Heterologous Expression of Three Ammopiptanthus mongolicus Dehydrin Genes Confers Abiotic Stress Tolerance in Arabidopsis thaliana

Ammopiptanthus mongolicus, a xerophyte plant that belongs to the family Leguminosae, adapts to extremely arid, hot, and cold environments, making it an excellent woody plant to study the molecular mechanisms underlying abiotic stress tolerance. Three dehydrin genes, AmDHN132, AmDHN154, and AmDHN200 were cloned from abiotic stress treated A. mongolicus seedlings. Cytomembrane-located AmDHN200, nucleus-located AmDHN154, and cytoplasm and nucleus-located AmDHN132 were characterized by constitutive overexpression of their genes in Arabidopsis thaliana. Overexpression of AmDHN132, AmDHN154, and AmDHN200 in transgenic Arabidopsis improved salt, osmotic, and cold tolerances, with AmDHN132 having the largest effect, whereas the growth of transformed plants is not negatively affected. These results indicate that AmDHNs contribute to the abiotic stress tolerance of A. mongolicus and that AmDHN genes function differently in response to abiotic stresses. Furthermore, they have the potential to be used in the genetic engineering of stress tolerance in higher plants.


Introduction
Dehydrins (DHNs), also known as group 2 late embryogenesis abundant (LEA) proteins [1][2][3][4][5][6], accumulate in response to dehydration stresses and play protective roles under stress conditions [7][8][9]. They function to resist drought, low temperature, high salinity, and abscisic acid stress. Dehydrins are typically highly hydrophilic and possess a large fraction of polar and charged amino acids [10]. The DHN protein family is characterized by the existence of a K-segment [11], while other conserved motifs, such as S-, Y-, and Φ-segments, have also been found in this protein family. However, the number of common amino acid sequences and the specific motifs in these proteins differ [12,13]. All plant dehydrins contain 1-15 K-segments, whose sequence is EKKGIMDKIKEKLPG. K-segments are lysine-rich and are located near the C terminus of dehydrins [14,15]. K-segments in dehydrins play an essential role, mainly stabilizing membranes [16][17][18]. Another conserved motif near the N-terminus of dehydrins is the tyrosine-rich Y-segment, which consists of 1-35 tandem repeats of the consensus sequence, (V/T)D(E/Q)YGNP. Another relatively conserved dehydrin sequence is the Accumulating evidence shows a positive correlation between dehydrin proteins and abiotic stress tolerance. Overexpression of the Opuntia streptacantha OpsDHN1 gene in Arabidopsis thaliana increased its tolerance to freezing treatment [20]. The dehydrin gene, OesDH, isolated from oleaster, the wild form of olive, enhanced drought tolerance in Arabidopsis transgenic plants [21]. Moreover, overexpression of durum wheat DHN-5 in Arabidopsis confers salinity tolerance [22]. Overexpression of RcDhn5 from Rhododendron catawbiense in Arabidopsis plants improved its freezing tolerance [23].
Although dehydrins participate in stress tolerance in cyanobacteria [24], Physcomitrella patens [25], and the necrotrophic fungus Alternaria brassicicola [26], the functions of DHN orthologs are poorly characterized in non-model plants, particularly perennial woody plants. Ammopiptanthus mongolicus, an evergreen broadleaf shrub plant [27,28], plays important ecological roles in the Inner Mongolia desert of China, and is an ideal species for research into abiotic tolerance of trees. We previously cloned and assessed the function of a cold-induced dehydrin-like gene, AmCIP, from A. mongolicus [6,29]. Here, we cloned AmDHN132, AmDHN154, and AmDHN200 from A. mongolicus via their high homology with AmCIP. Overexpression of the three dehydrin genes increased the tolerance of transgenic Arabidopsis to salt, osmotic, and cold stresses.

Sequence Characterization of the Dehydrin Gene Family
On the basis of previous studies [6,29], PCR amplification and sequencing indicated that three dehydrin cDNAs were successfully cloned and that each had a full-length open reading frame. These clones were 399, 465, and 603 bp in length, encoding 132, 154, 200 amino acids, respectively ( Figure  1, Table 1) and were named AmDHN132, AmDHN154, and AmDHN200. The cDNAs were uploaded to GenBank with the login numbers MH512938 for AmDHN132, MH512939 for AmDHN154, and MH512941 for AmDHN200.
The AmDHNs were classified as SK-types because of the following structural characteristics. They contain one S-segment and one K-segment and no Y-segment. However, one segment containing 20 amino acid residues, TGVLHGLGGHKGES(H/R)GDYKG, repeats one, two, and four times in AmDHN132, AmDHN154, and AmDHN200, respectively.

Creation of Transgenic Arabidopsis and the Subcellular Localization of AmDHNs
AmDHNs overexpression vectors ( Figure S4) were constructed and transformed into Arabidopsis. Western blotting indicated that the three AmDHN genes were successfully expressed in transgenic Arabidopsis ( Figure 2). GFP fluorescence showed that AmDHN154 localized in the  The AmDHNs were classified as SK-types because of the following structural characteristics. They contain one S-segment and one K-segment and no Y-segment. However, one segment containing 20 amino acid residues, TGVLHGLGGHKGES(H/R)GDYKG, repeats one, two, and four times in AmDHN132, AmDHN154, and AmDHN200, respectively. Plants 2020, 9,193 3 of 11

Creation of Transgenic Arabidopsis and the Subcellular Localization of AmDHNs
AmDHNs overexpression vectors ( Figure S4) were constructed and transformed into Arabidopsis. Western blotting indicated that the three AmDHN genes were successfully expressed in transgenic Arabidopsis ( Figure 2). GFP fluorescence showed that AmDHN154 localized in the nucleus, AmDHN200 localized in the cell membrane, and AmDHN132 localized in the cytoplasm and nucleus ( Figure 3).
Plants 2020, 9, x FOR PEER REVIEW  3 of 11 nucleus, AmDHN200 localized in the cell membrane, and AmDHN132 localized in the cytoplasm and nucleus (Figure 3).

Heterologous Expression of AmDHNs in Arabidopsis Enhances Abiotic Stress Tolerance
RT-PCR showed that AmDHNs were expressed in transgenic plants ( Figure 4). To demonstrate whether AmDHNs are involved in tolerance to abiotic stresses, wild-type and transgenic Arabidopsis overexpressing AmDHN132, AmDHN154, or AmDHN200 were treated with NaCl, mannitol, or low temperature. Wild-type and transgenic seeds were germinated on MS medium containing 0 (control), 50, 100, 150, or 200 mM NaCl. Under normal conditions, there was no difference between the transgenic and wild-type plants, but the seed germination rate of transgenic plants was significantly higher than that of wild-type plants on NaCl-containing medium (Figure 5a). After 10 days of treatment with 200 mM

Heterologous Expression of AmDHNs in Arabidopsis Enhances Abiotic Stress Tolerance
RT-PCR showed that AmDHNs were expressed in transgenic plants ( Figure 4). To demonstrate whether AmDHNs are involved in tolerance to abiotic stresses, wild-type and transgenic Arabidopsis overexpressing AmDHN132, AmDHN154, or AmDHN200 were treated with NaCl, mannitol, or low temperature. Wild-type and transgenic seeds were germinated on MS medium containing 0 (control), 50, 100, 150, or 200 mM NaCl. Under normal conditions, there was no difference between the transgenic and wild-type plants, but the seed germination rate of transgenic plants was significantly higher than that of wild-type plants on NaCl-containing medium (Figure 5a). After 10 days of treatment with 200 mM NaCl, most of the wild-type Arabidopsis leaves became white and gradually died, but overall the

Heterologous Expression of AmDHNs in Arabidopsis Enhances Abiotic Stress Tolerance
RT-PCR showed that AmDHNs were expressed in transgenic plants ( Figure 4). To demonstrate whether AmDHNs are involved in tolerance to abiotic stresses, wild-type and transgenic Arabidopsis overexpressing AmDHN132, AmDHN154, or AmDHN200 were treated with NaCl, mannitol, or low temperature.

Heterologous Expression of AmDHNs in Arabidopsis Enhances Abiotic Stress Tolerance
RT-PCR showed that AmDHNs were expressed in transgenic plants ( Figure 4). To demonstrate whether AmDHNs are involved in tolerance to abiotic stresses, wild-type and transgenic Arabidopsis overexpressing AmDHN132, AmDHN154, or AmDHN200 were treated with NaCl, mannitol, or low temperature. Wild-type and transgenic seeds were germinated on MS medium containing 0 (control), 50, 100, 150, or 200 mM NaCl. Under normal conditions, there was no difference between the transgenic and wild-type plants, but the seed germination rate of transgenic plants was significantly higher than that of wild-type plants on NaCl-containing medium (Figure 5a). After 10 days of treatment with 200 mM NaCl, most of the wild-type Arabidopsis leaves became white and gradually died, but overall the Wild-type and transgenic seeds were germinated on MS medium containing 0 (control), 50, 100, 150, or 200 mM NaCl. Under normal conditions, there was no difference between the transgenic and wild-type plants, but the seed germination rate of transgenic plants was significantly higher than that of wild-type plants on NaCl-containing medium (Figure 5a). After 10 days of treatment with 200 mM NaCl, most of the wild-type Arabidopsis leaves became white and gradually died, but overall the transgenic Arabidopsis grew better than wild-type Arabidopsis (Figure 5b). The seedling survival rate of the transgenic Arabidopsis was higher than that of the wild type ( Figure 5c). The data also indicated that AmDHN132 was better than AmDHN154 and AmDHN200 in conferring salt resistance.
Plants 2020, 9, x FOR PEER REVIEW 4 of 11 transgenic Arabidopsis grew better than wild-type Arabidopsis (Figure 5b). The seedling survival rate of the transgenic Arabidopsis was higher than that of the wild type ( Figure 5c). The data also indicated that AmDHN132 was better than AmDHN154 and AmDHN200 in conferring salt resistance. To reveal the role of AmDHNs in resistance to osmotic stress, mannitol (0-300 mM) was added to MS medium and seed germination was detected. AmDHNs improved seed germination under osmotic stress (Figure 6a). After 10 days of treatment with 200 mM mannitol, the number of lateral roots of transgenic Arabidopsis was much higher than that of the wild type. When grown on normal medium, the roots of AmDHN200 transgenic Arabidopsis were well developed and the true leaves were larger than those of the wild type (Figure 6b). The number of lateral roots of transgenic Arabidopsis was higher than that of the wild type after osmotic stress (Figure 6c). Overall, AmDHN132 was more effective than AmDHN154 and AmDHN200 in improving osmotic resistance.
Freezing tolerance was also evaluated. After treatment at −5 °C followed by recovery, the growth phenotype, electrolyte leakage, and survival rate showed that overexpression of AmDHN132 and AmDHN200 significantly improved the freezing resistance of transgenic Arabidopsis (Figure 7). To reveal the role of AmDHNs in resistance to osmotic stress, mannitol (0-300 mM) was added to MS medium and seed germination was detected. AmDHNs improved seed germination under osmotic stress (Figure 6a). After 10 days of treatment with 200 mM mannitol, the number of lateral roots of transgenic Arabidopsis was much higher than that of the wild type. When grown on normal medium, the roots of AmDHN200 transgenic Arabidopsis were well developed and the true leaves were larger than those of the wild type (Figure 6b). The number of lateral roots of transgenic Arabidopsis was higher than that of the wild type after osmotic stress (Figure 6c). Overall, AmDHN132 was more effective than AmDHN154 and AmDHN200 in improving osmotic resistance. Freezing tolerance was also evaluated. After treatment at −5 • C followed by recovery, the growth phenotype, electrolyte leakage, and survival rate showed that overexpression of AmDHN132 and AmDHN200 significantly improved the freezing resistance of transgenic Arabidopsis (Figure 7). overexpressing lines with and without osmotic stress. (c) Lateral root numbers of wild-type and transgenic plants after osmotic treatments. AmDHN154 transformed lines: 2-10-2, 2-10-39, and 2-11-2. AmDHN132 transformed lines: 4-10-19, 4-10-30, and 4-8-18. AmDHN200 transformed lines: 7-4-29, 7-5-47, and 7-6-59. Each column is the mean of three independent experiments and bars represent the standard error of the mean. The different letters denote significant differences (p < 0.05) according to ANOVA results.

AmDHNs Are Conserved in A. mongolicus
Dehydrins are classified as group II LEA family proteins and they play a fundamental role in resisting abiotic and biotic stresses [30]. The DHNs is a redundant family with 4 to 13 or more genes in different plant species [31]. The sequence of dehydrin proteins must have at least one copy of the lysine-rich K-segment [9]. Accordingly, in this study, we amplified three dehydrin genes from A. mongolicus based on previous findings [29]. AmDHN members are highly homologous and contain one S-segment and one K-segment. Unlike most dehydrins, the K-and S-segments are only present in single copies in the AmDHN sequences and the S-segment is located at the C-terminus, making them KS-type dehydrins [32]. In addition, the AmDHN sequences differ in the number of repeats of a 20 amino acid residue sequence, TGVLHGLGGHKGES(H/R)GDYKG.

Overexpression of AmDHNs Increases Abiotic Stress Tolerance of Arabidopsis
Research indicates that the role of DHNs is to defend the cell from dehydration damage caused by environmental stresses and cellular dehydration [33]. Stress tolerance was improved in tobacco by overexpression of SbDhn1 from Sorghum bicolor [34], in bananas by constitutive overexpression of MusaDHN-1 [35], and in rice by overexpression of OsDhn1 [36]. Here, we found that constitutive expression of AmDHN genes from A. mongolicus in Arabidopsis increased salt, osmotic, and cold tolerance, which is consistent with these previous studies. However, functional differences exist among the AmDHNs. Previously, Falavigna et al. [37] found that apple MdDHN proteins have different functions and overlapping levels of expression, and that their expression is fine-tuned by the environment during the dormancy process. In this study, Arabidopsis seedlings overexpressing three AmDHN genes have different abiotic stress tolerances, which may result from their differences in structure, homology, and location.

AmDHNs Are Conserved in A. mongolicus
Dehydrins are classified as group II LEA family proteins and they play a fundamental role in resisting abiotic and biotic stresses [30]. The DHNs is a redundant family with 4 to 13 or more genes in different plant species [31]. The sequence of dehydrin proteins must have at least one copy of the lysine-rich K-segment [9]. Accordingly, in this study, we amplified three dehydrin genes from A. mongolicus based on previous findings [29]. AmDHN members are highly homologous and contain one S-segment and one K-segment. Unlike most dehydrins, the K-and S-segments are only present in single copies in the AmDHN sequences and the S-segment is located at the C-terminus, making them KS-type dehydrins [32]. In addition, the AmDHN sequences differ in the number of repeats of a 20 amino acid residue sequence, TGVLHGLGGHKGES(H/R)GDYKG.

Overexpression of AmDHNs Increases Abiotic Stress Tolerance of Arabidopsis
Research indicates that the role of DHNs is to defend the cell from dehydration damage caused by environmental stresses and cellular dehydration [33]. Stress tolerance was improved in tobacco by overexpression of SbDhn1 from Sorghum bicolor [34], in bananas by constitutive overexpression of MusaDHN-1 [35], and in rice by overexpression of OsDhn1 [36]. Here, we found that constitutive expression of AmDHN genes from A. mongolicus in Arabidopsis increased salt, osmotic, and cold tolerance, which is consistent with these previous studies. However, functional differences exist among the AmDHNs. Previously, Falavigna et al. [37] found that apple MdDHN proteins have different functions and overlapping levels of expression, and that their expression is fine-tuned by the environment during the dormancy process. In this study, Arabidopsis seedlings overexpressing three AmDHN genes have different abiotic stress tolerances, which may result from their differences in structure, homology, and location.
DHNs are localized in different cell compartments, such as the cytosol, nucleus, mitochondria, vacuoles, and close to the plasma membrane, but they are mainly localized in the cytoplasm and nucleus [38]. In some cases, dehydrin proteins are limited to certain areas of an organ, such as guard cells, meristem cells, and pollen sacs [38]. Correspondingly, differences in dehydrin localization may be one of the reasons for its functional differentiation. AmDHN132 and AmDHN200 proteins in the transgenic Arabidopsis were located in the cell membrane ( Figure 2). The plants transformed by these genes showed high survival rates and lower conductivity values after freezing ( Figure 6). The degree of extravasation of electrolyte reflects the relative integrity of the cell membrane, indicating that AmDHN132 and AmDHN200 protected cell membranes from freezing injury. Some studies reported that DHN accumulates or migrates to the plasma membrane during stress [39,40]. The membrane damage of DHNA and DHNB knockout mutants of P. patens increased under stress [41]. Our findings support the membrane-protective effect of dehydrin proteins.
Although we have no information on the upstream cis-acting elements of the three AmDHN genes, they will be important in the functional differentiation of the three genes. Agarwal et al. [42] found that PpDHNA and PpDHNB were resistant to drought and cold, and they identified upstream low-temperature response cis-acting elements in the genes. By contrast, PpDHNC showed microbial resistance, and its upstream cis-acting elements were related to biological stress and not low-temperature. The difference among the upstream sequences of these three genes therefore led to functional differentiation. The identification of upstream cis-acting regulatory elements of the AmDHN genes is therefore important and will help reveal the molecular mechanism of AmDHNs underlying abiotic stress in A. mongolicus.

Potential Use of AmDHNs and Further Research
In summary, overexpression of AmDHNs improves the abiotic tolerance in the transgenic Arbidopsis plants, where the growth of transformed plants is not negatively affected. We, therefore, believe that AmDHNs could potentially be used in diverse crop and tree species to confer abiotic stress tolerance. For further research into the biological functions of AmDHNs, visualization of their changing localization under stress conditions will be highly valuable, because the differences in dehydrin localization and migration may be one of the reasons for its functional differentiation.

Plant Material and Normal and Stress Growth Conditions
A. mongolicus seeds were sown in pots with potting perlite matrix and grown in a culture chamber (25 • C, 60% relative humidity, 16

Construction and Transformation of Eukaryotic Expression Vectors
Using gene specific primers, PCAN and PCBP, the open reading frames of AmDHNs were amplified by PCR. To construct the expression vectors, the pEASY ® -Blunt Simple Cloning vector containing the ORF of AmDHNs was used as a template and a Hind III restriction site was added to the 5'end of the sense primers, a Pst I site was added to the 5'end of the antisense primer. The PCR product was digested with Hind III/Pst I and was subcloned into pCAMBIA2300-GFP vector, which uses the cauliflower mosaic virus 35S promoter to drive expression ( Figure S4). Recombinant and empty plasmids were transferred to the Agrobacterium strain LBA4404 and used to transform A. thaliana by the floral dip method [43].

Screening of Homozygous Lines
T1 seeds were collected, surface sterilized, and then seeded on sterile media containing 50 µg/mL kanamycin to select positive seedlings. A. thaliana was grown in a culture chamber after a 2-day vernalization period. Plants were then transferred to soil for cultivation to obtain T2 seeds. T3 seeds were acquired in the same way. Finally, T3 generation seeds grown on medium containing kana were considered to be homozygous strains if they grew normally.

RT-PCR Analysis
RT-PCR was performed according to the manufacturer's instructions (Tiangen, Biotech Co. LTD, Beijing, China). Sample and control reactions were amplified for 30 cycles. Primers used are listed in Table S1. Actin was used as an internal control in semi-quantitative RT-PCR analysis of dehydrin genes.

Western Blot Analysis
AmDHN protein levels were detected by Western blotting [44]. Proteins were extracted from two-week-old leaves, separated by 12% SDS-PAGE, and then transferred to nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). Blots were probed using an anti-GFP tag monoclonal antibody HRP-goat anti-mouse IgG (Augct, Beijing, China). Signals were detected by chemiluminescence using the chemiluminescent horseradish peroxidase (HRP) substrate system (Millipore) [7].

Subcellular Localization of AmDHN
Arabidopsis seeds were vernalized at 4 • C for 2 days and then grown on MS solid medium in the culture room for 4 days. To localize AmDHNs, fluorescence of GFP in the root cells was observed by laser confocal microscopy (Ti-E/Ti-U/Ti-S, Nikon, Tokyo, Japan). For GFP, the excitation wavelength was 488 nm.

Abiotic Stress Tolerance Analysis
Sterilized control and transgenic Arabidopsis seeds were vernalized at 4 • C on square MS (pH 5.8) medium for 2 days and then grown in a culture chamber under long-day conditions at 22 • C. For freezing tolerance analysis, 10-day-old wild-type and homozygous transgenic Arabidopsis plants grown under normal culture conditions were transferred to −5 • C for 4 h and then survival rate was calculated after 3 days of recovery at 22 • C. For electrolyte leakage analysis, eight pieces of leaf samples with the same area from each group were washed with deionized water and then immersed in 10 mL of deionized water. After vacuum filtration, the electrical conductivity of the supernatant (S1) was detected using a DDS-307 detector. The samples were then lethally boiled to detect the ultimate conductivity (S2, maximum conductivity of tissues). The relative leakage degree was calculated by the ratio S1/S2. For salt and drought stress tolerance evaluation, wild-type and transgenic Arabidopsis seeds were cultured on MS medium containing 0 (control), 50, 100, 150, 200 mM NaCl or 100, 200, 300 mM mannitol for 10 days. Germination rates were then measured. Eight-day-old seedlings were moved to MS medium containing 200 mM NaCl or 200 mM mannitol and cultured for 10 days. The survival rate was calculated after stress treatment. The experiment was repeated three times.

Data Analysis
Experiments were conducted in three biological replicates with each replicate consisting of three plants. All data were analyzed using SPSS (version 19.0; IBM Corp., Armonk, NY, USA) and were expressed as the mean ± SE. Multigroup comparisons of the means were carried out by one-way analysis of variance (ANOVA) test with post hoc contrasts by Student-Newman-Keuls test. The statistical significance for all tests was set at p < 0.05. All charts were plotted using Microsoft Excel.