasm. To verify the role of ZmDHN15 in cold stress, we overexpressed ZmDHN15 in yeast and Arabidopsis. We found that the expression of ZmDHN15 can significantly improve the cold resistance of yeast. Under cold stress, ZmDHN15-overexpressing Arabidopsis showed lower MDA content, lower relative electrolyte leakage, and less ROS (reactive oxygen species) when compared to wild-type plants, as well as higher seed germination rate, seedling survival rate, and chlorophyll content. Furthermore, analysis of the expression patterns of ROS-associated marker genes and cold-response-related genes indicated that ZmDHN15 genes play an important role in the expression of these genes. In conclusion, the overexpression of the ZmDHN15 gene can effectively improve the tolerance to cold stress in yeast and Arabidopsis. This study is important for maize germplasm innovation and the genetic improvement of crops.


Introduction
Maize (Zea mays L.) is an important food crop and industrial raw material crop, ranking first in the world's total food production [1]. Maize originates from the subtropical zone and is a thermophilic crop affected by low-temperature stress [2]. The germination and seedling stages of maize in the northern spring sowing area are extremely vulnerable to low temperatures [3]. Therefore, mining the cold-resistance genes of maize and studying their mechanisms have become a hot issue. The impact of low temperature on corn is divided into chilling damage (below 15 • C) and freezing damage (below 0 • C) [4]. Chilling damage can destroy the metabolic balance of water, inhibit respiration and photosynthesis, change the microstructure of cells, aggravate protein decomposition and metabolism, produce toxic substances, and disrupt hormone synthesis balance [5][6][7][8][9][10][11]. Furthermore, like all other stresses, cold stress causes oxidative stress due to an imbalance between the production of reactive oxygen species (ROS) and the ability of enzymatic and non-enzymatic antioxidant defense systems to process ROS [12]. sequence. The results showed that the promoter contained multiple functional elements related to the response to cold, light, hormone, and non-stress stress ( Figure 1B).

Bioinformatics Analysis of Maize Dehydrin Gene
The amino acid sequence homology alignment of five dehydrin genes in maize showed that all five dehydrin genes had S-type and Y-type conserved sequences ( Figure  1A). The 2000 bp upstream of the initiation codons (ATG) of these five dehydrin genes was used as the promoter sequence, and the cis-acting element analysis was carried out on the promoter sequence. The results showed that the promoter contained multiple functional elements related to the response to cold, light, hormone, and non-stress stress ( Figure 1B).

Analysis of the Expression Pattern of the Maize Dehydrin Gene under Cold Stress
In order to screen the functional genes of cold resistance among the above five maize dehydrin genes. qRT-PCR results showed that ZmDHN15 was significantly upregulated among these five genes ( Figure 2). In other words, ZmDHN15 may play an important role in the response of maize to cold stress.

Analysis of the Expression Pattern of the Maize Dehydrin Gene under Cold Stress
In order to screen the functional genes of cold resistance among the above five maize dehydrin genes. qRT-PCR results showed that ZmDHN15 was significantly upregulated among these five genes ( Figure 2). In other words, ZmDHN15 may play an important role in the response of maize to cold stress.

Subcellular Localization of ZmDHN15 Protein
Understanding the subcellular localization of gene expression products is important for the functional analysis of genes. ZmDHN15 CDS (coding sequences) was cloned into the transient expression vector pA7-YEP. The subcellular localization of ZmDHN15 protein was observed under a confocal microscope using the yellow fluorescence properties of YEP. The result shows that ZmDHN15 protein is localized in the cytoplasm of tobacco epidermal cells ( Figure 3).

Low-Temperature-Tolerance Assays of Yeast Transformants
In order to determine the effect of the ZmDHN15 protein on the survival rate of yeast recombinants under osmotic stress, we investigated the plaque growth of yeast cell lines transformed with a pYES2-ZmDHN15 vector and a control strain containing empty vector (pYES2) under cold stress at 4 • C. Under optimal conditions, the growth of the transformed yeast and the control yeast was not significantly different. Under the coldstress condition of 4 • C, the transformed yeast showed stronger growth ability than the qRT-PCR analysis of the expression profiles of the maize dehydrin gene family. The expression level was normalized to that of maize ZmACTIN1. 0: cold stress 0 h, 2: cold stress 2 h, 4: cold stress 4 h, 8: cold stress 8 h, 12: cold stress 12 h. Data were expressed as the mean of triplicate values, and the error represented the SD. Asterisks indicate statistically significant differences: p < 0.05 (*) and p < 0.01 (**).

Subcellular Localization of ZmDHN15 protein
Understanding the subcellular localization of gene expression products is important for the functional analysis of genes. ZmDHN15 CDS (coding sequences) was cloned into the transient expression vector pA7-YEP. The subcellular localization of ZmDHN15 protein was observed under a confocal microscope using the yellow fluorescence properties of YEP. The result shows that ZmDHN15 protein is localized in the cytoplasm of tobacco epidermal cells (Figure 3).

Low-Temperature-Tolerance Assays of Yeast Transformants
In order to determine the effect of the ZmDHN15 protein on the survival rate of yeast recombinants under osmotic stress, we investigated the plaque growth of yeast cell lines transformed with a pYES2-ZmDHN15 vector and a control strain contain- cell lines transformed with a pYES2-ZmDHN15 vector and a control strain containing empty vector (pYES2) under cold stress at 4 °C. Under optimal conditions, the growth of the transformed yeast and the control yeast was not significantly different. Under the cold-stress condition of 4 °C, the transformed yeast showed stronger growth ability than the control group ( Figure 4). Therefore, we believe that overexpression of ZmDHN15 can improve the tolerance of yeast transformants.

Generation of Transgenic Plants and Molecular Identification
The recombinant plasmid pCAMBIA3301-ZmDHN15 was constructed and introduced into wild-type Arabidopsis Col-0 lines (non-transgenic plant lines); ZmDHN15 was overexpressed in Arabidopsis, and eight transgenic lines were generated ( Figure 5A). Molecular detection was conducted on T3 transgenic plants. Three homozygous lines (OE1, OE3, and OE6) with the highest expression level were selected for follow-up experiments ( Figure 5C-E).

Generation of Transgenic Plants and Molecular Identification
The recombinant plasmid pCAMBIA3301-ZmDHN15 was constructed and introduced into wild-type Arabidopsis Col-0 lines (non-transgenic plant lines); ZmDHN15 was overexpressed in Arabidopsis, and eight transgenic lines were generated ( Figure 5A). Molecular detection was conducted on T3 transgenic plants. Three homozygous lines (OE1, OE3, and OE6) with the highest expression level were selected for follow-up experiments ( Figure 5C-E).

Overexpression of ZmDHN15 Enhances Cold Resistance in Transgenic Arabidopsis
In order to investigate the function of ZmDHN15, cold-tolerance identification was performed on the identified OE lines (OE1, OE3, and OE6) and Col-0 lines during germination. Under normal conditions, the seed germination rate of Col-0 lines was similar to that of OE lines (overexpressing plant lines) but significantly decreased at 4 • C ( Figure 6A,B). Furthermore, we found that the growth potential of Col-0 seedlings was weaker than that of OE strains under normal conditions. However, the growth potential of Col-0 seedlings was more affected than OE strains with the decrease of temperature ( Figure 6C showed higher survival rates than Col-0 lines ( Figure 6E,F). These results suggest that the OE lines of ZmDHN15 in Arabidopsis improve the cold tolerance of plants.   In order to further understand the response of transgenic Arabidopsis under cold stress, the plants of the Col-0 and OE lines were tested for cold tolerance. When exposed to 4 • C for 24 h and allowed to recover at room temperature for 7 d, the transgenic plants showed higher survival rates than Col-0 lines ( Figure 6E,F). These results suggest that the OE lines of ZmDHN15 in Arabidopsis improve the cold tolerance of plants.

Phenotypic Characterization of Overexpressed ZmDHN15 at the Mature Stage
The further phenotypic identification of mature plants of Col-0 and OE lines was performed. We observed no significant difference in the plant height of the OE lines compared with wild-type Col-0 plants ( Figure 7A,B); however, the root lengths of the OE lines were significantly different ( Figure 7C,D). In addition, the silique length and seed dry weight per plant were also significantly higher in the OE line than in the wildtype Col-0 plant ( Figure 7E-G). These phenomena suggest that the OE of ZmDHN15 in Arabidopsis thaliana improves the cold resistance of plants that are subjected to cold stress at the seedling stage and reduced the damage to plants caused by low temperature. Therefore, the introduction of this gene can effectively affect the yield of plants.

Overexpression of ZmDHN15 Reduces the ROS Accumulation
In order to study the effect of ZmDHN15 on plant physiology and biochemistry, the chlorophyll (CHL), malondialdehyde (MDA) contents, and electrolytic leakage rate (EL) of Col-0 plants and OE lines were determined. Under cold stress, the CHL content of OE lines was significantly increased, and the content of MDA and the EL rate was significantly decreased compared with Col-0 lines ( Figure 8A-C). Cold conditions usually cause oxidative damage to plants. In order to explore whether ZmDHN15 can reduce ROS accumulation, antioxidant enzyme activities, peroxidase activities, and the contents of H2O2 and O2 − were measured. O2 − expression levels were detected by the NBT (Ni-

Overexpression of ZmDHN15 Reduces the ROS Accumulation
In order to study the effect of ZmDHN15 on plant physiology and biochemistry, the chlorophyll (CHL), malondialdehyde (MDA) contents, and electrolytic leakage rate (EL) of Col-0 plants and OE lines were determined. Under cold stress, the CHL content of OE lines was significantly increased, and the content of MDA and the EL rate was significantly decreased compared with Col-0 lines ( Figure 8A-C Figure 8D-I). The NBT staining results were consistent with O 2 − assay results ( Figure 8J). Under cold-stress conditions, all OE lines exhibited less damage and accumulated less ROS than Col-0 lines. Therefore, it is speculated that the expression of ZmDHN15 may reduce ROS accumulation by increasing antioxidant enzymes and peroxidase activities in leaves.  The expression patterns of ROS-related marker genes and cold-response-related genes were investigated to further investigate the mechanism of ZmDHN15. The selected nine marker genes were expressed in Col-0 and OE lines under cold stress (Figure 9). The results show that the ZmDHN15 gene plays an important role in the increased expression of nine marker genes in transgenic plants. ZmDHN15 is a positive regulator of plant cold resistance and may enhance cold resistance through a CBF-dependent pathway in Arabidopsis. Notably, compared with Col-0 plants, all OE lines showed a minimal injury with a similar trend in physiological indices and the expression of genes related to cold resistance.

Discussion
Dehydrin (DHN) proteins have irregular structures that can effectively resist freezing. At low intracellular water potential, DHN proteins adsorb water molecules and act as osmoregulators [36]. DHN proteins are closely related to plant growth and development, except for their roles in stress response and distribution in different plant organs [37]. In this study, we identified five genes in the maize dehydrin gene family by bioin- Figure 9. Analysis of ROS-related marker genes and cold-response-related genes in transgenic plants.
(A-C) ROS-related marker genes expression analysis. (D-I) Cold-response-related genes expression analysis. Control: before the low-temperature stress at 4°C, cold stress: low-temperature stress at 4°C for 24 h. The expression level was normalized to that of AtACTIN1. Data were expressed as the mean of triplicate values, and the error represented the SD. Asterisks indicate statistically significant differences: Non-significant (ns), p < 0.05 (*) and p < 0.01 (**).

Discussion
Dehydrin (DHN) proteins have irregular structures that can effectively resist freezing. At low intracellular water potential, DHN proteins adsorb water molecules and act as osmoregulators [36]. DHN proteins are closely related to plant growth and development, except for their roles in stress response and distribution in different plant organs [37]. In this study, we identified five genes in the maize dehydrin gene family by bioinformatics and verified the biological function of ZmDHN15 under cold stress by a series of experiments. Multiple sequence alignment results showed that the five DHN proteins contained highly conserved DHN domains and different distributions of K and S segments. The findings were also confirmed in previous studies [20,38]. The promoter sequences of these five genes all have cold-responsive cis-acting elements. qPT-PCR was performed on these five genes under cold stress, and the results showed that ZmDHN15 was significantly upregulated under cold stress. Previous studies have shown that the homologous or heterologous expression of the DHN gene plays an important role in plant abiotic stress. Ju et al. heterologously expressed the maize ZmDHN11 gene in yeast and tobacco, which enhanced the tolerance of transgenic yeast and tobacco to osmotic stress [35]. Zhang et al. overexpressed pepper CaDHN4 in Arabidopsis thaliana, enhancing salt-and cold-stress tolerance [39]. In this study, the subcellular localization results showed that the ZmDHN15 protein was localized in the cytoplasm. The finding was consistent with previous reports [40]. The eukaryotic Saccharomyces cerevisiae is an ideal model organism for studying the stress-resistance function of genes, which has the advantages of speed and accuracy [39,40]. Its cell structure is more suitable for studying eukaryotic gene function. In this study, the stress-resistance function of ZmDHN15 was investigated using the Saccharomyces cerevisiae expression system. The results showed that the tolerance of ZmDHN15 transgenic yeast to cold stress was significantly improved. Arabidopsis thaliana has the advantages of a small plant size, more fruit, short life cycle, simple genome, and easy genetic manipulation [41,42] and is usually considered a model organism in various biological research disciplines based on plant material. In order to investigate the function of ZmDHN15, we generated transgenic Arabidopsis constitutively overexpressing ZmDHN15. We observed that, under cold-stress conditions, the germination rate and root length of OE lines were also higher than those of Col-0 lines. The measurement of some physiological and biochemical indicators showed that, under cold stress, the CHL content of the OE lines was significantly increased, and the content of MDA and EL rate was significantly decreased compared with the Col-0 lines. The results show that the overexpression of ZmDHN15 can significantly enhance the cold resistance of yeast and Arabidopsis.
ROS is important for species homeostasis and signal transduction [31]. The production and scavenging of ROS in plants are in a state of dynamic equilibrium under normal conditions [4,43]. More ROS, including superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ), etc., will be accumulated when plants are under abiotic stress, which may cause oxidative damage to biomolecules in plants [44]. Studies have shown that increasing the expression of ROS scavenging-related genes can improve plant stress tolerance [42]. Mbukeni Nkomo et al. found that higher levels of H 2 O 2 could aggravate plant oxidative damage [45]. Wang et al. found that salt stress would decrease the photosynthetic efficiency of Arabidopsis, damage the membrane, and accumulate more ROS [46]. Although ROS molecules mediate plant growth by activating many stress-related genes [31,46], the effect of DHN genes on ROS accumulation is unclear. In this study, the activities of CAT, SOD, POD, and APX were measured under cold-stress conditions, and the ROS accumulation in Arabidopsis leaves was also analyzed to explore the effect of the ZmDHN15 gene on the scavenging mechanism of ROS. Compared with Col-0 lines, the SOD, CAT and APX activities of OE lines increased, POD activities decreased, the accumulation of H 2 O 2 and O 2 − decreased, and the NBT staining results became light. These results indicate that the ZmDHN15 gene can reduce the ROS accumulation in vivo, thereby enhancing the tolerance of plants to cold stress. We also investigated ROS-related marker genes [10,47]. The expression levels of the selected three marker genes were induced in Col-0 and OE lines under cold stress. We analyzed the expression of several widely reported cold-related genes [2] (AtRD29A, AtCOR15B, and AtCOR47) in Col-0 and OE lines to further analyze the function of ZmDHN15 in plant cold resistance. The results showed that all genes were upregulated under low-temperature stress, and the changes in OE lines were more drastic than in Col-0 lines. The expression trends of physiological indicators and cold-resistancerelated genes were similar. The above results indicate that ZmDHN15 is a positive regulator of plant cold resistance. According to a previous analysis, the ZmDHN15 promoter sequence has a CRT/DRE response element, indicating that the expression of ZmDHN15 may be regulated by CBF/DREB transcription factors in response to ICE-CBF-COR cascading pathways to improve plant cold resistance [6,[48][49][50][51]. Furthermore, the expression patterns of CBF1, CBF2, and CBF3 in Col-0 and OE lines showed that all genes were expressed under low-temperature stress. Therefore, it is predicted that cold resistance may be enhanced through a CBF-dependent pathway in Arabidopsis.

Plant Materials and Growth Conditions
In this study, the wild-type Col-0 of Arabidopsis thaliana and the maize inbred line H8069 provided by the Plant Biology Center of Jilin Agricultural University were used as materials. Maize plants were grown under long-day conditions (light 16 h, dark 8 h) at 24 • C and relative humidity of 46%, and transgenic Arabidopsis plants were grown at 24 • C and relative humidity of 65% under long-day conditions.
The corn inbred line H8069 growing to the four-leaf stage was treated with a low temperature. Samples were collected at 0 h and 12 h of treatment, immediately frozen in liquid nitrogen, and fully ground. Then, total RNA was extracted by the Trizol method and reverse-transcribed into cDNA using a reverse transcription kit. The qPCR amplification was performed on the five genes of the DHN as mentioned above gene family using the SYBR Green qRT-PCR SuperMix instructions. Furthermore, 2 −∆∆CT was used for data processing with three replicates per sample. All primers used in the experiments are listed in Supplementary Table S1.

Subcellular Localization of ZmDHN15
The ZmDHN15 CDS without the stop codon was amplified by Vector Cloning Kit and cloned into the plant expression vector pA7-YEP (restriction sites: Xho I and Hind III) to construct the recombinant plasmid pYES2-ZmDHN15. The recombinant plasmid was expressed in tobacco leaves by the Agrobacterium-mediated method, and the expression of YEP was observed by fluorescence confocal microscopy with the empty vector pA7-YEP as the control.

Expression of ZmDHN15 in Saccharomyces Cerevisiae INVSc1
The ZmDHN15 cloning vector was inserted into the yeast expression vector pYES2 (restriction sites: Hind III and Bam HI) to construct the recombinant plasmid pYES2-ZmDHN15 and transform Saccharomyces cerevisiae INVSc1. The pYES2 empty vector was transformed as a negative control. The transformation was verified by bacterial liquid PCR results [53].

Low-Temperature Tolerance Assay of Yeast Transformants
Saccharomyces cerevisiae INVSc1 carrying the pYES2-ZmDHN15 recombinant plasmid and confirmed positive empty vector pYES2 were induced with galactose and then undiluted and diluted 10 −1 , 10 −2 , 10 −3 , and 10 −4 times, respectively. Then, 2 µL samples were taken and spotted on an S-U (containing 2% glucose) solid medium. After culturing at 4 • C for 48 h, the growth differences between the two strains were analyzed. The two strains cultured at 30 • C for 48 h were used as the control.

Generation of Transgenic Plants and Phenotypic Analysis
The pCAMBIA-3301-ZmDHN15 vector was constructed by inserting the ZmDHN15 gene cloning vector into the plant high expression vector pCAMBIA-3301(enzyme cleavage sites: Bgl II and BstE II). The product was introduced into wild-type Col-0 lines by the Agrobacterium-mediated floral dipping method to obtain transgenic lines. Homozygous plants were screened with 1/2 MS solid medium, and the subculture was continued. For the T3 generation, three high expressing lines were selected for further study. All primers used in the experiments are listed in Supplementary Table S1.
Col-0 and three highly expressed T3 generation transgenic Arabidopsis seeds were sterilized by 70% and 5% in turn, then sown on 1/2 MS medium and placed in a 4 • C incubator under long-day light conditions (light for 16 h). The control group was grown under long-day conditions (light 16 h, dark 8 h) in a 24 • C incubator. After 14 days, the germination of seeds was recorded, and the germination rate and root length of seedlings were counted.
In order to further understand the response of transgenic Arabidopsis to cold stress, three-week-old Col-0 and three high-expressing T3 transgenic Arabidopsis lines were subjected to cold stress at 4 • C for 24 h, followed by recovery at room temperature for 7 d. The phenotypic changes of Arabidopsis plants were photographed, and the survival rate was counted during the treatment period. The three-week-old Col-0 and three highly expressed T3 transgenic Arabidopsis lines were treated with cold stress at 4 • C for 24 h, and the following physiological and biochemical indicators were measured before and after cold stress: chlorophyll, electrolytic leakage (EL) rate, MDA, H 2 O 2 , and O 2 − . The contents were detected by the method proposed by Jing et al. [54,55], and the activities of SOD, POD, CAT, and APX were determined by Yu and Zhang et al. [39,56,57]. Three replicates were performed for each sample.

NBT (Nitro-Blue Tetrazolium Chloride) Staining Assay
Three-week-old Col-0 and three high-expressing T3 transgenic Arabidopsis lines were treated with cold stress at 4 • C for 24 h. The third unfolded rosette leaf was taken from each plant for NBT staining [21,36]. First, an NBT solution was prepared. A total of 0.05 g NBT and 0.5 mL 1 M phosphate buffer (pH = 7.8) was added into a 50 mL centrifuge tube, and ddH 2 O was added to make the solution reach 50 mL. The sampled leaves were then dyed in NBT solution for 0.5-1.0 h and taken out. The stained leaves were decolorized with 95% alcohol until the chlorophyll was completely degraded for easy observation and photography. Three replicates were performed for each sample.

Analysis of Expression Patterns of ROS-Related Marker Genes and Cold-Responsive Genes in Transgenic Plants
In order to study the response of ZmDHN15 to cold, the expression patterns of ROSrelated marker genes and cold-responsive genes in Arabidopsis thaliana were analyzed using AtACTIN1 as an internal reference gene. Three replicates were performed for each sample, and all primers used in the experiments are listed in Supplementary Table S1.

Statistical Analysis
All results in this study were performed in more than three replicates. Data were expressed as the mean of triplicate values, and the error represented the SD(standard deviation). Statistical analyses and plotting were performed using GraphPad Prism 8 (V8.4.3, GraphPad, Changchun, China). The statistical significance of the difference was confirmed by a Student's t-test; asterisks indicate statistically significant differences: p < 0.05 (*) and p < 0.01 (**).

Conclusions
In conclusion, ZmDHN15 is a positive regulator of cold tolerance in yeast and Arabidopsis. According to the subcellular localization results, the ZmDHN15 fusion protein is localized in the cytoplasm. Under cold-stress conditions, the overexpression of ZmDHN15 significantly improves the cold resistance of yeast. Subsequent analysis shows that ZmDHN15 can promote the growth of Arabidopsis plants and accumulate less ROS. Furthermore, under cold-stress conditions, the overexpression of ZmDHN15 in Arabidopsis can activate the expression of key genes in the ROS-signaling-and CBF-dependent pathways. Our findings advance our understanding of the function of DHN genes in maize.