GAI Functions in the Plant Response to Dehydration Stress in Arabidopsis thaliana

Abstract

DELLA (GAI/RGA/RGL1/RGL2/RGL3) proteins are key negative regulators in GA (gibberellin) signaling and are involved in regulating plant growth as a response to environmental stresses. It has been shown that the DELLA protein PROCERA (PRO) in tomato promotes drought tolerance, but its molecular mechanism remains unknown. Here, we showed that the gai-1 (gibberellin insensitive 1) mutant (generated from the gai-1 (Ler) allele (with a 17 amino acid deletion within the DELLA domain of GAI) by backcrossing gai-1 (Ler) with Col-0 three times), the gain-of-function mutant of GAI (GA INSENSITIVE) in Arabidopsis, increases drought tolerance. The stomatal density of the gai-1 mutant was increased but its stomatal aperture was decreased under abscisic acid (ABA) treatment conditions, suggesting that the drought tolerance of the gai-1 mutant is a complex trait. We further tested the interactions between DELLA proteins and ABF2 (abscisic acid (ABA)-responsive element (ABRE)-binding transcription factors) and found that there was a strong interaction between DELLA proteins and ABF2. Our results provide new insight into DELLA proteins and their role in drought stress tolerance.

Keywords: ABF2; DELLA; GA; drought; protein–protein interaction.

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 yeast two-hybrid assay. The yeast cells expressing the indicated constructs were spotted as a series of three dilutions. The yeast 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 yeast 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 (YFP^N) and YFP C-terminal (YFP^C) 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 yeast cells expressing the indicated constructs were spotted as a series of three dilution. The yeast 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 yeast cells expressing ABF2R-BD/RGA-AD, ABF2R-BD/GAI-AD, ABF2Q-BD/ RGA-AD, and ABF2Q-BD/GAI-AD.