Isolation and Functional Analysis of VvWRKY28, a Vitis vinifera WRKY Transcription Factor Gene, with Functions in Tolerance to Cold and Salt Stress in Transgenic Arabidopsis thaliana

The grape (Vitis vinifera L.) not only has a long history of cultivation, but also has rich nutritional value and high economic value. However, grapes often face many threats in the growth process. For example, low temperature and salt stress restrict the growth status, yield, and geographical distribution of grapes. WRKY, as one of the largest transcription factor (TF) families in plants, participates in the response of plants to stress. VvWRKY28, a new zinc finger type transcriptional regulator gene, was isolated from Beichun (V. vinifera × V.amurensis) in this study. From the subcellular localization results, it can be concluded that VvWRKY28 was localized in the nucleus. The expression of VvWRKY28 was enriched in leaves (young and mature leaves), and cold and high salt conditions can induce high expression of VvWRKY28. After being transferred into Arabidopsis, VvWRKY28 greatly improved the tolerance of Arabidopsis to low temperature and high salt and also changed many physiological and biochemical indicators of transgenic Arabidopsis to cope with cold and high salt stimulation. The content of malondialdehyde (MDA) was decreased, but for chlorophyll and proline, their content increased, and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were improved. In addition, under cold stress, binding with cis-acting elements promotes the expression of downstream genes related to cold stress (RAB18, COR15A, ERD10, PIF4, COR47, and ICS1). Moreover, it also plays an active role in regulating the expression of genes related to salt stress (NCED3, SnRK2.4, CAT2, SOD1, SOS2, and P5CS1) under salt stress. Therefore, these results provide evidence that VvWRKY28 may play a role in the process of plant cold and salt stress tolerance.


Introduction
In order to adapt and resist low temperature, high temperature, drought, salinization, nutrient deficiency, and other abiotic stresses, plants have formed a set of unique and complex physiological and molecular networks. When encountering abiotic stress, plants will undergo a series of changes at the physiological level, the plant cell membrane and antioxidant system will be destroyed, the photosynthetic function will be affected, and the accumulation of osmotic regulatory substances will change [1]. At the molecular level, the expression of transcription factors (TFs) plays a key role. TFs can combine with cisacting elements of target sites to regulate the transcription level of target gene [2], that is, plays an important role in improving the cold tolerance of plants [28]. Overexpression of RmICE1 in tobacco enhances cold tolerance by regulating ROS scavenging and expression of stress-responsive genes [29]. Genes associated with salt stress are expressed in two ways: the ABA-dependent pathway and the ABA-independent pathway [30]. Sucrose-nonfermentation-1 related protein kinase-2 proteins (SnRK2s) family members play important roles in plant ABA signaling pathways and responses to stress. Studies have shown that SnRK2.4 affects the growth of Arabidopsis taproot salt concentration (when too high) and thus regulates the response of Arabidopsis to salt stress [31]. Studies have shown that overexpression of ClWRKY20 can up-regulate many genes related to low temperature stress, such as COR27, COR413, ERD7, ERD15, ERD10,and so on [32].
During the growth and development of Beichun, it will inevitably be affected by the natural environment, resulting in failure to achieve the production target [33]. Therefore, it is particularly important to improve the stress resistance of grapes. Based on the important role of WRKY TFs in plant stress response, this study isolated and cloned a novel WRKY TF gene named VvWRKY28 from the grape Beichun and verified the main functions of VvWRKY28 under low temperature and salt stress. This finding lays a certain foundation for improving the cold and salt tolerance of grapes by using the regulatory effect of TFs.

Cloning and Sequence Analysis of VvWRKY28
Figure S1 showed the analysis results of ProtParam (http://www.expasy.org/tools/ protparam.html, accessed on 1 October 2021). It can be concluded that the VvWRKY28 gene encoded 319 amino acids, and the complete open reading frame (ORF)was 960 bp. The theoretical pIof the predicted protein was 6.76 and the predicted molecular weight (MW) was 35.00 kDa. The average hydrophilic coefficient was −0.782, which was a hydrophilic protein. The VvWRKY28 protein contained 22 kinds of amino acids, of which Ser, Gly, Pro, Lys, and Asp were more abundant, accounting for 11.6%, 8.8%, 8.5%, 6.6%, and 6.3%, respectively. The underlined part was a WRKY domain and a C 2 H 2 -type zinc finger structure, indicating that it belonged to class II.

Phylogenetic Relationship of VvWRKY28 with Other WRKY Proteins
In order to explore the evolutionary relationship between plant WRKY proteins, WRKY proteins of 14 other species were compared with VvWRKY28 using DNAMAN. These genes were obtained by similarity analysis using blast functions in the NCBI website, including the genes JrWRKY28 (Juglans regia, XP_035543967.1), CiWRKY28 (Carya illinoinensis, XP_042940078.1), CaWRKY28 (Coffea arabica, XP_027088028.1), CsWRKY28 (Camellia sinensis, AYA73393.1), Hb-WRKY28 (Hevea brasiliensis, XP_021644344.1), MeWRKY28 (Manihot esculenta, XP_021598441.1), ArWRKY28 (Actinidia rufa, GFY88621.1), QlWRKY28 Quercus lobata, XP_030968701.1), AtWRKY28 (Arabidopsis thaliana, NP_193551.1), TcWRKY28 (Theobroma cacao, EOY27787.1), RcWRKY28 (Ricinus communis, XP_002519733.1), DzWRKY28 (Duriozibethinus, XP_022715624.1), and PtWRKY28 (Populus trichocarpa, XP_024436877.1). The comparison results were shown in Figure 1A. The sequence in the red box was the conserved amino acid sequence of the VvWRKY protein, which was unique to the WRKY family. WRKY amino acid sequences of various species had high homology only in their conserved domains. According to the homology of the phylogenetic tree ( Figure 1B), VvWRKY28 had the highest homology with JrWRKY28 and Ci WRKY28 and has the closest evolutionary relationship. Figure 2 showed the subcellular localization results of the VvWRKY28 protein. It can be seen that GFP, which was used as a control, was distributed in the whole cytoplasm ( Figure 2B), while VvWRKY28 GFP fusion protein only appeared in the nucleus ( Figure 2E). Meanwhile, the position of the nucleus was also confirmed after 4 ,6-diamino-2-phenylindole (DAPI) staining ( Figure 2F). These results indicated that the VvWRKY28 protein was localized in the nucleus.  Figure 2 showed the subcellular localization results of the VvWRKY28 protein. It can be seen that GFP,which was used as a control, was distributed in the whole cytoplasm ( Figure 2B), while VvWRKY28 GFP fusion protein only appeared in the nucleus ( Figure 2E). Meanwhile,the position of the nucleus was also confirmed after 4′, 6-diamino-2-phenylindole (DAPI) staining ( Figure 2F). These results indicated that the VvWRKY28 protein was localized in the nucleus.

Expression Specificity Analysis of VvWRKY28
Under control conditions, the expression levels of the VvWRKY28 gene in different tissues can be seen from Figure 3. The results of qRT-PCR showed that VvWRKY28was expressed differently in various tissues,and the expression level from high to low was in new leaves, roots, mature leaves, and stems. Among them, the expression levels in new leaves were 2.08-fold, 2.48-fold, and 2.74-fold higher than those in roots, stems, and mature leaves, respectively. This suggested that the expression of VvWRKY28was tissue-specific.

Expression Specificity Analysis of VvWRKY28
Under control conditions, the expression levels of the VvWRKY28 gene in different tissues can be seen from Figure 3. The results of qRT-PCR showed that VvWRKY28 was expressed differently in various tissues, and the expression level from high to low was in new leaves, roots, mature leaves, and stems. Among them, the expression levels in new leaves were 2.08-fold, 2.48-fold, and 2.74-fold higher than those in roots, stems, and mature leaves, respectively. This suggested that the expression of VvWRKY28 was tissue-specific.
The VvWRKY28 gene was subjected to stress treatment under the conditions of cold, high salt, water shortage, and high temperature. The expression of the VvWRKY28 gene after treatment for 1, 3, 6, 12, 24, and 48 h was shown in Figure 3B,C. Under cold conditions, in new leaves, the expression of VvWRKY28 began to be up-regulated after 1 h of stress, and reached the maximum at 3 h, about 9-fold that of the control group (CK). After that, the expression began to decline, and in roots, it reached the maximum at 12 h. Under salt stress, the time point when the expression of VvWRKY28 reached the highest level in new leaves and roots was 12 h and 6 h, respectively, and the expression amount was 10-fold and 6-fold that of the untreated group. Under drought and heat stress, the expression of VvWRKY28 in new leaves reached the maximum at 24 h and 12 h, respectively, whilein the root, it reached the peak at 12 h and 24 h. However, when the ambient temperature was low or the salt concentration was high, the maximum expression level of VvWRKY28 in new leaves and roots was higher than that in drought or high temperature environments. under normal conditions, low temperature, salt, drought, and heat stress. The data were the average of 3 repeated trials. The asterisk above the column indicated that there were significant and extremely significant differences between the treatment group and the control group (control) (* p ≤ 0.05; ** p ≤ 0.01).
The VvWRKY28gene was subjected to stress treatment under the conditions of cold, high salt, water shortage, and high temperature. The expression of the VvWRKY28 gene after treatment for 1, 3, 6, 12, 24, and 48h was shown in Figure 3B,C. Under cold conditions, in new leaves, the expression of VvWRKY28 began to be up-regulated after 1h of stress, and reached the maximum at 3h, about 9-fold that of the control group (CK). After that, the expression began to decline, and in roots, it reached the maximum at 12h. Under salt stress, the time point when the expression of VvWRKY28 reached the highest level in new leaves and roots was 12h and 6h, respectively, and the expression amount under normal conditions, low temperature, salt, drought, and heat stress. The data were the average of 3 repeated trials. The asterisk above the column indicated that there were significant and extremely significant differences between the treatment group and the control group (control) (* p ≤ 0.05; ** p ≤ 0.01).

Analysis of the Ability of VvWRKY28 Transgenic A. thaliana to Cold Stress
The results of qRT-PCR showed that VvWRKY28 was more sensitive to the stimulation of cold and salt, so the transgenic A. thaliana was obtained through the control of the CaMV 35S promoter, and it was placed in cold and high salt environment to explore the role of VvWRKY28 under these two stresses. Using wild-type plants (WT) as controls, qRT-PCR was performed on T 2 generation transgenic line A. thaliana. The result showed that eight lines were successfully transformed, and the expression level of VvWRKY28 was relatively high in Strain 2, Strain 6, and Strain 7 (S2, S6 and S7) ( Figure 4A).   t-test showed that under cold stress, there was a very significant difference between transgenic lines and WT, ** p ≤ 0.01. Figure 4B showed the phenotype of WT A. thaliana and transgenic A. thaliana (S2, S6, S7) after low-temperature treatment. It can be seen that there was no obvious difference in the growth trend of all lines without cold treatment. However, after growing at low temperature (−4 • C) for 12 h, the growth trend of WT became weak, and the leaf wilting was obvious, while the leaf damage of transgenic lines was not obvious.
The survival rate of all plants (WT, S2, S6, S7) was calculated after being put back to normal conditions for five days ( Figure 4C). Under the control condition, the survival rates of WT and transgenic A. thaliana were basically at the same level-97.1%, 96.5%, 97.4%, and 98.3%, respectively. After growing at −4 • C for 12 h, the survival rate of WT decreased significantly (only 18.57% of the plants survived), while the survival rates of transgenic lines were 77.8%, 68.8%, and 82.3%, respectively. There was a significant difference between the WT and transgenic A. thaliana. This result indicated that transgenic plants were more tolerant to cold damage.
The content of some physiological indicators and the activity of related enzymes were also measured. The purpose of this was to further clarify the role of VvWRKY28 under low temperature stress. As shown in Figure 5, the contents of MDA, chlorophyll, and proline, as well as the activities of CAT, POD, and SOD of all plants, were almost equal under control conditions in all plants. After cold stress treatment, the MDA content of WT A. thaliana was significantly higher than that of transgenic lines, while the content and activity of other indicators were higher in transgenic A. thaliana. The above results indicatedthat the transgenic plants had a stronger antioxidant capacity, a lower degree of plasma membrane peroxidation, a stronger ability to scavenge ROS, and can survive better at low temperature, while WT plants had no such ability.  Figure 4B showed the phenotype of WT A. thaliana and transgenic A. thaliana (S2, S6, S7) after low-temperature treatment. It can be seen that there was no obvious difference in the growth trend of all lines without cold treatment. However, after growing at low temperature (−4 °C) for 12h, the growth trend of WT became weak, and the leaf wilting was obvious, while the leaf damage of transgenic lines was not obvious.
The survival rate of all plants (WT, S2, S6, S7) was calculated after being put back to normal conditions for five days ( Figure 4C). Under the control condition, the survival rates of WT and transgenic A. thaliana were basically at the same level-97.1%, 96.5%, 97.4%, and 98.3%, respectively. After growing at −4 °C for 12h, the survival rate of WT decreased significantly (only 18.57% of the plants survived), while the survival rates of transgenic lines were 77.8%, 68.8%, and 82.3%, respectively.There was a significant difference between the WT and transgenic A. thaliana. This result indicated that transgenic plants were more tolerant to cold damage.
The content of some physiological indicators and the activity of related enzymes were also measured.The purpose of this was to further clarify the role of VvWRKY28 under low temperature stress. As shown in Figure 5, the contents of MDA, chlorophyll, and proline, as well as the activities of CAT, POD, and SOD of all plants, were almost equal under control conditions in all plants. After cold stress treatment, the MDA content of WTA. thaliana was significantly higher than that of transgenic lines, while the content and activity of other indicators were higher in transgenic A. thaliana. The above results indicatedthat the transgenic plants had a stronger antioxidant capacity, a lower degree of plasma membrane peroxidation, a stronger ability to scavenge ROS, and can survive better at low temperature, while WT plants had no such ability. The standard error was the average of 3 repeated tests. The asterisk above the column indicated that there was a very significant difference between the transgenic lines and the WT lines, ** p ≤ 0.01.

Expression Analysis of Cold-Resistant Downstream Genes in VvWRKY28-OE A. thaliana
WRKY TFs can specifically bind to the W-box of the promoter of the target gene, thereby playing an active or inhibitory regulatory role on the expression of downstream genes. WRKY TFs play an important regulatory role in the response of plants to low and AtICS1 (Arabidopsis thaliana, AT1G74710). A. thaliana was grown at −4 • C for 12 h, and it was found that these six genes had higher expression levels in the VvWRKY28 transgenic line. The expression of AtRAB18, AtCOR15A, AtERD10, AtPIF4, AtCOR47,and AtICS1 was actively up-regulated, thereby further enhancing the adaptability of plants to cold. temperature stress. Therefore, the expression changes of six key genes located downstream of WRKY TF were analyzed (Figure 6), including AtRAB18 (Arabidopsis thaliana, U75603.1), AtCOR15A (Arabidopsis thaliana, NM_001336986.1), AtERD10 (Arabidopsis thaliana, BG732218.1), AtPIF4 (Arabidopsis thaliana, NM_001337007.1), AtCOR47(Arabidopsis thaliana, NM_101894.4), and AtICS1 (Arabidopsis thaliana, AT1G74710). A. thaliana was grown at −4 °C for 12 h, and it was found that these six genes had higher expression levels in the VvWRKY28 transgenic line. The expression of AtRAB18, AtCOR15A, AtERD10, AtPIF4, AtCOR47,and AtICS1 was actively up-regulated, thereby further enhancing the adaptability of plants to cold.

Analysis of the Ability of VvWRKY28 Transgenic A. thaliana to Resist Salt Stress
Only 200mm NaCl solution was used to irrigate WT and transgenic A. thaliana transplanted into nutrient soil every day. After a week, the growth of all plants was observed. It was found that the phenotypes of WT and transgenic plants were healthy under control conditions. After seven days of salt stress treatment, the leaves of all plants appeared yellowing, but the withering of transgenic plants was not obvious, and the yellowing of wild-type plants was serious ( Figure 7A).

Analysis of the Ability of VvWRKY28 Transgenic A. thaliana to Resist Salt Stress
Only 200mm NaCl solution was used to irrigate WT and transgenic A. thaliana transplanted into nutrient soil every day. After a week, the growth of all plants was observed. It was found that the phenotypes of WT and transgenic plants were healthy under control conditions. After seven days of salt stress treatment, the leaves of all plants appeared yellowing, but the withering of transgenic plants was not obvious, and the yellowing of wild-type plants was serious ( Figure 7A). Figure 7B showed the survival rates of all A. thaliana strains grown in a high salt environment. When the growth conditions were normal, the survival rates of WT and transgenic lines (S2, S6, and S7) were 97.3%, 96.7%, 96.4%, and 97.2%, respectively. The survival rates of WT and transgenic plants (S2, S6 and S7) were significantly different after surviving in a high salt environment for one week, and the survival rates decreased to 22.5%, 85.4%, 84.7%, and 84.6%, respectively. Compared with WT plants, the survival rate of transgenic plants under salt stress was significantly higher.
The changes of various physiological indexes of A. thaliana after salt stress treatment were shown in Figure 8. Under control conditions, the contents of chlorophyll, MDA, and proline, and the activities of SOD, POD, and CAT of all plants, were almost at the same level. However, under high salt conditions, the overexpression of VvWRKY28 led to a significant increase in the activities of SOD, POD, and CAT in transgenic A. thaliana, a significant increase in the content of proline, and a slight decrease in the content of chlorophyll, but it was still much higher than the that of WT plants. Moreover, it was obvious that the MDA content of transgenic lines was lower than that of WT lines. Therefore, it can be concluded that the high expression level of VvWRKY28 had a significant effect on improving the salt tolerance of transgenic A. thaliana.  liana, a significant increase in the content of proline, and a slight decrease in the content of chlorophyll,but it was still much higher than the that of WT plants. Moreover, it was obvious that the MDA content of transgenic lines was lower than that of WT lines.Therefore, it can be concluded that the high expression level of VvWRKY28 had a significant effect on improving the salt tolerance of transgenic A. thaliana.

Expression Analysis of Salt-Resistant Downstream Genes in VvWRKY28-OE A. thaliana
The signal transmission of salt stress is the core issue of plant salt stress response. TFs play a key role in the signal transmission pathway. No matter which pathway, the response of TFs to salt stress can be through ABA-dependent and ABA-independent pathways.Therefore, in order to investigate the effect of WRKY TFs on the expression of downstream salt stress-related genes, the expression levels of six salt stress-related genes

Expression Analysis of Salt-Resistant Downstream Genes in VvWRKY28-OE A. thaliana
The signal transmission of salt stress is the core issue of plant salt stress response. TFs play a key role in the signal transmission pathway. No matter which pathway, the response of TFs to salt stress can be through ABA-dependent and ABA-independent pathways. Therefore, in order to investigate the effect of WRKY TFs on the expression of downstream salt stress-related genes, the expression levels of six salt stress-related genes were examined. These six genes were AtNCED3  Figure 9. There was no obvious difference in the expression amount of these genes without salt stress treatment. After growing in a high salt environment (200 mm NaCl) for seven days, AtNCED3, AtSOS2, AtSnRK2.4, AtP5CS1, AtCAT2, and AtSOD1 in VvWRKY28-OE linesincreased more. These changes can explain that VvWRKY28 can improve the salt tolerance of plants through a variety of signal transduction pathways. The SOS signal transduction pathway is an important pathway for plants to respond to salt stress, which can regulate ion homeostasis at the cellular level to improve plant salt tolerance. SOS2 is a key gene in this pathway.

Discussion
In recent years, more and more TFs have been identified and studied, proving that they play a key role in plant abiotic stress. Whether at the cellular or individual level, transcription factor regulation is a very important way to control gene expression [26,34]. As one of the largest TF families in plants, WRKY TFs play an indispensable role in regulating plant responses to stress.Numerous studies have shown that WRKY TFs play an important role in the regulatory mechanism and can significantly enhance the resilience of plants to cope with complex environments [26]. Understanding the important regulatory role of WRKY genes in plant defense against adverse environments is critical for plant breeding and crop improvement.
In this study, the WRKY gene fragment was obtained by designing specific primers using homologous cloning technology, and the cloned target gene was analyzed by bio-informatics to explore the basic information of the gene and predict its biological function.After sequence analysis with DNAMAN, it was found that the amino acid sequence of the TF contains a highly conservative heptapeptide sequence WRKYGQK, and only contained a WRKY domain and a C2H2 zinc finger structure, indicating that it was a class II member of the WRKY family. Although the length of the VvWRKY28

Discussion
In recent years, more and more TFs have been identified and studied, proving that they play a key role in plant abiotic stress. Whether at the cellular or individual level, transcription factor regulation is a very important way to control gene expression [26,34]. As one of the largest TF families in plants, WRKY TFs play an indispensable role in regulating plant responses to stress. Numerous studies have shown that WRKY TFs play an important role in the regulatory mechanism and can significantly enhance the resilience of plants to cope with complex environments [26]. Understanding the important regulatory role of WRKY genes in plant defense against adverse environments is critical for plant breeding and crop improvement.
In this study, the WRKY gene fragment was obtained by designing specific primers using homologous cloning technology, and the cloned target gene was analyzed by bio-informatics to explore the basic information of the gene and predict its biological function. After sequence analysis with DNAMAN, it was found that the amino acid sequence of the TF contains a highly conservative heptapeptide sequence WRKYGQK, and only contained a WRKY domain and a C 2 H 2 zinc finger structure, indicating that it was a class II member of the WRKY family. Although the length of the VvWRKY28 protein was not completely the same as that of other homologous WRKY proteins, they differed greatly in non-conservative sequences, and they contained highly similar conservative sequences, which indicated that the WRKY gene family was highly conservative in the evolution process. The proteins with the highest homology and closest evolutionary relationship with VvWRKY28 were JrWRKY28 and CiWRKY28 were considered, which meant they had the same function. Class II WRKY TFs are generally involved in regulating plant growth and evolution and also play a role in plant resistance to their stress environment. It has been found that CiWRKY can respond to at least one abiotic stress (drought, cold, salt, high pH, or ABA) [35]. We also found that most of the homologous genes of VvWRKY28 are related to abiotic stresses such as low temperature and high salt. Therefore, we speculate that the target genes studied are also likely to participate in the process of plants responding to stress.
The WRKY TF geneisactively involved in the regulation of plant growth and metabolism and the transduction of various stress signals to regulate plant responses to stress [24,36]. At present, the research on the regulation of WRKY TFs on abiotic stress in plants is still in progress. In a previous study, we found that we found that overexpression of the Mb-WRKY1 gene isolated from M. baccata led to enhanced tolerance of transgenic tobacco to drought stress [37]. He et al. found that TaWRKY33 from the wheat subgroup IIa played an active regulatory role in ABA and drought-responsive signaling networks [38]. Multiple CmWRKY genes were rapidly up-regulated under drought and NaCl treatment, responding significantly to these stresses [39]. Therefore, the target gene VvWRKY28 in this study is very likely to play a similar role in the network of plant stress tolerance mechanisms.
As can be seen from Figure 3A, VvWRKY28 can be induced in roots, stems, new leaves, and mature leaves, but the expression levels are different, and higher expression levels are found in new leaves and roots. This indicated that the expression of the gene was tissue-specific, which may be because VvWRKY28 mainly senses stress signals in active organs.
In new leaves and roots, the expression of the VvWRKY28 gene after low temperature, high salt, water shortage, and high temperature stress was shown in Figure 3B,C. When the new leaves were grown under these four growth conditions, the VvWRKY28 gene expression level reached the peak time of 3 h, 12 h, 24 h, and 12 h, respectively. Under the same conditions, VvWRKY28 gene expression in roots reached the highest level at 12, 6, 12, and 24 h, respectively. This situation may be because new leaves were more sensitive to low temperature and high temperature stress than roots, indicating that new leaves were more susceptible to temperature, while roots respond faster to high salt and drought stress. Moreover, the maximum expression level of VvWRK28 in new leaves and roots under cold and salt stress was higher than that under other stresses. These phenomena indicated that VvWRK28 gene expression could be induced by low temperature, high salt, drought, and high temperature stress, but VvWRKY28 might play a more obvious role in regulating plant response to low temperature and salt stress.
Abiotic stress will not only change the appearance of plants and affect their survival rate, but it will also interfere with the normal metabolic pathways of plants, making various physiological indicators of plants not at normal levels during their growth and development [40,41]. Free radicals are inevitably produced in aerobic biological cells. The excessive accumulation of free radicals will cause harmful effects on plants, accelerate cell aging and damage, and lead to diseases [42]. The protective enzyme system is an important way to scavenge reactive oxygen species. This system mainly includes SOD, POD, and CAT. Their activities can be used as very key indicators in the study of plant resistance [43]. The growth environment of plants will affect the content of chlorophyll, which can reflect the growth status of plants in adversity. Therefore, the content of chlorophyll is of great significance to study the response of plants to adversity [44][45][46]. The accumulation of osmotic substances helps to improve the tolerance of plants to stress to a great extent. As a typical osmotic substance, proline plays an indispensable role in regulating osmotic balance and protecting cell structure and participates in the response process of plants to stress [47,48]. The accumulation of MDA will damage the plant cell membrane and cells to a certain extent. There is a close relationship between its content and the damage degree of plants. The more MDA that is accumulated, the more serious the damage to cell membranes will be [49,50]. The overexpression of VvWRKY28 in transgenic A. thaliana improvedthe survival rate of these plants under the survival pressure of cold and high salt ( Figures 5C and 7B), and also significantly increased the activities of SOD, CAT, POD, and the content of proline. Although the content of chlorophyll decreased slightly, it was significantly higher than that of WT. The content of MDA decreased greatly. These results all indicated that, when plants were subjected to low temperature and salt stress, VvWRKY28 can increase the accumulation level of enzymatic antioxidants (SOD, CAT, and POD) and proline, prevent the excessive accumulation of reactive oxygen species, and maintain cell osmotic balance, thereby improving plant cold tolerance and salt tolerance. In addition, lower MDA content can reduce the amount of oxidative damage to plant cells, so transgenic lines will be more efficient than WT lines.
As a plant-specific TF, the WRKY family is widely involved in plant response to stress. At the same time, the expression of genes can be regulated by binding to cisacting elements in the promoter regions of downstream genes to improve abiotic stress tolerance [51]. After low temperature treatment, it was found that the expression levels of the cold stress-related genes AtRAB18, AtCOR15A, AtERD10, AtPIF4, AtCOR47, and AtICS1 located downstream of VvWRKY28 were higher than those without cold stress, especially in VvWRKY28-OE plants (this is more obvious in Figure 6). The SOS signal transduction pathway is an important pathway for plants to respond to salt stress, which can regulate ion homeostasis at the cellular level to improve plant salt tolerance. SOS2 is a key gene in this pathway [26]. ROS is a key signaling molecule for cells to rapidly respond to different stimuli. Many studies have demonstrated that WRKY TFs can regulate the homeostasis of ROS by regulating the expression of antioxidant enzymes (CAT, SOD, POD) genes and improve the salt tolerance of plants [52,53]. TFs regulate salt stress mainly through ABAdependent and ABA-independent pathways. Compared with WT Arabidopsis, under salt stress, the expression levels of SOS2 and P5CS1 genes and the correlation in ABA signal transduction pathway genes NCED3 and SnRK2.4, as well as the antioxidant enzyme genes CAT2 and SOD1, were more significantly up-regulated in VvWRKY28-OE A. thaliana. Thus, the adaptability of plants to salt stress was improved. The expression levels of these genes were generally consistent with previous studies [54,55]. These results suggested that plants can not only improve their tolerance to low temperature by combining with cis-acting elements of downstream target genes, but also regulate the expression of downstream genes related to salt stress through ABA-dependent and ABA-independent pathways, as well as ROS and SOS pathways, so as to make plants more adapted to high salt environment. The high-level expression of these related downstream genes proved that VvWRKY28 plays an important regulatory role in the complex mechanism of plant response to salt and cold stress, which laid a foundation for further studies on the function of WRKY TFs. Besides, further studies are needed to understand the molecular mechanism of VvWRKY28 in regulating plant stress responses.
Therefore, according to previous reports and the results of this experiment, a possible model was established to show the response mechanism of VvWRKY28 gene to low temperature and salt stress ( Figure 10). The expression level of VvWRKY28 was significantly increased after cold stress, and it was bound to the W-box on the promoters of downstream target genes and regulated their expression, resulting in a significant increase in the expression levels of RAB18, COR15A, ERD10, PIF4, COR47, and ICS1. In addition, salt stress can also induce the high-level expression of VvWRKY28, activate the ABA signaling pathway and the salt stress response process, and promote the expression of NCED3 and SnRK2.4 in the ABA-dependent pathway. CAT2, SOD1, and SOS2 were rapidly expressed to regulate the content of ROS and ion balance in cells, and the expression level of ABA-independent P5CS1 gene was significantly increased.

Plant Material and Growth Conditions
The tissue culture seedlings of Beichun(it is a hybrid of Muscat Hamburg and Vitis amurensisRupr by the Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences) were placed in a MS growth medium containing agar for rapid propagation. The concentration of indole-3-butyric acid (IBA) and 6-benzylaminopurine (6-BAP) added to the medium was 0.6 mg/L.After 35 days of culture, tissue culture seedlings with good growthwere transferred to a MS rooting medium for continuous culture to promote their rooting. At this time, the concentration of IBA added to the medium was 1.2 mg/L [23]. After growing roots, the tissue cultured seedlings were transplanted to Hogland nutrient solution for growth, and the hydroponic solution was changed every 3 days. The environment of the tissue culture room remained stable (temperature of about 25 °C, relative humidity of 80-85%). When the tissue culture seedlings grow to have 7-10 complete leaves (fully expanded leaves) and relatively strong roots, the 50 seedlings with good morphology and basically the same growth trend were divided into 5 groups, which were respectively placed under low temperature (incubator temperature of 4 °C), high salt (200 mM NaCl high saline culture conditions), water shortage (Hogland nutrient solution containing PEG6000 with a concentration of 20%), and high temperature (incubator temperature of 37 °C). The other group, as the CK, was grown in a tissue culture chamber at 25 °C and hydroponically cultured with normal Hoagland solution. According to our previous study, the new leaves, roots, mature leaves, and stems of all seedlings were sampled at 0, 1, 3, 6, 12, 24, and 48 h after treatment [15]. After sealing,

Plant Material and Growth Conditions
The tissue culture seedlings of Beichun(it is a hybrid of Muscat Hamburg and Vitis amurensis Rupr by the Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences) were placed in a MS growth medium containing agar for rapid propagation. The concentration of indole-3-butyric acid (IBA) and 6-benzylaminopurine (6-BAP) added to the medium was 0.6 mg/L. After 35 days of culture, tissue culture seedlings with good growthwere transferred to a MS rooting medium for continuous culture to promote their rooting. At this time, the concentration of IBA added to the medium was 1.2 mg/L [23]. After growing roots, the tissue cultured seedlings were transplanted to Hogland nutrient solution for growth, and the hydroponic solution was changed every 3 days. The environment of the tissue culture room remained stable (temperature of about 25 • C, relative humidity of 80-85%). When the tissue culture seedlings grow to have 7-10 complete leaves (fully expanded leaves) and relatively strong roots, the 50 seedlings with good morphology and basically the same growth trend were divided into 5 groups, which were respectively placed under low temperature (incubator temperature of 4 • C), high salt (200 mM NaCl high saline culture conditions), water shortage (Hogland nutrient solution containing PEG6000 with a concentration of 20%), and high temperature (incubator temperature of 37 • C). The other group, as the CK, was grown in a tissue culture chamber at 25 • C and hydroponically cultured with normal Hoagland solution. According to our previous study, the new leaves, roots, mature leaves, and stems of all seedlings were sampled at 0, 1, 3, 6, 12, 24, and 48 h after treatment [15]. After sealing, they were immediately placed in liquid nitrogen and stored at −80 • C to prepare for RNA extraction.

Cloning and qRT-PCR Expression Analysis of VvWRKY28
The EasyPure Plant RNA Kit (TransGen Biotech, Beijing, China) was used to extract total RNA. The extracted parts were roots, stems, young leaves, and mature leaves. The first strand of cDNA was synthesized by Trans Script ® First-Strand cDNA Synthesis Super Mix (transgen, Beijing, China). With the CDs region of VvWRKY28 as the reference sequence, two pairs of specific primers were designed with Primer 5.0 software, named VvWRKY28-F and VvWRKY28-R, respectively. The specific sequences were shown in Table S1. The complete sequence of the gene was obtained by PCR, and the amplification template was the first strand cDNA of Beichun. After gel purification, the obtained DNA fragment was cloned into pEASY-T1 Cloning Kit (TransGen Biotech, Beijing, China) and sequenced (BGI, Beijing, China).
With reference to the method of Han et al. [56], qRT-PCR was performed on VvWRKY28 to analyze its expression under abiotic stress. The housekeeping gene VvActin (AF369524) was used as an internal control, and the expression of this gene can remain stable under almost all conditions. 25 µL of qPCR amplification mixture contained 12.5 µL 2xMix, 9 µL ddH 2 O, 1.5 µL cDNA, 1 µL VvWRKY28-F, and 1 µL VvWRKY28-R. The PCR reaction procedure was: pre denaturation at 95 • C for 5min, denaturation at 95 • C for 45 s, annealing at 56 • C for 1 min, and extension at 72 • C for 1 min. After 35 cycles, it continued to extend at 72 • C for 5 min [57]. The relative expression of the gene was obtained bythe 2 −∆∆CT method [58].

Subcellular Localization Analysis of the VvWRKY28 Protein
The VvWRKY28 ORF was cloned between the SacI and BamHI sites of the pSAT6-GFP-N1 vector, and the modified red-shifted green fluorescent protein (GFP) was present between these two sites. The constructs were injected into onion epidermal cells, the nuclear markers in the nuclear assay were stained with DAPI, and the transient expression of VvWRKY28-GFP fusion protein was observed under confocal microscopy (LSM 510 Meta, Zeiss, Jena, Germany).

Obtaining Transgenic A. thaliana
The 5 and 3 ends of VvWRKY28 cDNA were added to the restriction sites of SacI and BamHI by PCR to construct an expression vector for the transformation of A. thaliana. The primers used were shown in Table S1 (q-PCR-F, q-PCR-R). The PCR product and PCAM3011 were linked together by GUS gene replacement after being digested by SacI and BamHI. Transformation of the Agrobacterium, mediated by GV3101, into the Columbia ecotype A. thaliana was completed by the inflorescence-mediated method. Transgenic A. thaliana was selected for the MS medium (the concentration of kanamycin added was 50 mg/L). After qRT-PCR analysis, the transgenic line was finally determined, and the CK was WT. Further analysis was carried out with T 3 generation plants as experimental materials.

Determination of Relevant Physiological Indexes
The seeds of WT and transgenic lines (S2, S6, and S7) were sown on the culture medium and then transferred to the nutrient soil containing vermiculite two weeks later (vermiculite: nutrient soil ratio was 1:2). After 14 days of growth, all A. thaliana was divided into two groups, one group was treated at the low temperature of −4 • C for 12 h, and the other group was treated at high salt conditions irrigated with 200 mM NaCl solution for 7 days. Then, the survival rate was calculated with 15 nutrient pots [59].
CK and Arabidopsis thaliana, after stress treatment, were collected for the measurement of physiological indicators. The measurement of chlorophyll content referred to the method of Lu et al. [60]. Proline content was measured according to the method of Ou et al. [61]. MDA content was analyzed at 530 nm by the TBA method [62]. CAT activity was measured according to the method described by Zhang et al. [63]. SOD activity was determined with nitroblue tetrazolium (NBT) [64]. The activity of POD was measured according to the method described by Wang et al. [65].

Analysis of the Downstream Gene Expression of VvWRKY28
The RNA of WT and transgenic Arabidopsis, without stress treatment and after cold and salt stress treatment, was extracted. The RNA was reverse transcribed into the first strand cDNA as an amplification template. The internal reference was VvActin. According to the reaction system in Section 2.2, the expression levels of several downstream target genes (RAB18, COR15A, ERD10, PIF4, COR47, ICS1, NCED3, SnRK2.4, CAT2, SOD1, SOS2, and P5CS1) of WRKY TFs were detected by QRT PCR. The specific primers used were listed in Table S1.

Statistical Analysis
The one-way analysis of variance was completed by SPSS21.0 software (IBM, Chicago, IL, USA). All data were obtained after 3 repeated tests, and then thestandard deviation (SD) was measured separately. Statistical differences were referred to as significant (* p ≤ 0.05, ** p ≤ 0.01).

Conclusions
In this study, a WRKY-TF named VvWRKY28 was isolated from Beichun. The subcellular results showed that the VvWRKY28 protein was localized in the nucleus. From the phylogenetic tree results, it can be concluded that the genetic relationship between the VvWRKY28 protein and the VvWRKY71 protein was the closest. Low temperature, high temperature, high salt, and drought stress can induce the high-level expression of VvWRKY28, but VvWRKY28 was more sensitive to low temperature and high salt signals, and its expression in roots and new leaves was higher. After it was introduced into Arabidopsis, it caused changes in many physiological and biochemical indicators of Arabidopsis under low temperature and high salt environment. For example, the content of MDA was significantly lower than that of WT Arabidopsis, but the content of proline and chlorophyll and the activities of CAT, SOD, and POD were higher in transgenic A. thaliana. In addition, the overexpression of VvWRKY28 can also play a certain regulatory role in the expression of downstream target genes. Under low temperature stress, binding with cis-acting elements promotes the expression of downstream genes related to cold stress, such as RAB18, COR15A, ERD10, PIF4, COR47, and ICS1. Moreover, it can also positively regulate the expression of genes related to salt stress such as NCED3, SnRK2.4, CAT2, SOD1, SOS2,and P5CS1 under salt stress. The results of this study comprehensively showed that the overexpression of VvWRKY28 played a key role in improving the cold tolerance and salt tolerance of plants.