Chemical activation of ABA signaling in grapevine through the iSB09 and AMF4 ABA receptor agonists enhances water use efficiency

Abstract

Grapevine (Vitis vinifera L.) is the world's third most valuable horticultural crop, and the current environmental scenario is massively shifting the grape cultivation landscape. The increase in heatwaves and drought episodes alter fruit ripening, compromise grape yield and vine survival, intensifying the pressure on using limited water resources. ABA is a key phytohormone that reduces canopy transpiration and helps plants to cope with water deficit. However, the exogenous application of ABA is impractical because it suffers fast catabolism, and UV-induced isomerization abolishes its bioactivity. Consequently, there is an emerging field for developing molecules that act as ABA receptor agonists and modulate ABA signaling but have a longer half-life. We have explored the foliar application of the iSB09 and AMF4 agonists in the two grapevine cultivars cv. 'Bobal' and 'Tempranillo' to induce an ABA-like response to facilitate plant adaptation to drought. The results indicate that iSB09 and AMF4 act through the VviPYL1-like, VviPYL4-like, and VviPYL8-like ABA receptors to trigger stomatal closure, reduce plant transpiration, and increase water use efficiency. Structural and bioinformatic analysis of VviPYL1 in complex with ABA or these agonists revealed key structural determinants for efficient ligand binding, providing a mechanistic framework to understand receptor activation by the ligands. Physiological analyses further demonstrated that iSB09 has a more sustained effect on reducing transpiration than ABA, and agonist spraying of grapevine leaves protected PSII during drought stress. These findings offer innovative approaches to strengthen the vine's response to water stress and reduce plant consumptive water use under limited soil water conditions.

FIGURE 1

Phylogenetic relationships among PYL family members from Vitis vinifera and Arabidopsis thaliana. A maximum‐likelihood tree was built from full protein sequences (MEGAx), using an LG model and 1000 bootstraps. Clades represent subfamilies I (PYL8‐like), II (PYL4‐like) and III (PYL1‐like), respectively. Protein sequences were translated from VCOST.v3 liftoff gene models in the T2T genome assembly, found in the GRAPEDIA portal. Bootstrap values are indicated in each tree node.

FIGURE 2

Leaf and root expression of grape PYL genes across public transcriptomic data. RNA‐Seq raw data (Illumina) corresponding to (A) leaf (3772 runs) and (B) root (367 runs) tissues were downloaded from the SRA‐NCBI and reanalyzed. Violin plots show the overall frequency distribution of data points, while the inner boxplots mark interquartile ranges, mean (dotted line), and median (solid line) values.

FIGURE 3

IR thermography and LI‐600 measurements show that iSB09 treatment induces stomatal closure in well‐watered Bobal grapevine plants. (A) iSB09 treatment is more effective than exogenous ABA treatment. IR thermography reveals a higher increase in leaf temperature in 50 μM iSB09‐ than in 100 μM ABA‐treated plants at 24 h posttreatment. (B) Imaging quantification was conducted using FLIR Thermal Studio; bars show the mean ± SD of leaf temperature, and points represent individual data (n = 2 replicates, 12 plants per treatment). (C, D) The effect of iSB09 on transpiration persists for at least one week after foliar spraying. Transpiration measurements at (C) 96 h and (D) one week after mock‐ or foliar spraying treatment with 30 μM ABA or 30 μM iSB09 solution. Bold lines show the mean ± SD, representing individual values as points (n = 2 replicates, three plants per treatment). Different letters indicate a significant difference. (E) Photograph of plants used in (C) and (D).

FIGURE 4

Physiological measurements in the Bobal grapevine cultivar under well‐watered conditions reveal increased water use efficiency after agonist application. (A, B) The effect of mock, 30 μM iSB09, or 30 μM ABA treatment on stomatal conductance (gs), photosynthesis (A_N), leaf transpiration (E), and water use efficiency (WUE; A_N/gs) was determined at 3 (A) and 96 (B) hours after the foliar application of the agonist under optimal irrigation conditions. N = 2 replicates, three plants per treatment. Different letters indicate a significant difference. (C) Photographs of the vines in the greenhouse and the LI‐6800 device used for the physiological measurements.

FIGURE 5

Physiological measurements in the Tempranillo grapevine cultivar under well‐watered conditions reveal increased water use efficiency after 20 μM AMF4 application. (A, B). The effect of mock or AMF4 treatment on stomatal conductance (gs), leaf transpiration (E), photosynthesis (A_N), and water use efficiency (WUE; A_N/gs) was determined at 3 (A) and 96 (B) hours after the foliar application of the agonist under optimal irrigation conditions. N = 2 replicates, three plants per treatment. Student's t‐test was used to compare agonist‐treated to their corresponding mock‐treated samples. The asterisks indicate p ≤ 0.01. (C) Photographs of the vines grown outdoors at ICVV and the LI‐6400 device used for the physiological measurements.

FIGURE 6

iSB09 treatment protects PSII after drought stress followed by rehydration. Measurements of (A) gs, (B) E, (C) ΦPSII and (D) ETR in mock and 30 μM iSB09‐treated Bobal grapevine plants under greenhouse conditions. Three plants per treatment were grown in greenhouse conditions for 2.5 months. At 0 h, plants were sprayed with mock (0.1% DMSO and 0.05% Tween20) or 30 μM iSB09. On the 6.5 days, plants were watered with 25% of field capacity (blue drop). Continuous measurements were taken with an LI‐600 porometer (LI‐COR). Points show the mean of four measures per plant (12 measurements per treatment) at each time, and asterisks reveal statistical significance between agonist‐ and mock‐treated plants in a two‐tailed t‐test (* p ≤ 0.05, ** p ≤ 0.01).

FIGURE 7

PP2C inhibition assays reveal that iSB09 and AMF4 activate VviPYL1‐like, VviPYL4‐like and VviPYL8‐like ABA receptors. (A, B) In vitro PP2C assays in which the indicated Vitis vinifera receptors were incubated with ∆N‐HAB1 PP2C in the absence (no drug, 0.1% DMSO) or presence of 10 μM ABA, iSB09, or AMF4. Bars show mean ± SD, and asterisks represent different levels of statistical significance (* p ≤ 0.05, *** p ≤ 0.001) in a one‐tailed t‐test analyzing each drug against no drug treatment. (C) The pattern of interactions of ABA (light blue, left), iSB09 (pink, middle), and AMF4 (wheat, right) into the VviPYL1 pocket (green). The experimental structure of VviPYL1:ABA fulfills all the necessary requirements for proper ABA binding within the pocket. The VviPYL1:ABA complex (PDB code 9GNM) shows structural similarity to the CsPYL1:ABA complex (PDB code 5MMX) reported by Moreno‐Alvero et al. (2017). (D) Superposition of the iSB09 (pink) and AMF4 (wheat) agonists into the ligand binding pocket of the VviPYL1 receptor (green).