Combining chemical and genetic approaches to increase drought resistance in plants

Abstract

Drought stress is a major threat to crop production, but effective methods to mitigate the adverse effects of drought are not available. Here, we report that adding fluorine atoms in the benzyl ring of the abscisic acid (ABA) receptor agonist AM1 optimizes its binding to ABA receptors by increasing the number of hydrogen bonds between the compound and the surrounding amino acid residues in the receptor ligand-binding pocket. The new chemicals, known as AMFs, have long-lasting effects in promoting stomatal closure and inducing the expression of stress-responsive genes. Application of AMFs or transgenic overexpression of the receptor PYL2 in Arabidopsis and soybean plants confers increased drought resistance. The greatest increase in drought resistance is achieved when AMFs are applied to the PYL2-overexpression transgenic plants. Our results demonstrate that the combining of potent chemicals with transgenic overexpression of an ABA receptor is very effective in helping plants combat drought stress.

Fig. 1

Two-dimensional chemical structure of AMFs and AMC1β. As halide derivatives of AM1, AMFs and AMC1β share a similar AM1 structural motif with one or more halogen atoms (fluorine or chloride) added in the 4-menthylphenyl tail. The three hydrogen bond-forming groups of ABA, carbonyl, hydroxyl, and carboxyl, and their counterparts in AM1 and AMF4, are highlighted in red, blue, and green, respectively

Fig. 2

AMFs are potent PYL receptor agonists. a–d Agonist dose–response curves for AMFs and AMC1β. Dose-dependent interactions between HAB1 and PYR1 (a), PYL1 (b), PYL2 (c), or PYL7 (d) in the presence of all five AMFs, AMC1β, AM1 and (+)-ABA are determined in AlphaScreen assays. EC₅₀ values for the interactions are listed below the curves (n = 3, error bars = SD). e Inhibition of HAB1 activity induced by AMFs and mediated by the 11 PYLs in phosphatase activity assays. The working concentration is 1 μM for AMF1β, AMF2α, and (+)-ABA. Values are means ± SD (n = 3). f Dose-dependent inhibition of HAB1 activity resulting from the binding of AMFs to the PYL2–HAB1 complex. The working concentrations are 1 and 0.1 μM for all five AMFs and (+)-ABA. Values are means ± SD (n = 3)

Fig. 3

Structural comparison of AM1 and AMF4 within the PYL2–HAB1 complex. a, b Two-dimensional structural schematics of interactions between AM1 (a) or AMF4 (b) and residues in the PYL2 ligand-binding pocket (A) or in HAB1 (B). The schematics show an increase in the number of hydrogen bonds (dashed lines) between the fluorine atoms (green-filled circles) of AMF4 and the nitrogen atoms (blue-filled circles) of the Asn173 residue in the PYL2-binding pocket. Red-, yellow-, blue-, and turquoise-filled circles represent oxygen atoms, sulfur atoms, nitrogen atoms and water molecules, respectively. The number represents the distance (Å) between two atoms/molecules. The PYL2–AM1–HAB1 schematic model is from previously published paper. c Overlay of three-dimensional structural schematics of AM1 (purple) with AMF4 (green) in the PYL2-binding pocket, with hydrogen bonds (dashed lines) and water molecules (filled circles) for AM1 and AMF4 in purple and green colors, respectively. d A 2F _o–2F _c electron density map of bound AMF4 and its surrounding residues contoured at 1.0σ. N173 of PYL2 is highlighted in yellow and the white dotted line represents the hydrogen bond between the fluorine atom of AMF4 and N173 of PYL2

Fig. 4

AMFs regulate ABA-responsive genes in Arabidopsis plants. Col-0 plants are sprayed with 10 μM (+)-ABA (ABA or A), AMF1β (AMF1 or 1), or AMF4 (AMF4 or 4) are sampled 6, 24, and 72 h post treatment, and DMSO (DMSO or C) is used as the control. Gene expression profiles are based on RNA-seq results. a Overlap of ABA- and AMFs-induced DEGs. AMF1β or AMF4 induce highly correlated responses at the transcript level compared with ABA. Ontology analysis of ABA- or AMF4-specific DEGs shows that some ABA- or abiotic stress-related processes are still among the most enriched biological processes (based on their P values) at 3 days after AMF4 treatment but not at 3 days after ABA treatment. b Heat map of ABA- or abiotic stress-related DEGs in Col-0 plants. DEGs are clustered based on expression profiles. Each row contains an ABA- or abiotic stress-related DEG based on gene ontology analysis. Each column represents a chemical-time combination. The DEGs showing divergent time-course response to ABA or AMFs are highlighted. The color in each cell indicates its relative expression level compared to the mean expression level in all samples of the same row according to the color bar, with red and green representing upregulated and downregulated genes, respectively

Fig. 5

AMF sprays increase leaf temperature. a Leaf surface temperature is increased by treatment with AMFs. Four-week-old Arabidopsis aba2-1 mutants are sprayed with 5 μM indicated chemical solutions, and DMSO is used as the control. Plants are photographed with an IR camera before treatment (0 days) and at 2 or 4 days after treatment. b AMF sprays increase leaf temperature in a dose-dependent manner. Four-week-old Arabidopsis aba2-1 mutants are treated with corresponding chemicals at 5, 2, or 1 μM, and DMSO is used as the control. Plants are photographed with an IR camera before treatment (0 days) and at 2 or 4 days after treatment. c AMF4 increases soybean leaf temperature. Soybean plants are treated with 20 μM (+)-ABA or AMF4, and DMSO is used as the control. Plants are photographed with an IR camera before treatment (0 days) and at 1, 2, or 3 days after treatment

Fig. 6

AMFs increase the drought resistance of Arabidopsis and soybean plants. a AMF treatments increase drought resistance of Arabidopsis. Wild-type (Col-0) plants are grown under short-day conditions for 2 weeks before watering is stopped. The plants are subsequently treated with DMSO (control), or 10 μM (+)-ABA, AM1, or AMFs once per week for another 2 weeks before watering was resumed. The plants are photographed before watering was stopped (top panel) and 14 days after watering is stopped (bottom panel). b AMF4 treatments increase drought resistance of soybean plants. Williams 82 soybean plants at the triple trifoliate stage are subjected to drought (watering was stopped), and then are sprayed twice at 3-days intervals with DMSO (control) or 50 μM (+)-ABA, AMF2α, or AMF4 in 1 week period before watering is resumed. The plants are photographed before watering is stopped and 1 month after watering is resumed. c Survival rates of plants in b are calculated 1 month after watering is resumed; plants are considered to have survived if they have new leaves emerging. Values are the mean survival rates from 15 individual plants per treatment, and error bars indicate SD. d The growth of soybean is monitored by measuring the area of all leaves. Eight-day-old Williams 82 soybean plants are subjected to drought (watering was stopped), and their leaf areas are recorded by a camera. The plants are sprayed with DMSO or AMF4 (20 μM) at 3 and 8 days after watering is stopped. For the well-watered condition, the plants are watered every 3 days. Error bars indicate the standard deviation of four biological replicates

Fig. 7

The combining of chemical and genetic approaches provides the highest drought resistance. a Additive effects of AMF4 and stress-induced PYL2 expression in enhancing drought resistance in Arabidopsis. Wild-type plants (Col-0) and transgenic plants transformed by Arabidopsis RD29a promoter-driven PYL2 (pRD29a-PYL2) are grown under short-day conditions for 2 weeks before watering is stopped. The plants are subsequently treated with DMSO (control) or 10 μM (+)-ABA or AMF4 once per week for another 2 weeks. The plants are photographed before treatment (0d) and at 23, 26 or 28 days after first chemical treatment. b–e Additive effects of AMF4 and stress-induced PYL2 expression in enhancing drought resistance in soybean. b Wild-type (Ws82) and transgenic plants transformed by RD29a promoter-driven PYL2 (PYL2ox) at the triple trifoliate stage are subjected to drought (watering was stopped) and are treated every 3 days with DMSO (control), 50 μM (+)-ABA, or 50 μM AMF4 for 9 days before watering is resumed. The plants are photographed before watering is stopped (0 days) and 7, 9, and 10 days after watering is stopped. c Survival rates are calculated daily from the 7th day until the 9th day after watering is stopped, and plants are considered to have survived if their leaves had not wilted. Values are the mean survival rates from three replicates with 15 individual plants per treatment, and error bars indicate SD. d Soybean plants treated with 20 μM (+)-ABA, AMF4, or DMSO (the control) are photographed with an IR camera before they are treated (0 days) and 1 or 2 days after treatment. e The relative soil water content in b is determined as the mass of water in soil/mass of oven-dry soil*100%, and values are the mean soil water content after 7 days without watering, when DMSO-treated wild-type soybean plants begin to wilt. Each combination of treatment and plant line is represented by six replicate pots, and error bars indicate SD