**3. Discussion**

GA is an important phytohormone that regulates plant growth. Increasing evidence has demonstrated that GA plays a role in the response to environmental stresses. DELLA proteins are key negative regulators in GA signaling, and our results showed that DELLA proteins increased drought tolerance by interacting with ABF2, a positive regulator in the ABA signaling pathway.

The cellular mechanism of the drought tolerance of the *gai-1* mutant seems complex and confusing. The stomatal density of the *gai-1* mutant is higher than that of the wild type, which makes water loss occur more quickly. Indeed, we found that the water loss rate of the detached leaves in the *gai-1* mutant was higher than that of the wild type (Figure 1B). The stomatal aperture in ABA treatment was found to be smaller in the *gai-1* mutant than that of the wild type sample, which may be responsible for the drought tolerance. The stomata's phenotype, density, and aperture of the *gai-1* mutant are similar to those of the gain-of-function of *PRO* in tomato [8], suggesting that it is conserved for DELLA proteins in regulating plant development and stress response.

As DELLA proteins do not have a DNA binding domain, it is common for them to interact with other transcription factors to regulate downstream target genes. For example, DELLA proteins interact with ABI3 and ABI5 to activate *SOM* (*SOMNUS*) expression at high temperatures [15]; RGA interacts with BZR1 (Brassinazole-resistant 1) to inhibit its transcriptional activities to downstream genes to regulate cell growth [16]; RGA interacts with WRKY6 (WRKYGQK) to block its transcriptional activities on its downstream genes, *SAG13* (Senescence-associated gene13) and *SGR* (Stay green), to regulate senescence [17]; and RGL2 interacts with the NF-YC (NUCLEAR FACTOR-Y C) homologues NF-YC3, NF-YC4, and NF-YC9 to activate the downstream gene ABI5 to regulate seed germination [18]. Here, we showed that DELLA proteins interact with ABF2 to regulate drought tolerance. AREB/ABF (abscisic acid-responsive element binding) proteins play pivotal roles in the regulation of plant responses to abiotic stresses. By binding to the ABRE element in the promoter region of stress-responsive genes, AREB/ABF factors regulate gene expression under drought stress [19,20]. In Arabidopsis, four AREB/ABF factors, namely, AREB1/ABF2, AREB2/ABF4, ABF1, and ABF3, are induced by ABA and osmotic stress [21,22]. Overexpressing *AREB1*/*ABF2*, *AREB2*/*ABF4*, or *ABF3* promotes drought tolerance, and a loss-of-function of these genes enhances drought sensitivity [14,16,23,24]. Many stress-inducible genes, including *RD29B* and *RAB18*, were downregulated in the *areb1 areb2 abf3 abf1-2* quadruple mutant [20]. Our results showed a strong interaction between DELLA proteins and the ABF2 protein (Figure 2A,B). It is reasonable that the interaction of DELLA proteins with ABF2 could activate ABF2 transcriptional activity to promote drought tolerance. A further expression assay of the downstream gene in the *gai-1* mutant and the binding assay of ABF2 to the promoter of *RD29B* or *RAB18* in the presence or absence of DELLA proteins would allow for the determination of the role of the interaction between DELLA proteins and ABF2. We also cannot exclude the possibility that DELLA proteins interact with other AREB/ABF factors, such as AREB2/ABF4, ABF1, or ABF3. A further interaction assay between the DELLA proteins and other AREB/ABF factors will deepen our understanding of the role of DELLA proteins in drought tolerance.

In summary, our results showed that GAI increased drought tolerance in Arabidopsis. GAI had conserved functions in increasing the stomatal density and decreasing the stomatal aperture under ABA treatment conditions. Further, we showed that GAI interacted with ABF2, especially the

N-terminal end P domain and the bZIP domain of ABF2. Our results provide new insight into DELLA protein functions in drought stress tolerance and the crosstalk between ABA and GA in response to drought tolerance.

#### **4. Materials and Methods**

#### *4.1. Plant Materials and Growth Conditions*

*Arabidopsis thaliana* ecotype Col-0 was used as the wild type in this study. The *gai-1* mutant was kindly gifted by Dr. Xiangdong Fu (Institute of Genetics and Developmental Biology, CAS). The seeds were germinated and grown on MS (Murashige & Skoog) medium and transplanted into soil at 10 days after germination. The plants were grown under a 16 h light/8 h dark photoperiod at 23 ◦C.

#### *4.2. Drought Treatment, Water Loss Analysis, and Stomatal Aperture Measurement*

For measurement of drought tolerance, water was withheld from 21 day old wild-type and *gai-1* mutant plants. After 21 days of drought treatment, the plants were rewatered; the plants were photographed 6 days after re-watering. For measurement of water loss, eight rosette leaves from eight plants were detached from 3 week old well-watered plants and weighted at the indicated times. For the stomatal function, rosette leaves from well-watered plant were incubated in a solution containing 50 mM KCl, 10 mM CaCl2, and 10 mM MES (pH 6.15) for 2 h under light. ABA was then added to the solution to a final concentration of 10 μM. After ABA treatment for 2 h, stomatal apertures were measured as described previously [2].

## *4.3. Protein–Protein Analysis*

The constructs were created in two pairs of Gateway-compatible destination vectors: pGBKT7-DEST (BD) with pGBAD7-DEST (AD) and pEarleyGate201-YN (N-terminal YFP) with pEarleyGate202-YC (C-terminal YFP) [25]. The coding sequences of *GAI*, *RGA*, *RGL1*, *RGL2*, *RGL3*, *ABF2*, and di fferent deletion fragments of *ABF2* were amplified from Col-0 cDNA, inserted into pDONR207, and then recombined in the appropriate destination vector. Yeast two-hybrid and BiFC assays were performed as previously described [2]. For Y2H, *Saccharomyces cerevisiae* strain AH109 was used for co-transformation of the AD and BD constructs. A series of 5 μL aliquots of diluted co-transformed AH109 culture was spotted onto SD plates lacking Trp, Leu, His, and Ade, and incubated at 30 ◦C for 2–5 days. Plasmids pGBKT7 and pGADT7-Rec were used as negative controls. For the BiFC assay, *Agrobacterium tumefaciens* GV3101 carrying the YFP N-terminal and YFP C-terminal fusion constructs was infiltrated into *N. benthamiana* leaves, as described by Luo et al. [2]. The reconstituted YFP signals were observed using confocal imaging 48 h after infiltration. Empty vectors were used as negative controls.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/21/3/819/s1.

**Author Contributions:** Conceptualization, X.L.; methodology, Z.W., C.C., L.L., Z.R., and S.X.; validation, Z.W., and Z.R.; formal analysis, Z.W. and L.L; investigation, X.L.; writing—original draft preparation, Z.W.; writing—review and editing, X.L.; funding acquisition, X.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Program of China, gran<sup>t</sup> number 2016YFA0500503; Fundamental Research Funds for Central Universities, gran<sup>t</sup> number 2662018PY075; and Huazhong Agricultural University's Scientific and Technological Self-innovation Foundation, gran<sup>t</sup> number 2015RC014.

**Acknowledgments:** We thank Xiangdong Fu (Institute of Genetics and Developmental Biology, CAS) for providing *gai-1* mutant seed.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

*Int. J. Mol. Sci.* **2020**, *21*, 819
