**2. Results**

To study the function of DELLA proteins in drought tolerance in Arabidopsis, we firstly analyzed the phenotype of the *gai-1* mutant under the condition of drought treatment. Three-week-old seedlings of wild type (Col-0) and mutant *gai-1* were withheld from water for 21 days. The wild type plants were severely wilted, whereas the *gai-1* mutant did not wilt and continued to grow. After rewatering, all of the *gai-1* plants recovered, whereas none of the wild type plants survived (Figure 1A), indicating that the *gai-1* mutant is more tolerant to drought and that GAI is a positive regulator in the plant response to drought tolerance. The function of GAI in drought tolerance is consistent with that of PRO in tomato, suggesting that this tolerance is a conserved function of DELLA proteins in the plant kingdom.

Changes in transpiration rate could account for the altered tolerance to drought. We then tested the water loss rate of the detached leaves. Leaves of 3 week old seedlings were cut and exposed to air and were weighted at regular time points. To our surprise, the *gai-1* mutant leaves lost their water at a much higher rate than the wild type leaves (Figure 1B). The water loss of the wild type sample was only 20%, whereas the water loss of the *gai-1* mutant was over 30% at 4 h after exposure to air, suggesting that the *gai-1* mutant is sensitive to dehydration when detached leaves are exposed to air.

The stomata are key channels that control gas exchange and water evaporation. We then tested the stomatal density and aperture from leaves of wild type and *gai-1* plants grown in soil. The stomatal density of the *gai*-*1* mutant was significantly higher than that of the wild type plant (2.6×) (Figure 1C,D). This may be the reason for the higher rate water loss in the *gai-1* mutant for the detached leaves. For the stomatal apertures, the wild type and *gai-1* mutant were comparable under KCl-treated control conditions. However, under ABA treatment, the stomatal aperture of the *gai-1* mutant was much smaller than that of the wild type (Figure 1E,F). The stomatal density and aperture of the *gai-1* mutant under stress conditions are consistent with those of the *PRO* gain-of-function mutant, suggesting that this is a conserved mechanism for DELLA proteins in regulating plant development and environmental adaption.

Generally, DELLA proteins function by interacting with other transcriptional factors. As ABF2 (abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors) is a key regulator in drought tolerance, we hypothesized that GAI interacts with ABF2 to increase drought tolerance. To test this hypothesis, we tested the interaction between GAI and ABF2 via yeas<sup>t</sup> two-hybrid and BiFC (bimolecular fluorescent complimentary) assays. In the yeas<sup>t</sup> two-hybrid assay, GAI and ABF2 were recombined to the gateway-compatible destination vectors pGADT7-DEST (AD) and pGBKT7-DEST (BD), respectively. The AD and BD constructs were cotransformed to the yeas<sup>t</sup> strain AH109, and their interaction was determined by the growth on the SD (synthetic dropout) medium lacking

Trp (tryptophan), Leu (leucine), His (histidine), and Ade (adenine). There was a strong interaction between GAI and ABF2 in the yeas<sup>t</sup> two-hybrid assay (Figure 2A). For the BiFC assay, GAI and ABF2 were recombined to pEarleyGate201-YN (N-terminal YFP (yellow fluorescent protein)) and pEarleyGate202-YC (C-terminal YFP), respectively. Both constructs were coinfiltrated into *Nicotiana benthamiana* leaves. The YFP signal was observed in the nucleus of the plant cell coexpressing GAI-YFP<sup>N</sup> and ABF2-YFP<sup>C</sup> (Figure 2B), but no YFP signal was detected in the plant cell coexpressing GAI-YFP<sup>N</sup> and empty YFP<sup>C</sup> or ABF2-YFP<sup>C</sup> and empty YFP<sup>N</sup> (Figure S1), indicating that GAI and ABF2 interacted in the nucleus of the plant cell. We also tested the interactions between other DELLA proteins, including RGA, RGL1, RGL2, and RGL3, with ABF2. The yeas<sup>t</sup> two-hybrid and BiFC assays both showed that all of the DELLA proteins interacted with ABF2 (Figure 2A,B).

**Figure 1.** The *gai-1* (gibberellin insensitive 1) mutant is more tolerant to drought stress than WT (wild type). (**A**) *gai-1* mutant plants showed tolerance to dehydration stress. *gai-1* mutant plants showed the ability to withstand long drought conditions without negative effects whereas the wild type under the same conditions completely wilted. (**B**) *gai-1* plants showed increased water loss compared to WT. Data shown are the means ± SDs from three biological repeats (*n* = 3, eight leaves from eight plants were used for each repeat, *p* < 0.001). (**C**,**D**) Stomatal density of WT and *gai-1* mutant. Stomatal density was observed from comparable age leaves of 3 week old wild type and *gai-1* plants. The stomatal density was represent by number of stomata per millimeters squared. Data shown are the means ± SDs from three biological repeats (*n* = 3, five leaves from five plants were used for each repeat, *p* < 0.001). (**E**) Representative stomata of the WT and *gai-1* mutant under control and abscisic acid (ABA) treatment conditions. Leaves of the WT and *gai-1* mutant were treated with 10 μM ABA for 2 h (+), and (−) represents leaves without ABA treatment. (**F**) Stomatal apertures of the WT and *gai-1* mutant corresponding to (**E**). Values are mean ratios of width to length ± SDs of three independent experiments. Letters indicate significant differences from the WT (0 ABA treatment) according to the Student's Newman–Kuels test (\*\*\* *p* < 0.05).

**Figure 2.** DELLA (GAI/RGA/RGL1/RGL2/RGL3) proteins interacted with ABF2 (abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors). (**A**) DELLA proteins interacted with ABF2 in yeas<sup>t</sup> two-hybrid assay. The yeas<sup>t</sup> cells expressing the indicated constructs were spotted as a series of three dilutions. The yeas<sup>t</sup> cells expressing the constructs of ABF2-pGBKT7-DEST (BD)/GAI-pGADT7-DEST (AD), ABF2-BD/RGA (REPRESSOR OR GAI3)-AD, ABF2-BD/RGL1 (RGA-LIKE1)-AD, ABF2-BD/RGL2-AD, and ABF2-BD/RGL3-AD grew better on the SD medium than that of yeas<sup>t</sup> growth cells expressing the control's constructs. (**B**) BiFC (bimolecular fluorescent complimentary) assay between DELLA proteins and ABF2. *Nicotiana benthamiana* leaves were co-transformed with the constructs containing the indicated YFP (yellow fluorescent protein) N-terminal (YFPN) and YFP C-terminal (YFPC) fusions, and YFP was imaged 48 h after transformation. Bars = 50 μm. (**C**) Interaction assay between GAI and RGA with ABF2 fragments. P: 1–60 amino acid; Q: 61–116 amino acid; R: 117–199 amino acid; bZIP (basic region/leucine zipper): 200–417 amino acid. The yeas<sup>t</sup> cells expressing the indicated constructs were spotted as a series of three dilution. The yeas<sup>t</sup> cell expressing the constructs of ABF2-BD/GAI-AD, ABF2-BD/RGA-AD, ABF2P-BD/GAI-AD, ABF2P-BD/RGA-AD, ABF2bZIP-BD/GAI-AD, ABF2bZIP-BD/RGA-AD, grew more effectively on the SD medium than that of yeas<sup>t</sup> cells expressing ABF2R-BD/RGA-AD, ABF2R-BD/GAI-AD, ABF2Q-BD/ RGA-AD, and ABF2Q-BD/GAI-AD.

There are three conserved motifs in the N-terminal of ABF2: P, Q, and R. The P motif is responsible for transactivation activity and activates downstream gene expression [14]. To test which motif is responsible for interacting with GAI, we tested the interaction between GAI and the P, Q, R, and bZIP (basic region/leucine zipper) motifs of ABF2. Our yeas<sup>t</sup> two-hybrid assay showed that there was a strong interaction between the GAI and the P and bZIP motifs, whereas the interaction between GAI and the Q and R motifs was much weaker (Figure 2C). We also tested the interaction between RGA and the P, Q, R, and bZIP motifs of ABF2. RGA also showed a strong interaction with the P and bZIP motifs but a weak interaction with the Q and R motifs (Figure 2C).
