Loss of ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation

TL;DR

This study finds that the ASFT protein negatively affects drought tolerance in Arabidopsis by inhibiting OST1 kinase activity, which controls stomatal closure.

Contribution

The study reveals a new regulatory role for ASFT in ABA signaling through OST1 autophosphorylation modulation.

Findings

ASFT interacts with and inhibits OST1 autophosphorylation, reducing stomatal aperture.

ASFT loss increases drought tolerance in Arabidopsis, while overexpression reduces it.

ASFT functions upstream of OST1 in ABA signaling, as confirmed by genetic evidence.

Abstract

Drought stress severely constrains plant growth and productivity. To mitigate water loss, plants primarily regulate stomatal aperture through the Abscisic acid (ABA) signaling pathway, where the Sucrose Nonfermenting 1-Related Protein Kinase 2 (SnRK2) family kinase Open Stomata 1 (OST1) acts as a central positive regulator. However, the upstream regulators that fine-tune OST1 activity remain incompletely characterized. Aliphatic Suberin Feruloyl Transferase (ASFT), a BAHD acyltransferase essential for suberin aromatic monomer biosynthesis, was previously uncharacterized regarding its function in leaves. Here, we report that ASFT negatively regulates drought tolerance in Arabidopsis thaliana by directly interacting with OST1 and inhibiting its autophosphorylation, thereby restricting stomatal aperture. Consistent with this, the asft mutant exhibited decreased water loss and enhanced survival under drought, whereas ASFT-overexpressing lines showed opposite phenotypes. BiFC, Co-IP and in vitro kinase assays confirmed that ASFT directly interacts with OST1 and suppresses its autophosphorylation, while dehydration-induced OST1 phosphorylation was elevated in the asft mutant. Genetic evidence confirmed that ASFT functions upstream of OST1. This study reveals a moonlighting role for this suberin biosynthetic enzyme in ABA signaling and provides a potential target for breeding drought-resistant crops.

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1. Introduction

Drought stress is one of the major abiotic stresses threatening global agricultural productivity and ecosystem stability [1,2]. It extensively disrupts plant physiological, biochemical, and molecular processes, thereby impairing plant growth and survival [3,4]. In response to water deficit, plants have evolved multi-layered adaptive strategies, including activating signal transduction pathways, remodeling metabolic networks, modifying root system architecture to enhance water uptake and restructuring leaf epidermal structures to minimize transpirational water loss [5,6,7,8,9,10]. Among these adaptive mechanisms, the regulation of stomatal movement is particularly critical. As the primary portals for gas exchange and transpirational water loss on leaf surfaces, stomata tightly regulate water evaporation by adjusting their aperture, which directly determines plant drought tolerance [11]. The Abscisic acid (ABA)-mediated signaling pathway is the core module governing stomatal closure under drought conditions [12,13]. Upon ABA perception, PYR/PYL receptors inhibit clade A protein phosphatase 2Cs (PP2Cs), which in turn release the inhibition of Sucrose Nonfermenting 1-Related Protein Kinase 2 (SnRK2) family kinases. Open Stomata 1 (OST1), a key SnRK2 family kinase, acts as a central regulator in this pathway. The released OST1 kinase is then activated via autophosphorylation and phosphorylates downstream targets such as anion channels, K^+^ influx channels, transcription factors and NADPH oxidases, ultimately facilitating stomatal closure [14,15,16,17].

Beyond stomatal regulation, plants also rely on hydrophobic barrier polymers to limit non-stomatal water loss and resist environmental stresses [18,19]. Suberin, a complex polyester composed of aliphatic and aromatic monomers, forms a critical barrier in roots, stems and other aerial tissues, regulating water transport and preventing pathogen invasion [18,20]. Aliphatic Suberin Feruloyl Transferase (ASFT), a key member of the BAHD acyltransferase family, catalyzes the transesterification between feruloyl-CoA and fatty alcohols to produce alkyl ferulate, a crucial precursor for suberin biosynthesis. In Arabidopsis thaliana ASFT loss-of-function mutants, the content of ferulic acid monomers in root and seed coat suberin is significantly reduced, leading to increased permeability [21,22]. Subsequent studies on its homologous genes in potato, poplar and kiwifruit have been further confirmed [23,24,25]. Interestingly, although suberin content in Arabidopsis leaves is significantly lower than that in roots, the expression level of ASFT in leaves is basically comparable to that in roots [21]. This suggests that ASFT may exert an unknown biological function independent of suberin biosynthesis in aerial tissues.

Here, we investigated the potential non-metabolic function of ASFT in Arabidopsis. We found that ASFT negatively regulates drought tolerance by modulating stomatal aperture. Through a combination of genetic, biochemical and physiological approaches, we obtained evidence that ASFT directly interacts with the OST1 kinase and inhibits its autophosphorylation activity. Genetic evidence further confirmed that ASFT functions upstream of OST1. Our study thus proposes a novel ‘moonlighting’ role for ASFT, bridging the synthesis of a hydrophobic barrier with the regulation of stomatal movement by signal transduction. This work not only expands the molecular network upstream of OST1 but also provides a new perspective on the integration of structural and signaling strategies in plant drought adaptation.

2. Results

2.1. Overexpression of ASFT Enhances Water Loss and Stomatal Aperture in Arabidopsis

Although Aliphatic Suberin Feruloyl Transferase (ASFT) is well established as a key enzyme for root suberin biosynthesis, its high transcript levels in leaves prompted us to investigate its role in aerial tissue (Figure S1). Our initial observation revealed that asft mutants wilted more slowly than wild type (WT) under drought conditions (Figure S2). To validate this, we performed a controlled drought assay on 21-day-old soil-grown plants of WT, the asft mutant and two independent ASFT-overexpressing lines (ASFT OE#5 and ASFT OE#6) (Figure S3 and Figure 1a). After approximately 16 days of water deprivation—until over 50% of plants showed evident wilting—watering was resumed for 3 days to assess recovery. Consistent with our initial finding, the asft mutant demonstrated a significantly higher survival rate compared to WT, whereas the overexpression lines were markedly more susceptible (Figure 1a,b).

To further corroborate the role of ASFT in regulating plant water loss, rosette leaves were collected from 24-day-old seedlings of each genotype for in vitro dehydration assays. Leaves were dehydrated under laboratory conditions for 6 h, with continuous monitoring of the water loss rate. Consistent with the drought stress phenotypes, the in vitro water loss rate of the asft mutant was significantly lower than that of WT, whereas ASFT-OE lines exhibited a substantially higher water loss rate (Figure 1c). Additionally, leaf surface temperature—an indirect indicator of transpirational water loss [26]—exhibited a consistent trend: leaf temperature of the asft mutant was significantly higher than that of WT, while ASFT-OE lines showed significantly lower leaf temperatures (Figure 1d,e). Collectively, these findings indicate that ASFT expression level is tightly associated with plant water loss efficiency. To determine the cause of altered transpiration, we examined stomatal aperture. Stomata were significantly narrower in the asft mutant and wider in ASFT-OE lines compared to WT (Figure 1f), indicating that ASFT expression level negatively regulates stomatal opening. This change in stomatal aperture likely explains the observed differences in leaf water loss and drought tolerance.

Suberin, a critical compound involved in water transport [18,20], requires ASFT as a key enzyme for its biosynthesis. Mutations in ASFT might induce changes in suberin composition or content, thereby potentially impacting water transport. Nevertheless, hydraulic conductivity measurements revealed no significant difference between the asft mutant and WT (Figure S4), further supporting that the drought-tolerant phenotype of the asft mutant is not attributed to altered water transport from underground to aerial tissues.

2.2. ASFT Specifically Interacts with OST1 and Regulates Its Autophosphorylation

To elucidate how ASFT regulates stomatal aperture, we performed a yeast two-hybrid screen using ASFT as bait against an Arabidopsis cDNA library. Screening yielded five candidate interactors, among which we focused on the core Abscisic acid (ABA) signaling kinase Open Stomata 1 (OST1) due to its central role in stomatal control (Figure 2a and Figure S5). To validate the interaction between ASFT and OST1, we further constructed the recombinant vectors pXY106-nYFP*-OST1* (N-terminal YFP fusion with OST1) and pXY104-cYFP*-ASFT* (C-terminal YFP fusion with ASFT). These were co-transformed into Agrobacterium tumefaciens, separately with the pXY106-nYFP empty vector control, and then used to infiltrate Nicotiana benthamiana leaves. After one day of cultivation, observation under a confocal laser scanning microscope revealed that a specific YFP fluorescence signal was detected in the group co-infiltrated with pXY106-nYFP*-OST1* and pXY104-cYFP*-ASFT*, while no fluorescence signal was observed in the empty vector control group, indicating that ASFT and OST1 interact in plant cells (Figure 2b). To further verify the direct interaction in vivo, we employed an Agrobacterium-mediated transient expression system to co-express Myc-tagged ASFT (ASFT-Myc) and His-tagged OST1 (OST1-His) in N. benthamiana. Total protein was extracted from tobacco leaves three days after infiltration, and co-immunoprecipitation (Co-IP) was performed using Anti-Myc agarose beads. Western blot analysis of the precipitated complexes using an Anti-His antibody showed a specific OST1-His band in the ASFT-Myc immunoprecipitate (Figure 2c), indicating a direct interaction between ASFT and OST1 in planta. Furthermore, confocal microscopy revealed clear co-localization of ASFT-GFP and OST1-mRuby2 at the subcellular level, supporting the spatial feasibility of their interaction (Figure S6).

The ASFT-OST1 interaction appeared specific, as ASFT did not interact with other SnRK2 family members in yeast two-hybrid assays (Figure S7). To investigate the molecular basis of the ASFT-OST1 interaction, we first utilized GRAMM docking. This analysis predicted the formation of hydrogen bonds involving three residues from OST1 and five from ASFT (Figure 2d). Crucially, one of the OST1 residues involved was Ser175 (S175), a site essential for its phosphorylation [27,28]. This structural insight suggested that ASFT might influence OST1 phosphorylation. To test this hypothesis, we performed an in vitro phosphorylation assay using prokaryotically expressed ASFT-GST and OST1-His. The results demonstrate that ASFT specifically modulates the autophosphorylation state of OST1 (Figure 2e). In contrast, FACT (Fatty Alcohol:caffeoyl-CoA Caffeoyl Transferase), a BAHD acyltransferase involved in Arabidopsis wax biosynthesis but not in ABA signaling, did not exhibit a similar effect. In summary, these findings indicate that the specific and direct interaction between ASFT and OST1, a key ABA signaling kinase in Arabidopsis, has functional consequences for OST1 autophosphorylation.

2.3. OST1 Mutation in the asft Mutants Enhances Water Loss and Reduces Drought Tolerance

The OST1 protein undergoes autophosphorylation or phosphorylates downstream transcription factors and proteins in response to various stimuli [29,30,31]. It is also a key protein that regulates stomatal movement under stress conditions [32]. To investigate whether ASFT regulates stomatal aperture and drought tolerance through OST1, we generated an asft ost1 double mutant through genetic hybridization (Figure S8). Using the WT, asft and ost1 single mutants as controls, we systematically analyzed the stomatal phenotypes, water loss characteristics, and drought stress responses of each genotype. Stomatal-related traits were first quantified in 25-day-old soil-grown plants of each genotype. Stomatal aperture measurements revealed that the asft had a significantly lower stomatal aperture than WT. In contrast, both the ost1 single mutant and the asft ost1 double mutant exhibited significantly higher stomatal apertures than WT, with no statistical difference between them (Figure 3a,b). No significant differences in stomatal density were observed among any of the genotypes compared to WT (Figure 3c). To investigate the impact of these stomatal aperture differences on plant water loss, the above-ground parts of 25-day-old seedlings from each genotype were excised for detached water loss rate assays, with fresh weight recorded every 5 min. The results showed that the water loss rate of the asft ost1 double mutant was similar to that of the ost1 single mutant, both being significantly higher than those of WT and the asft (Figure 3d). Leaf surface temperature was measured using a FLIR-530 thermal camera (FLIR Optoelectronic Technology, Santa Barbara, CA, USA) infrared camera, and the results were consistent with the water loss analysis: the leaf temperatures of the ost1 mutant and the asft ost1 double mutant were significantly lower than that of WT, whereas the asft mutant had a significantly higher leaf temperature than WT (Figure 3e,f). This confirms the regulatory role of stomatal aperture in controlling plant water loss. To determine the contribution of the ASFT-OST1 pathway to drought tolerance, we subjected different genotypes to a severe drought assay. Twenty-one-day-old seedlings, grown initially under well-watered conditions, were deprived of water for approximately 16 days, until over 50% exhibited clear wilting of leaves and stem bases. After rewatering for three days, survival rates were recorded. The asft mutant exhibited a significantly higher survival rate than the WT. In contrast, the ost1 single mutant and the asft ost1 double mutant both showed significantly lower survival rates than WT. Critically, there was no significant difference in survival between the ost1 and asft ost1 genotypes (Figure 3g,h).

In summary, our genetic epistasis analysis indicates that OST1 function is necessary for the manifestation of the drought-resistant phenotype in the asft mutant. The asft ost1 double mutant, which lacks functional OST1, fails to retain the water-saving traits of the asft parent and instead displays a drought-sensitive phenotype similar to that of the ost1 single mutant. These findings support a model in which ASFT acts upstream in the pathway, influencing stomatal aperture largely through the OST1 kinase, which in turn affects whole-plant water economy and drought resilience in Arabidopsis.

2.4. ASFT Regulates Stomatal Aperture Through Modulating OST1 Autophosphorylation

To determine whether ASFT regulates stomatal movements through OST1, we performed stomatal aperture assays using epidermal peels from 21- to 23-day-old plants of the following genotypes: WT, asft, ost1 and asft ost1 mutants. Stomata were pre-opened under high-intensity light for 2–2.5 h and then treated with ABA (0, 20, or 50 μM) for 2 h before measurement. Under control conditions (0 μM ABA), stomata of the asft mutant were significantly narrower than those of all other genotypes. ABA treatment induced dose-dependent stomatal closure in all lines. Notably, at 50 μM ABA, the stomatal aperture of the asft ost1 double mutant was significantly larger than that of WT and indistinguishable from the ost1 single mutant (Figure 4a). Additionally, asft stomata remained consistently smaller than WT stomata across all ABA concentrations, indicating both an altered baseline aperture and enhanced responsiveness to ABA (Figure 4a).

Given the central role of OST1 in ABA-mediated stomatal closure [14,16], we sought to investigate whether the stomatal phenotype of asft was associated with altered OST1 activity. We therefore examined the in vivo autophosphorylation status of OST1 (at the key S175 residue) in response to dehydration stress using a phospho-specific antibody. Consistent with its established function, dehydration induced OST1 phosphorylation in WT plants (Figure 4b). In contrast, the asft mutant exhibited increased OST1 phosphorylation compared to WT following dehydration treatment (Figure 4b). To further validate this finding, we employed a heterologous tobacco overexpression system. When 35S::OST1-GFP was expressed alone, dehydration treatment induced a strong OST1 phosphorylation signal. By contrast, co-expression of 35S::ASFT-MYC significantly attenuated this phosphorylation signal (Figure S9). Together, these results suggest that loss of ASFT function is associated with elevated OST1 kinase activity in vivo. The observed enhancement of OST1 activity likely underlies the constitutively enhanced stomatal closure response of the asft mutant.

3. Discussion

ASFT is well established for its role in synthesizing aromatic monomers for suberin biosynthesis, primarily in roots and seeds [21,22]. Its expression is tissue-specific and stress-inducible [33,34,35,36,37,38]. However, our results further corroborate a previously noted yet functionally unexplained phenomenon: despite the low suberin content in Arabidopsis leaves, ASFT transcript levels remain high and comparable to those in roots (Figure S1) [21]. This decoupling between gene expression and metabolic output suggests that ASFT may perform a non-canonical function in aerial tissues.

Phenotypic screening revealed that the asft mutant exhibited significantly enhanced survival under drought stress (Figure S2). This improved performance could stem from either enhanced water uptake or reduced water loss. To distinguish between these possibilities, we measured root hydraulic conductivity. The asft mutant was expected to have reduced root suberin. However, its hydraulic conductivity was unchanged, indicating that altered root water transport is not the driver of its drought-tolerant phenotype (Figure S4). This finding prompted us to shift our focus to water relations in aerial tissues. Physiological analyses indicated that detached leaves from asft mutants lost water more slowly and maintained higher leaf surface temperatures, suggesting reduced transpiration. Importantly, these changes were associated with a significant reduction in stomatal aperture, whereas ASFT-overexpressing lines displayed larger stomatal apertures (Figure 1). This observation aligns with recent findings in tomato, where asft mutants also showed lower transpiration rates and stomatal conductance under water-limiting conditions [39]. Collectively, these results support the notion that the enhanced drought tolerance of the asft mutant is primarily attributed to a stomatal mechanism that limits transpirational water loss.

To explore the molecular link between ASFT and stomatal regulation, we examined OST1, the core kinase of the ABA signaling pathway, as a candidate interacting protein of ASFT (Figure S5). Yeast two-hybrid screening, bimolecular fluorescence complementation, and co-immunoprecipitation assays indicated a specific physical interaction between ASFT and OST1 in plant cells. Structural modeling further suggested that ASFT may bind near the S175 residue of OST1, a site known to be critical for OST1 autophosphorylation (Figure 2). Consistent with this inference, in vitro kinase assays showed that ASFT could inhibit OST1 autophosphorylation (Figure 2e). Moreover, under osmotic stress, OST1 phosphorylation levels were elevated in the asft mutant compared to the wild type, supporting a potential role for ASFT as an endogenous inhibitor of OST1 kinase activity in vivo (Figure 4b). Together, these findings reveal a potential “moonlighting” function for ASFT as a direct regulator of OST1. To further investigate the functional relationship between ASFT and OST1, we generated an asft ost1 double mutant and performed genetic epistasis analysis. The results showed that the double mutant exhibited phenotypes highly similar to those of the ost1 single mutant: stomatal apertures were significantly larger and statistically indistinguishable from ost1; water loss rates and leaf temperature profiles followed trends consistent with ost1; and drought tolerance was similarly compromised (Figure 3). Furthermore, the enhanced ABA sensitivity of stomatal closure observed in the asft single mutant was absent in the asft ost1 double mutant background (Figure 4a). These results collectively suggest that the ost1 mutation is epistatic to asft, supporting a model in which ASFT likely functions upstream of OST1 to negatively regulate stomatal closure and plant drought tolerance.

Our study indicates that ASFT, a metabolic enzyme involved in suberin biosynthesis, moonlights as a novel negative regulator of drought tolerance in Arabidopsis leaves by directly interacting with and inhibiting the OST1 kinase (Figure 4c). This finding places ASFT among other multifunctional Arabidopsis proteins, such as metabolic enzymes known to coordinate both catalytic and non-catalytic roles [40,41,42]. We propose that this dual functionality may enable the plant to synchronize the formation of water-retaining suberin barriers with the regulation of stomatal transpiration, thereby providing a novel mechanism for the systemic integration of drought responses.

4. Materials and Methods

4.1. Plant Materials, Growth Conditions and Drought Resistance

The asft (SALK_048898) and ost1–3 (SALK_008068) mutants in the Col-0 background were used in this study. Seeds of the asft mutant were generously provided by Drs. Isabel Molina and Mike Pollard (Michigan State University, East Lansing, MI, USA) [22]. The asft ost1 double mutant was generated by genetic crossing. The transgenic lines pASFT::GUS, 35S::OST1-GFP and 35S::OST1-GFP in the asft mutant background were obtained via Agrobacterium-mediated floral dip transformation. Primer sequences are listed in Supplemental Table S1. The Col-0 ecotype served as the wild-type (WT) control. All seeds used in this study were incubated at 4 °C in the dark for 3 d. After cold stratification, the seeds were sown on agar germination medium (GM) and grown in a growth chamber (16 h light/8 h dark cycle, 80 µmol m^−2^ s^−1^ photon flux density, 22 °C). To compare the differences in drought resistance among plants of different genotypes, one-week-old agar seedlings were transferred to pots containing the same weight of soil and water. Trays were re-watered when more than 15 plants of one genotype in one tray exhibited wilting leaves and stem bases. Three days after re-watering, survival rates in all trays were assessed [43].

4.2. Analysis of Stomatal Aperture and Stomatal Closure in Response to ABA

Stomatal aperture was assayed as previously described [44]. Epidermal peels from the fourth leaves of 24-day-old WT plants were incubated in MES-KCl buffer (pH 6.15) under high light for 2 h, then treated with varying ABA concentrations for another 2 h. Three independent experiments (four to six plants each) were performed. Stomatal images were captured by light microscopy and analyzed using Fiji: Version 2.9.0 (https://fiji.sc).

4.3. Leaf Surface Temperature and Leaf Water Loss Assay

Thermal imaging was performed as previously described [45]. Thermal images of 30-day-old WT, asft, ost1, and asft ost1 plants were captured inside a growth chamber using a FLIR-530 thermal camera (FLIR Optoelectronic Technology, Santa Barbara, CA, USA). The thermal data were processed and quantified using FLIR Tools (v6.4). For water loss assays, rosette leaves from 30-day-old plants were excised, weighed immediately, and then weighed periodically at room temperature.

4.4. Yeast Two-Hybrid Screening and Yeast Two-Hybrid Assay

For large-scale yeast two-hybrid screening, an Arabidopsis cDNA library (Clontech Laboratories, Inc., Mountain View, CA, USA) in Y187 was mated with Y2H Gold harboring pGBKT7-ASFT at 30 °C for 24 h. Diploids were selected on SD/-Leu/-Trp/-Ade/-His medium supplemented with X-α-Gal and Aureobasidin A. For pairwise interaction assays, pGBKT7-OST1 and pGBKT7 constructs for the other nine SnRK2 family members (SnRK2.1, SnRK2.2, SnRK2.3, SnRK2.4, SnRK2.5, SnRK2.7, SnRK2.8, SnRK2.9, and SnRK2.10) were each co-transformed with pGADT7-ASFT into Y2H Gold using the LiAc-PEG method. The following controls were included: pGADT7-ASFT co-transformed with empty pGBKT7 (to exclude ASFT autoactivation), and empty pGADT7 co-transformed with pGBKT7-OST1 (to exclude OST1 autoactivation). Transformants were first selected on SD/-Leu/-Trp medium, and interactions were subsequently tested on SD/-Leu/-Trp/-Ade/-His medium containing 15 mM 3-AT. All primers used are listed in Supplemental Table S1.

4.5. BiFC Assay and Subcellular Localizations of ASFT and OST1

For the BiFC analysis as described previously [46], ASFT and OST1 CDS were cloned into pXY106-YFP^N^/pXY104-YFP^C^, generating ASFT-YFP^N^ and YFP^C^-OST1. For localization, they were cloned into pCAMBIA1300-35S-GFP/mRuby2. Constructs were transformed into A. tumefaciens GV3101 and infiltrated into N. benthamiana (OD600 = 1.0). Fluorescence was imaged by confocal microscopy (Nikon A1plus; Nikon, Tokyo, Japan): GFP/YFP (488/490–550 nm) and mRuby2 (561/570–620 nm). Primers are listed in Supplemental Table S1.

4.6. Co-IP Assays

ASFT and OST1 CDS were PCR-amplified with MYC/Flag-tagged primers and cloned into pCAMBIA1300 (35S promoter, Sal I/Pst I). Constructs were transformed into A. tumefaciens GV3101 and infiltrated into N. benthamiana. Three days post-infiltration, total proteins were extracted, immunoprecipitated with anti-MYC beads, and immunoblotted with anti-His antibody. Primers are listed in Supplemental Table S1.

4.7. Root Hydraulic Conductivity (Lpr-h) Measurements

Root hydraulic conductivity (Lpr-h) was measured as described in [47] using 21–23-day-old hydroponic plants. Decapitated root systems were sealed in a pressure chamber with hydroponic solution (pH 6.5). Roots were pressurized at 350 kPa for 10 min, then at 320, 240, and 160 kPa for 2 min each. Sap flow (Jv) was recorded with flow meters (Bronkhorst, France) and LabVIEW software (Version 1.03, https://gramm.compbio.ku.edu). (Version 2020, National Instruments, Austin, TX, USA). Root dry weight (D_Wr_) was measured after 80 °C drying. Lpr-h (mL g^−1^ h^−1^ MPa^−1^) = Jv/(D_Wr_ × P). Two independent experiments were conducted, each with 15 biological replicates.

4.8. Protein Purification and In Vitro Kinase Assay

Recombinant proteins were expressed and purified following established protocols [48]. The coding sequences of ASFT, FACT and OST1 were individually cloned into the pGEX-2TK or pET28a vectors. The resulting constructs were transformed into Escherichia coli BL21 (DE3) for protein expression. For the kinase assay according to previous reports [49], 1 µg of OST1 was incubated alone or with 2 µg of ASFT or FACT-GST in kinase buffer containing 100 µM ATP and 1 µCi of [γ-^32^P] ATP. After 30 min at 25 °C, reactions were terminated and proteins were separated by SDS-PAGE. The gel was stained with CBB to confirm uniform protein loading (lower panel). The phosphorylation signals were captured from the dried gel using a phosphor imager (Typhoon FLA 7000, GE Healthcare, Chicago, IL, USA). Primer sequences are listed in Supplemental Table S1.

4.9. Protein Docking

Protein docking was performed essentially as described in [50]. The structure of OST1 was obtained from the PDB (ID: 3UJG). The ASFT structure was modeled based on its UniProt sequence (Q94CD1) using the SWISS-MODEL server (SIB Swiss Institute of Bioinformatics, Basel, Switzerland; https://swissmodel.expasy.org). Protein-protein docking was performed with GRAMM (Version 1.03, https://gramm.compbio.ku.edu) to predict the interaction complex. The top-ranking model, selected based on docking score and cluster analysis, was used to identify key interfacial residues. All structural visualizations and analyses were conducted in PyMOL (Version 3.1.6.1, Schrödinger, LLC, New York, NY, USA) [51,52].

4.10. Detection of OST1 Phosphorylation by Immunoblotting

Protein was extracted from dehydrated (0, 1 and 2 h) or ABA-treated (50 μM, 30 min) 4-week-old 35S::OST1-GFP and 35S::OST1-GFP asft plants. Tissue was homogenized in buffer (100 mM HEPES pH7.5, 5 mM EDTA/EGTA, 10 mM Na_3_VO_4_/NaF, 50 mM β-glycerophosphate, 10 mM DTT, 10 μg/mL leupeptin/antipain/aprotinin, 1 mM PMSF, 5% glycerol), centrifuged (12,000 rpm, 4 °C, 30 min), and supernatants collected [53,54]. Protein concentration was measured by Bradford assay. For immunoblotting, 80 μg protein/lane was separated, transferred, and probed with anti-phospho-S175-SnRK2.6 (ABclonal, 1:1000, Woburn, MA, USA) and HRP-conjugated secondary antibody (1:10,000) [55]. Signals were detected using Lumi-Light substrate (Roche, Basel, Switzerland).

4.11. Statistical Analysis

All experiments were performed with at least three independent biological replicates unless otherwise specified. The exact number of replicates for each experiment, including biological and technical replicates, is indicated in the corresponding figure legends. Data are presented as mean ± standard deviation (SD). Statistical significance between two groups was determined by two-tailed Student’s t-test. Comparisons among multiple groups were analyzed by one-way ANOVA followed by appropriate post hoc tests. A p-value < 0.05 was considered statistically significant (* p < 0.05; ** p < 0.01). All statistical analyses were performed using SPSS (Version 26.0, IBM Corp., Armonk, NY, USA).

5. Conclusions

In this study, we identified a novel moonlighting function of ASFT in regulating drought tolerance in Arabidopsis. Our key findings demonstrate that ASFT negatively regulates drought tolerance by modulating stomatal aperture, directly interacts with OST1, and inhibits its autophosphorylation at Ser175. Genetic epistasis analysis placed ASFT upstream of OST1, as the asft ost1 double mutant exhibited phenotypes similar to the ost1 single mutant in stomatal aperture, water loss, and drought tolerance. These findings reveal a previously unrecognized regulatory mechanism in which a suberin biosynthetic enzyme moonlights as a direct inhibitor of OST1, linking metabolic processes with ABA signaling to fine-tune stomatal responses under drought stress. This work provides a potential target for breeding drought-resistant crops through genetic manipulation of ASFT expression.