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. 2026 Mar 12;49(8):4688–4701. doi: 10.1111/pce.70488

An Engineered Abscisic Acid Receptor Enhances ABA Signaling and Improves Abiotic Stress Tolerance in Rice

Jaeeun Song 1, In‐Sik Song 1, Rigyeong Kim 1, Sangkyu Park 1, Eun‐Hye Kim 1, Hyo Lee 1, Saet Buyl Lee 1, Hyunwoo Cho 2, Sangho Lee 2, Ok Ran Lee 3, Beom‐Gi Kim 1,✉
PMCID: PMC13353701  PMID: 41820799

ABSTRACT

Abscisic acid (ABA) is a key phytohormone that orchestrates adaptive responses in plants exposed to abiotic stress. The ABA signaling cascade is triggered by ABA‐mediated binding of PYRABACTIN RESISTANCE1‐LIKE (PYL) receptors to clade A Type 2 C protein phosphatases (PP2CAs). In Arabidopsis thaliana, several constitutively active variants of ABA receptors have been described, offering valuable tools for improving plant tolerance to environmental stresses. To identify amino acid residues in the rice ABA receptor OsPYL5 that are involved in ABA‐independent interactions, we implemented a random mutagenesis strategy followed by yeast two‐hybrid (Y2H) screening. We identified residues L‐93 and N‐102 as key residues that influence the ABA independent interaction of OsPYL5 with OsPP2CA51. Substituting these residues with T or Y significantly enhanced the activity of an ABA‐responsive reporter in rice protoplasts, even in the absence of ABA treatment. We therefore engineered a double‐point mutant, OsPYL5L93W N102Y, which demonstrated a strong ABA‐independent interaction with OsPP2CA51 in Y2H assays, elevated activation of ABA‐responsive reporter in rice protoplasts, and suppression of PP2CA phosphatase activity in vitro in the absence of ABA. Transgenic rice lines overexpressing OsPYL5L93W N102Y showed delayed germination and growth retardation in the absence of ABA treatment. They also exhibited increased sensitivity to ABA during germination and in early seedling growth assays compared to an OsPYL5‐overexpressing transgenic rice line (OsPYL5‐OX). Moreover, compared to OsPYL5‐OX, they showed dramatic upregulation of ABA‐responsive genes both without ABA and with low concentrations of ABA. These transgenic lines also showed enhanced tolerance to drought and salt stress compared to both the control cultivar and OsPYL5‐OX. Taken together, our findings not only identify key residues of OsPYL5 that enable ABA‐independent receptor function, but also highlight the feasibility of engineering ABA receptors to improve abiotic stress tolerance.

Keywords: ABA, abiotic stress tolerance, Oryza sativa, OsPYL5, random mutagenesis

Summary statement

An engineered a double‐point mutant, OsPYL5L93WN102Y demonstrate the ABA‐independent interaction with OsPP2CAs and transgenic rice overexpressing OsPYL5L93WN102Y showed enhanced drought stress tolerance.

1. Introduction

The accelerating pace of climate change, coupled with the steady rise in global population, presents critical challenges to global food security. Climate change exacerbates these challenges by increasing the frequency and severity of abiotic stresses that negatively impact crop production. Among the abiotic stresses, drought and salinity are the most detrimental, exerting widespread effects on agricultural systems worldwide (Eze et al. 2022).

Oryza sativa (rice), which feeds nearly half of the global population, is notably vulnerable to abiotic stresses, especially drought and salinity (Maghboli Balasjin et al. 2022; Franco‐Navarro et al. 2025). As a semi‐aquatic crop with a shallow root system and high water demands, rice is particularly sensitive to fluctuations in water availability and soil ionic balance. This sensitivity has driven substantial research efforts to discover the genetic and molecular basis of stress tolerance in rice. Key regulatory genes such as dehydration‐responsive element‐binding proteins (DREBs), basic leucine zipper transcription factors (bZIPs), and abscisic acid (ABA) receptors have been identified as important contributors to adaptive stress responses (Dubouzet et al. 2003; Mukherjee et al. 2006; Hossain et al. 2010; Kim et al. 2014; Dittrich et al. 2019; Esmaeili et al. 2022). These genes are all related to ABA signaling and play central roles in orchestrating physiological responses to environmental stress.

ABA is a key phytohormone that mediates plant responses to drought, salinity, and cold by regulating stomatal closure, gene expression, and osmotic homeostasis (Lee et al. 2006; Tuteja 2007; Finkelstein 2013). ABA biosynthesis begins in plastids and is completed in the cytosol, after which it is conjugated into inactive ABA‐glucose esters in the endoplasmic reticulum. In response to environmental stress signals, these conjugates are rapidly hydrolyzed by β‐glucosidases to release active ABA (Burla et al. 2013). Active ABA is recognized through an ABA‐mediated interaction between an ABA receptor in the PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE‐LIKE/REGULATORY COMPONENT OF ABA RECEPTORS (PYL) family and a clade A Type 2 C protein phosphatase (PP2CA) (Miyakawa et al. 2013; Min et al. 2019; Takeuchi et al. 2021). The resulting ABA/PYL/PP2CA ternary complex activates SUCROSE NONFERMENTING1‐related protein kinase 2 (SnRK2s), which in turn phosphorylate key downstream targets such as SLOW ANION CHANNEL1 (SLAC1) and ABA‐RESPONSIVE ELEMENT‐BINDING (AREB) transcription factors (Park et al. 2009; Cutler et al. 2010).

Given their central role in ABA signaling and adaptation to abiotic stress, ABA receptors are promising targets for engineering stress‐tolerant crops (Seo and Koshiba 2002; Nambara and Marion‐Poll 2005). The molecular mechanisms of ABA perception and signal transduction have been extensively studied, and this knowledge is being leveraged to develop innovative crop varieties with improved stress tolerance (Park et al. 2015; Lozano‐Juste et al. 2023). Recent advances in structural biology have enabled the development of constitutively active (CA) variants of PYL receptors that bypass the requirement for ABA binding (Gonzalez‐Villagra 2023). These engineered receptors adopt an active conformation that allows stable ABA‐independent interactions with PP2CAs, maintaining downstream SnRK2 activity and inducing stress‐related gene expression even in the absence of external stress or ABA (Peterson et al. 2010; Mosquna et al. 2011; Pri‐Tal et al. 2024). Such CA receptors represent a powerful tool for enhancing plant stress responses, and their functional properties and potential for field application are increasingly being explored in both model plants and crops (Pri‐Tal et al. 2024).

Designing CA variants typically involves rational mutagenesis based on crystallographic structures, targeting residues at the gate–latch interface or hydrophobic cores to stabilize the closed conformation of the receptor. CA variants of PYL2 and PYL4 enhance drought tolerance in Arabidopsis, and tomato (Solanum lycopersicum) lines expressing CA receptors exhibit improved water‐use efficiency (Park et al. 2015; Gonzalez‐Villagra 2023). In rice, a CA ABA receptor has not been reported, even though the overexpression of native ABA receptors such as OsPYL5 and OsPYL10 has been shown to enhance drought and salinity tolerance (Kim et al. 2014; Verma et al. 2019).

In this study, we screened a random mutagenesis library using yeast two‐hybrid (Y2H) system to identify key residues of OsPYL5 that are involved in ABA‐independent activity. Based on this screening, we generated a double‐point mutant, OsPYL5L93W N102Y which exhibits strong ABA‐independent binding to PP2C51 and effectively inhibits its phosphatase activity. Transgenic rice plants overexpressing OsPYL5L93W N102Y demonstrated enhanced tolerance to drought and salinity stress. These findings provide insights into the molecular mechanisms govering ABA‐independent interactions of OsPYLs with downstream signaling components and offer a promising strategy to improve crop stress tolerance through targeted engineering of ABA receptor functions.

2. Materials and Methods

2.1. Construction of an Error‐Prone PCR‐Based Mutant Library and Yeast Two‐Hybrid Screening

Random PCR mutagenesis of OsPYL5(Os05g12260) was performed using TITANIUM Taq polymerase (Takara, Japan) in a 50 µL reaction containing MnSO₄, biased dNTPs, and a GC solution (NanoHelix, Korea). Mutagenized OsPYL5 and OsPP2C51(Os05g49730) were cloned into pGADT7 (prey) and pGBKT7 (bait), respectively, and transformed into yeast strains Y187 (MATα) and Y2HGold (MATa) respectively. Mating‐based yeast two‐hybrid (Y2H) screening was conducted on SD/–Leu/‐Trp plates, followed by interaction assays on triple dropout (SD/–Leu/–Trp/–His/+X‐α‐Gal/+AbA [Aureobasidin A]) and quadruple dropout (SD/–Leu/–Trp/–His/–Ade/+X‐α‐Gal/+AbA) media. Following plasmid isolation from yeast cells, positive clones were sequenced using Sanger methods.

To assess potential ABA‐independent interactions, we synthesized DNA encoding single amino acid point mutantion of OsPYL5 (Macrogen, Korea), namely L93W and N102Y. These DNA were then cloned into pBGKT7 and co‐transformed along with OsPP2C51 cloned into pGADT7 in the AH109 yeast strain. Transformants were selected on SD/–Leu/–Trp plates, then screened for interaction on SD/–Leu/–Trp/–His/–Ade plates. Transformant cultures were adjusted to an OD595 of 1.0, and 10 μL was spotted onto the plates. Photographs were taken 4 days after spotting and incubation at 30°C. Colony growth was evaluated after 4 days.

2.2. Isolation of Rice Protoplasts and Transient Expression of an ABA‐Responsive Reporter

As described in Min et al. (2022), rice seeds were germinated on half‐strength (½x) Murashige and Skoog (MS) medium (with 0.4% phytagel, pH 5.8) in the dark for 7–8 days, then transferred to light/dark (16 h/8 h) condition at 28°C for 2 days. To activate the enzymes, the enzyme solution (1.5% cellulase R‐10, 0.3% macerozyme R‐10, 0.4 M sucrose, pH 5.8) was incubated at 65°C for 10 min and then cooled to room temperature. After cooling, BSA, CaCl₂, β‐mercaptoethanol, and ampicillin were added. Rice stem segments were then incubated in the prepared enzyme solution at 28°C for 4 h to release protoplasts. The released protoplasts were filtered through mesh, purified using W5(0.1% Glucose, 0.9% NaCl, 2 mM MES, 0.08% KCl, 1.84% CaCl2·2H2O, pH 5.8) and a 22% sucrose gradient, and resuspended in MaMg buffer (600 mM Mannitol, 15 mM MgCl2, 5 mM MES monohydrate, pH 5.8). For transfection, rice protoplasts suspended in MaMg buffer were mixed with plasmid DNA depending on the experimental condition. For normalization, only 5 μg of the pABRE‐DRE::fLUC (fLUC) reporter and 0.5 μg of pUbi::rLUC (rLUC) were used (Min et al. 2022). A combination of 5 μg of OsPYL5 or its mutants, 1 μg of PP2C51, 5 μg of the fLUC reporter, and 0.5 μg of rLUC was used. A PEG solution (40% PEG 6000, 100 mM Ca(NO₃)₂, 400 mM mannitol) was then added, and the mixture was incubated for 1.5 h at 28°C. After incubation, PEG was gradually diluted with W5 solution, and the protoplasts were collected by gentle centrifugation and resuspended in W5 supplemented with ampicillin. Protoplasts were then treated with either ABA or DMSO and incubated at 28°C for 24 h. Luciferase activity was subsequently measured using the Dual‐Luciferase Reporter Assay System (Promega, USA).

2.3. Expression of Recombinant Proteins in Escherichia coli

His‐tagged OsPYL5 and GST‐tagged OsPP2C51 constructs were transformed into E. coli Rosetta 2 DE3 cells (Novagen, USA). The transformed cells were cultured in LB medium at 37°C until the OD600 reached approximately 1.0. Protein expression was induced by adding 0.1 mM IPTG, followed by incubation at 20°C for 20 h, with gentle shaking. Cells were harvested and resuspended in lysis buffer as follows. For His‐tagged proteins, the lysis buffer contained 20 mM Tris‐HCl (pH 8.0), 200 mM NaCl, 4 mM DTT, and 0.05% Triton X‐100. For GST‐tagged proteins, the same buffer was used with the addition of 5 mM MgCl2. After centrifugation to remove cell debris, the supernatant was applied to glutathione‐Sepharose resin (GE Healthcare, USA). The resin was washed with 200 mL of 1x PBS, and proteins were eluted with 20 mL of buffer B supplemented with 10 mM reduced glutathione. Protein concentrations were measured using the Bradford assay, with BSA as the standard.

2.4. GST Pull‐Down, Immunoblotting, and Phosphatase Inhibition Assays

Purified GST‐OsPP2C51, OsPYL5‐His, and OsPYL5L93W N102Y‐His were used for GST pull‐down assays. Glutathione‐Sepharose resin (GE Healthcare, USA) was washed with pull‐down buffer containing 20 mM Tris‐HCl (pH 8.0), 1 mM MgCl₂, 4 mM DTT, and 0.1 mg/mL BSA. Ten μg of GST‐OsPP2C51 was incubated with the resin in pull‐down buffer for 30 min at 4°C. After this, 10 μg of either OsPYL5‐His or OsPYL5L93W N102Y mutant protein was added, and the mixture was incubated for 1 h at 4°C with gentle agitation. The resin was washed three times with pull‐down buffer, and bound proteins were eluted using the same buffer supplemented with 10 mM reduced glutathione. Eluted samples were analyzed using SDS‐PAGE and immunoblotting. Phosphatase assays were performed as described by Han et al. (2017). Reaction mixtures contained 80 nM GST‐OsPP2C51, 400 nM OsPYL5‐His or mutant protein, and phosphatase buffer (20 mM Tris‐HCl (pH 8.0), 1 mM MnCl₂, 0.1% (v/v) 2‐mercaptoethanol, 0.1 mg/mL BSA). After 10 min of incubation at 37°C, the reactions were initiated with 15 mM pNPP. Absorbance at 405 nm was measured after 1 h to determine phosphatase activity.

2.5. Generation of Transgenic Rice Plants

The rice cultivar used in this study was Oryza sativa cv. Samkwang. To generate overexpressing (OX) rice plants, the coding sequence of OsPYL5 was cloned into the pGA2897 gateway vector containing the maize (Zea mays) ubiquitin promoter (Szostkiewicz et al. 2010; Kim et al. 2012; Kim et al. 2015). Agrobacterium tumefaciens strain LBA4404 was transformed with this construct by electroporation and used for subsequent plant transformation as described previously (Han et al. 2012). Transgenic plants were selected on ½x MS medium (Duchefa Biochemie, The Netherlands) supplemented with 40 μg/mL hygromycin.

2.6. Rice Growth and Abiotic Stress Treatments

Surface‐sterilized dehulled rice seeds were germinated on ½ × MS medium solidified with 0.4% Gelrite (Duchefa Biochemie, Netherlands) and supplemented with 0 or 5 μM ABA (Sigma‐Aldrich, USA). Germination was scored until 7 days based on radicle emergence and shoot greening. For growth assays, uniformly grown transgenic seedlings were transferred to plates of ½x MS medium containing either 0 (mock) or 5 μM ABA. Shoot lengths were measured 3 days after transfer for the mock treatment and 6 days after transfer for the ABA treatment. For drought and salt stress tolerance assays, seeds from plants overexpressing OsPYL5 or its mutants were germinated in water containing 40 μg/mL hygromycin for 5–6 days. Selected seedlings were transferred to soil and grown in a greenhouse for 21 days, with watering every 5 days. Drought stress was imposed by withholding water for 5 days, followed by rewatering. For salt stress, 30‐day‐old plants were treated with 200 mM NaCl for 10 days, and then allowed to recover for another 10 days. Survival rates and fresh weights were measured, with three biological replicates per plant line.

2.7. Gene Expression Analysis by RT‐qPCR

Rice tissue samples (50–100 mg) were ground using a TissueLyser II (Qiagen Inc., Germany), and total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Germany) following the manufacturer's protocol. RNA purity and concentration were measured using a NanoDrop1000 (Thermo Fisher Scientific, USA). The RNA was dissolved in DEPC‐treated water, incubated at 65°C for 10 min, and chilled on ice. First‐strand cDNA was synthesized from 5 μg of total RNA using an RNA to cDNA EcoDry premix (Takara, Japan) with incubation at 42°C for 60 min, followed by enzyme inactivation at 72°C for 10 min. Quantitative PCR was conducted using TOPreal qPCR 2 × premix (Enzynomics, Korea) and the primers listed in Table S2. OsUBIQUITIN 5 (OsUbi05) was used as an internal control, and relative gene expression was calculated using the 2–∆Ct or 2–∆∆Ct method.

2.8. Field Cultivation Conditions and Measurement of Agricultural Traits

Seeds from transgenic rice plants were germinated and grown for 1 week on ½x MS media containing hygromycin B (40 mg L–1). After acclimating for 2 days in the greenhouse, young seedlings were transferred to pots (16 × 6 × 5.5 cm) filled with nursery soil and grown at 24°C–30°C for 4 weeks in the greenhouse. The rice plants were transplanted into a paddy field in Jeonju, South Korea, in mid‐May, and seed harvesting was conducted annually at the end of October from 2022 to 2024. Agricultural traits of the transgenic rice plants were evaluated at the maturity stage (24‐week‐old plants), with measurements taken from seven individual plants per line in three independent transgenic lines. Traits assessed included total grain weight, culm length, panicle length, number of panicles, and internode length.

3. Results

3.1. Identification of the Amino Acid Residues That Enable ABA‐Independent Interaction Between OsPYL5 and OsPP2C51

The interaction between ABA receptors and PP2CA proteins is essential for regulating plant responses to abiotic stress, and several studies have identified critical amino acid residues involved in this process (Melcher et al. 2009; Mosquna et al. 2011). In rice, OsPP2C51 and several OsPP2CAs, interacts with OsPYL5 in an ABA‐dependent manner (Figure S1) (Bhatnagar et al. 2017). To generate ABA‐independent variants of OsPYL5, a random mutant library of OsPYL5 was constructed using error‐prone PCR, yielding approximately 2.5 × 10⁷ clones (Table S1). From this library, approximately 8 × 10⁶ clones were screened using a Y2H system to identify ABA‐independent interactions. In the Y2H assay, OsPP2C51 was used as bait, and the OsPYL5 variants were employed as prey. From the initial screen, 144 clones were selected, and a subsequent secondary screen narrowed these down to thirteen clones that exhibited ABA independent interaction with OsPP2C51. Subsequently, we analyzed in detail to determine their exact amino acid substitutions. Sequencing of the corresponding 13 yeast plasmids revealed a total of 33 single amino acid substitutions at 28 distinct residues in OsPYL5 (Figure 1A).

Figure 1.

Figure 1

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Identification of OsPYL5 mutants capable of ABA‐independent interaction with OsPP2C51. A. Interactions between OsPP2C51 and OsPYL5 mutants selected from the mutant library, as shown by a Y2H assay. B. Y2H screening to identify single amino acid mutants of OsPYL5 that interact with OsPP2C51 in an ABA‐independent manner. Interactions were determined by the presence of yeast growth on SD media lacking Leu, Trp, His, and Ade, in the presence of 0, 0.1, or 1 μM ABA. Various OsPYL5 mutants were used as bait, and OsPP2C51 was used as prey. C. Site‐directed mutants of OsPYL5, L93F, N102S, and N102Y, interact with OsPP2C51 in an ABA‐independent manner, as shown by Y2H. D. The effect of the mutations in OsPYL5 on OsRab16A promoter‐driven LUC activity in rice protoplasts in the absence of ABA. Control, protoplasts transfected with pABRE‐DRE::fLUC and pUbi::rLUC only. Data are presented as mean ± SD, n = 3. Significant differences compared to OsPYL5 were determined using one‐way ANOVA. **p < 0.01; ****p < 0.0001; ns, not significant.

To identify the specific amino acid residues critical for mediating the ABA‐independent interaction between OsPYL5 and OsPP2C51, each of the 33 amino acid substitutions identified from the Y2H screening was individually introduced into OsPYL5 to generate corresponding single‐point mutants. These mutants were subsequently evaluated in Y2H assays under absence of ABA. This initial single‐residue analysis allowed us to dissect the individual contributions of candidate residues to the ABA‐independent interaction, providing a foundation for subsequent combinatorial mutant analyses. Through this approach, six single‐point mutations (L93F, E100V, N102S, N102Y, A182V, and T189A) were found to confer an ABA‐independent interaction with OsPP2C51 (Figure 1B). Notably, L93F conferred the strongest interaction, and the repeated identification of substitutions at residue N102 (N102S and N102Y) suggested a pivotal role for this site in modulating ABA receptor‐PP2CA binding in the absence of ABA (Figure 1C). Thus, we focused on the L93 and N102 amino acid residues. Based on these observations, we focused on the L93 and N102 amino acid residues. Although A182V also conferred ABA‐independent interaction, this site has been previously reported. In contrast, the remaining mutants exhibited relatively weak activity and were therefore excluded from further analysis. In the absence of ABA, overexpression of the OsPYLL93F mutant exhibited a trend toward higher LUC reporter activity compared to wild‐type OsPYL5, although the difference was not statistically significant. In contrast, overexpression of OsPYLN102S and OsPYLN102Y significantly enhanced reporter activity, displaying approximately 1.5‐fold and two‐fold increases, respectively, relative to the wild type (Figure 1D).

3.2. OsPYL5 Interaction With OsPP2C51 was Enhanced by Specific Amino Acid Substitutions

To select the optimal alternative amino acid residues at L93 and N102 of OsPYL5 for ABA‐independent interaction with OsPP2C51, we substituted each of nine different amino acid residues at both positions. Y2H analysis revealed that substituting L93 with either Phe or Trp, both of which possess bulky hydrophobic side chains, enhanced ABA‐independent interaction with OsPP2C51 (Figure 2A). In addition, replacing N102 with Ser, Pro, Phe, Trp or Tyr also led to an ABA‐independent interaction with OsPP2C51 (Figure 2B). In rice protoplast assays, only one of the substitutions at L93, OsPYL5L93W, significantly enhanced LUC reporter activity, resulting in roughly a 1.7‐fold increase compared to the wild type OsPYL5 (Figure 2C). Several different amino acid substitutions at N102 significantly increased ABA‐independent signaling; among them, OsPYL5N102Y showed the highest reporter activity, resulting in a 3.1‐fold increase over wild type OsPYL5 (Figure 2D).

Figure 2.

Figure 2

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Identification of alternative amino acids at L93 and N102 that enhance the interaction between OsPYL5 and OsPP2C51. A, B. Y2H screening for point mutants of OsPYL5 at L93 (A) or N102 (B) that can interact with OsPP2C51 in the absence of ABA and assessment of auto‐activation. C, D. The effect of different point mutations of OsPYL5 at L93 (C) or N102 (D) on ABA‐responsive luciferase reporter activity in the absence of ABA treatment in rice protoplasts. Data are presented as mean ± SD, n = 3. Significant differences compared to OsPYL5 were determined by one‐way ANOVA. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant. Interaction was determined by Y2H on media lacking Leu, Trp, and His with 2 mM 3‐AT and containing different concentrations of ABA. Abbreviations: L (Leu), N (Asn), R (Arg), S (Ser), P (Pro), A (Ala), F (Phe), M (Met), W (Trp), Y (Tyr), I (Iso).

3.3. The Double‐Point Mutant OsPYL5L93W N102Y Significantly Enhanced ABA‐Independent Interactions Compared to Either Single‐Point Mutant

To evaluate whether combining the L93W and N102Y mutations could further enhance the ABA‐independent interaction between OsPYL5 and OsPP2C51, we generated an OsPYL5L93W N102Y double‐point mutant. A Y2H assay in the absence of ABA revealed that this double‐point mutant exhibited a much stronger interaction with OsPP2C51 than either single point mutant did (Figure 3A). Moreover, Y2H assays indicated that the WY double‐point mutant exhibited broadened ABA‐independent interaction specificity toward several clade A PP2Cs, including OsPP2C51, OsPP2C49, and OsPP2C30 (Figure S3). Luciferase reporter assays showed that OsPYL5L93W and OsPYL5N102Y led to 1.5‐fold and twofold increases in activity relative to wild‐type OsPYL5, respectively. In contrast, OsPYL5L93W N102Y displayed a highly synergistic increase of over six‐fold in the absence of ABA (Figure 3B). When 5 µM ABA was added, OsPYL5L93W did not show enhanced activity, whereas both OsPYL5N102Y and OsPYL5L93W N102Y exhibited an approximate two‐fold increase compared to wild‐type OsPYL5 (Figure 3C). These results suggest that the ABA‐independent signaling activity observed in the double‐point mutant is primarily attributable to the N102Y substitution.

Figure 3.

Figure 3

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The double‐point mutant OsPYLL93W N102Y strongly enhances ABA‐independent signaling compared to single‐point mutants. A. Y2H assay showing interactions between PP2C51 and OsPYL5L93W N102Y and two single‐point mutants. Interactions were determined by the presence of yeast growth on SD media lacking Leu, Trp, and His, containing 2 mM 3‐AT and 0, 0.1, or 1 μM ABA and assessment of auto‐activation. B, C. Comparative analysis of ABA signaling effects in OsPYL5 mutants without (B) or with (C) ABA treatment. Significant differences compared to OsPYL5 were determined using one‐way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. D. OsPYL5‐His and OsPYL5L93W N102Y‐His were co‐immunoprecipitated with GST‐OsPP2C51. E, F. GST‐OsPP2C51 phosphatase activity was measured in the presence of different molar ratios of GST‐OsPP2C51:OsPYL5‐His or GST‐OsPP2C51:OsPYL5L93W N102Y‐His (1:0, 1:1, 1:2), determined in the absence (E) or presence of 10 μM ABA (F). Reactions contained 500 nM OsPP2C51 and 0, 500, 1000 nM His‐tagged PYL proteins. The activity of OsPP2C51 was measured every minute for 130 min. Protein amounts were quantified by visual comparison to BSA standards following SDS‐PAGE analysis. Values represent the percentage of activity compared with OsPP2C51 without PYL proteins (1:0). Statistical significance was assessed using unpaired t‐tests with Welch's correction. Significance levels are indicated as follows: **p < 0.01, ns, not significant.

To validate these findings in vitro, His‐tagged OsPYL5 and OsPYL5L93W N102Y were expressed in E. coli and analyzed using GST pull‐down assays with OsPP2C51. In the absence of ABA, only OsPYL5L93W N102Y exhibited a detectable interaction, whereas wild‐type OsPYL5 showed only a very weak interaction. Upon treatment with 10 µM ABA, the interaction of OsPYL5L93W N102Y increased slightly compared to wild type OsPYL5, but the magnitude of the increase was considerably lower than the observed under ABA‐free conditions (Figure 3D). GST‐OsPP2C51 efficiently retained OsPYL5‐His, whereas GST alone did not, confirming the specific physical association between the two proteins in vitro (Fig. S4). Phosphatase inhibition assays confirmed the enhanced functional interaction (Figure 3E,F). OsPYL5L93W N102Y inhibited OsPP2C51 phosphatase activity more effectively than the wild‐type receptor, exhibiting approximately 20% greater inhibition at a 1:1 molar ratio in the absence of ABA (Figures 3E), and up to 5% greater inhibition at a 1:2 ratio in the presence of 10 μM ABA (Figure 3F). Taken together, these results demonstrate that OsPYL5L93W N102Y enhances ABA signaling, particularly under ABA‐free conditions, by strengthening its interaction with OsPP2C51 and more effectively suppressing its phosphatase activity.

3.4. Overexpression of OsPYL5 L93W N102Y Delayed Germination

Transgenic rice lines overexpressing either wild‐type OsPYL5 (OX) or the OsPYL5L93W N102Y mutant (WY) were generated using the pGA2897 binary vector containing the maize ubiquitin promoter. From the T₁ generation, one OX line and two WY lines (WY2 and WY4) were selected based on transgene expression levels confirmed by RT‐qPCR, and transgene copy number was subsequently examined in the T₀, T₁, and T₄ generations (Figure S6). T3 seeds were harvested and used in phenotypic assays. To evaluate the effect of OsPYL5L93W N102Y overexpression, seed germination assays were conducted under controlled conditions (28°C, 16 h light/8 h dark). At 4 days after sowing in the absence of ABA, all transgenic lines exhibited delayed germination compared to nontransgenic control plants, with WY2 and WY4 showing particularly severe inhibition (Figure 4A). At 48 h after sowing in the absence of ABA, the control line showed 82% germination, whereas OX, WY2, and WY4 had germination rates of 52%, 25%, and 3%, respectively (Figure 4B). Upon treatment with 5 μM ABA, the germination delay was further exacerbated. By 114 h, control seeds reached 100% germination, whereas OX seeds achieved just over 50%, and both WY lines remained below 32%. These results suggest that OsPYL5L93W N102Y overexpression confers increased ABA sensitivity and/or enhances ABA‐independent inhibition of seed germination in planta (Figure 4C,D).

Figure 4.

Figure 4

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Overexpression of a mutated ABA receptor delayed rice seed germination. A. Seed germination of control(Samkwang), OsPYL5 overexpression (OX), and OsPYL5L93W N102Y (WY) lines was examined under standard growth conditions. From left to right: Control, OX2, and WY2 are shown in the top row, and their corresponding lines (OX4, OX6 below OX2; WY4, WY5 below WY2) are arranged in the bottom row. Each panel represents germinated seedlings after 3 days of incubation on half‐strength MS medium. B. Seed germination of the same lines under 5 µM ABA treatment. The sample arrangement is identical to panel A: control, OX lines (OX2, OX4, and OX6), and WY lines (WY2, WY4, and WY5). Each panel shows germinated seedlings after 7 days on ½ MS medium supplemented with 5 µM ABA. Scale bar = 2 cm. C. Germination rates of control, OX lines, and WY line seeds in the absence of ABA. D. Germination rates of control, OX lines, and WY line seeds in the presence of 5 μM ABA. Transgenic and control seeds were sown on the same plate for direct comparison of germination. Two biological replicates each with 25 seeds for each treatment were set in seed germination.

3.5. Overexpression of OsPYL5 L93WN102Y Enhanced ABA Signaling Related to Both Plant Growth and Gene Expression

Under field conditions, OsPYL5L93W N102Y‐overexpressing plants showed reduced plant height and total seed yield compared to wild‐type and OX lines (Figure 5A,B), suggesting a growth penalty associated with the constitutive activation of the ABA pathway. To characterize the effects of OsPYL5 and OsPYL5L93W N102Y overexpression on ABA signaling and stress responses, post‐germination growth assays were conducted using the OX, WY2, and WY4 lines with or without ABA treatment. In the absence of ABA, all three overexpressing lines exhibited reduced shoot growth compared to the non‐transgenic control. However, a 5 μM ABA treatment resulted in more pronounced growth inhibition in the WY lines than in the OX line (Figure 5C,D). To assess the activation of downstream signaling, we analyzed the expression of three ABA‐responsive rice genes (Rab16A, LEA3, ABF1) in 14‐day‐old seedlings. In the absence of ABA, all three genes were significantly upregulated in WY2 and WY4 compared to OX, indicating constitutive activation of ABA signaling (Figure 5E, top row). In the presence of 0.1 µM ABA, expression levels of Rab16 A and LEA3 remained significantly higher in the WY lines than in the OX line (Figure 5E, bottom row). In contrast, ABF1 expression did not differ significantly between the control and OX lines. Although the increase was statistically significant for the WY lines, the magnitude of this increase was less pronounced than that of the other two genes. Together, these results demonstrate that OsPYL5L93W N102Y overexpression enhances ABA signaling at both the phenotypic and molecular levels, regardless of exogenous ABA treatment.

Figure 5.

Figure 5

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Overexpression of a mutated ABA receptor confers increased ABA sensitivity and ABA‐responsive gene expression. A. Representative mature T3 plants grown in a rice paddy. Plants overexpressing either OsPYL5‐OX (OX) or OsPYL5L93W N102Y (WY2), along with wild‐type control plants. B. Box plots showing plant height, panicle length, number of panicles, and total seed weight of OsPYL5 and OsPYL5 L93W N102Y overexpression plants (n = 7). The boxes represent the 25th to 75th percentiles, with the median indicated by a line in the middle and the mean marked by a + sign. The whiskers extend to the minimum and maximum values within each group. C. After 3 days of germination, OX, WY2, and WY4 seedlings were transferred to ½x MS medium either without ABA (upper row) or with 5 μM ABA (lower row). Scale bar, 2 cm. Representative images were recorded 3 days after transplantation for mock‐treated plants and 6 days after transplantation for ABA‐treated plants. B. Shoot lengths. Error bars represent the SE of 10 individuals. D. RT‐qPCR analysis using three ABA‐responsive genes. Similar expression levels were observed in two replicates. Twelve‐day‐old seedlings were used for the experiments. Data represent mean ± SD of three biological replicates. Significant differences were identified using one‐way ANOVA. Significant differences compared to OsPYL5 were determined using one‐way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

3.6. OsPYL5L93W N102Y‐OX Showed Increased Abiotic Stress Tolerance Compared to OsPYL5‐OX

To assess the impact of OsPYL5L93W N102Y on abiotic stress responses, drought and salinity tolerance assays were conducted under controlled greenhouse conditions. After 3 weeks of growth, plants were subjected to drought stress by withholding water for 7–10 days, followed by a 7‐day recovery period of rewatering. Before drought stress, all lines looked similar (Figure 6A, top). Upon rewatering, WY2 plants exhibited improved drought tolerance, with enhanced greenness and fewer wilted leaves compared to the control and OX plants (Figure 6A, bottom). Quantitative analysis showed that the survival rate of WY2 was 5.5‐fold higher than that of the control, and 2.4‐fold higher than that of OX (Figure 6B). Additionally, WY2 showed a 1.6‐fold increase in fresh weight relative to OX, which is a statistically significant improvement. WY4 showed an 8% higher survival rate relative to OX and a slight increase in fresh weight, though this was not statistically significant (Figure 6B). Under salt stress, the leaves of control plants gradually curled, turned yellow at the tips, and were drooping or collapsing after 3 weeks of salt treatment (Figure 6C). In contrast, OX and WY lines maintained better leaf integrity and exhibited significantly higher survival rates and fresh weight compared to control plants (Figure 6D). Specifically, WY2 showed a twofold increase in survival rate and a 1.5‐fold increase in fresh weight relative to OX, whereas WY4 showed 1.4‐fold and 1.5‐fold increases in survival rate and fresh weight, respectively (Figure 6D). These findings demonstrate that the overexpression of OsPYL5L93W N102Y enhances plant tolerance to both drought and salinity stress more effectively than the overexpression of wild‐type OsPYL5. The increased stress tolerance observed in the WY lines likely reflects an enhanced capacity for ABA signaling, highlighting their potential to improve crop resilience under adverse environmental conditions.

Figure 6.

Figure 6

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OsPYL5L93W N102Y‐OX showed improved abiotic stress tolerance compared to OsPYL5‐OX. A. Representative images of plants in the drought tolerance assay. The upper panel shows the plants before the drought stress treatment. The lower panel shows plants after 5 days of drought stress treatment followed by 7–10 days of rewatering. B. Survival rates (left panel, n = 6 pots) and fresh weights (right panel, n = 72 plants, 12 plants in one pot) measured after 5 days of drought treatment and 7–10 days of rewatering. Survival rates represent the percentage of surviving plants in each pot. Plants were subjected to drought treatment for 5 days, followed by 7–10 days of rewatering. Significant differences were determined by one‐way non‐parametric ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. C. Representative images of plants from the salt tolerance assay. The upper images show 3‐week‐old plants before the salt stress treatment. The lower images show plants after 10 days of a 200‐mM salt stress treatment followed by 10 days of rewatering. D. Survival rates (upper panel, n = 6) and fresh weights (lower panel, n = 36, six plants in one pot) after 6 days of rewatering. Survival rates represent the percentage of surviving plants in each pot. Significant differences were determined by one‐way non‐parametric ANOVA, *p < 0.05; **p < 0.01; ns, not significant. The Samkwang cultivar was used as a control.

4. Discussion

Through error‐prone PCR‐based random mutagenesis, we generated an OsPYL5 mutant library and screened it for altered PYL–PP2CA interactions using Y2H assays. This approach identified two novel residues in OsPYL5 (L93 and N102) that conferred ABA‐independent interaction with OsPP2C51 when mutated. These findings validate the use of Y2H‐based screening of mutagenesis libraries to discover residues not only critical for ABA signaling but also amenable to receptor engineering.

It has been reported that several amino acid residues can confer ABA‐independent activity. For example, the H60P and V83F mutations in AtPYR1 stabilize the receptor in an active conformation without ABA (Dorosh et al. 2014), and mutation of any two residues among V107, L111, and V192 in tomato PYL3 confers strong ABA‐independent inhibition of HAB1 (Wang et al. 2021). Saturation mutagenesis of AtPYR1 further identified additional residues (H60, V83, A89, F159, K170) with similar functional effects (Mosquna et al. 2011). Likewise, substitutions in the latch loop of tomato PYL1 enhance flexibility and receptor activity in both ABA‐dependent and independent modes (Infantes et al. 2022). Structural studies also show that conformational changes in the ABA‐binding pocket, gate loop, and C‐terminal helix are key determinants of receptor activation and ligand sensitivity (Yin et al. 2009; Szostkiewicz et al. 2010; Hao et al. 2011).

Among the OsPYL5 variants we identified here through random mutagenesis, several mutations occurred in residues that are structurally or functionally similar to previously characterized residues involved in ABA‐independent interactions with PP2CAs in Arabidopsis. For instance, the A182V substitution in OsPYL5 lies within the fifth α‐helix and aligns with T162 in AtPYR1, where the T162F mutation promotes constitutive PP2C interaction (Mosquna et al. 2011). Similarly, OsPYL5 T189A aligns with Q169 in AtPYR1, adjacent to the CA mutation site K170 (Figure S2). These results indicate that residues identified through random mutagenesis include conserved functional sites that are capable of mediating ABA‐independent interactions with PP2CAs (Miao et al. 2018; Tian et al. 2015; Kim et al. 2014).

To understand the structural context of these mutations, we performed a sequence alignment using four well‐characterized Arabidopsis ABA receptors (AtPYR1, AtPYL1, AtPYL2, AtPYL5) and three rice homologs (OsPYL3, OsPYL5, OsPYL10). Structural annotations based on the crystal structure of OsPYL3 served as a framework for mapping the mutation sites (Figure S2, S5). Residue N102 aligns with Vl82 in AtPYR1, a position adjacent to the gate loop and flanked by residues V81 and V83. These residues were previously identified as sites of CA mutations in Mosquna et al. (2011). Our mutagenesis results showed that diverse substitutions at this position, such as Tyr, Phe, Trp, Ser, and Pro, consistently conferred ABA‐independent activity. This indicates that the residue functions as a mutational hotspot (Figure 2). In contrast, L93 is in a surface‐exposed loop region outside the canonical gate and latch loops and has not been previously implicated in ABA receptor function. Nonetheless, its replacement with bulky hydrophobic residues such as Trp or Phe enabled ABA‐independent interactions with OsPP2C51 in Y2H assays (Figure 2B). These findings suggest that both conserved core motifs and peripheral, noncanonical regions can contribute to receptor activation, providing valuable targets for engineering ABA receptors with enhanced or novel properties.

The L93W and N102Y mutations in OsPYL5 exhibited a strong synergistic effect on receptor activation. The double mutant OsPYL5L93W N102Y inhibited OsPP2C51 even without ABA, indicating constitutive activation. This effect exceeded that of either single mutation. With ABA, the enhanced activity was mainly due to N102Y, suggesting its role in ABA‐dependent signaling, while L93W contributed primarily to ABA‐independent activation. These results show that the two mutations function through somewhat different mechanisms. N102Y enhances both basal and ABA‐induced activity, whereas L93W mainly promotes ABA‐independent signaling. The combined effect of these mutations highlights how manipulating structurally separate sites can produce synergistic outcomes, providing an effective approach to fine‐tuning receptor function through multi‐site engineering.

Mutational activation of ABA receptors can enhance ABA signaling independently of the hormone, leading to distinct physiological traits in plants. For example, the PYL4A194T mutant in Arabidopsis inhibits PP2Cs without ABA, resulting in constitutive activation of ABA‐responsive genes, enhanced stomatal closure, and improved drought resistance characterized by reduced water loss and increased survival under water deficit (Pizzio et al. 2013). Likewise, engineered AtPYR1 and AtPYL2 mutants that stabilize the ABA‐bound conformation show elevated ABA signaling in the absence of ABA, promoting hypersensitive responses and prolonged seed dormancy (Mosquna et al. 2011). In tomato, a Glu‐to‐Asp substitution in the latch loop of the dimeric receptor SlPYL1 enhances both ABA‐dependent and ABA‐independent PP2C inhibition, stabilizes the active conformation, and confers increased ABA sensitivity and drought tolerance (Infantes et al. 2022). Together, these findings demonstrate that mutations identified through screening or engineering can modulate plant development and stress responses by enhancing ABA sensitivity or mimicking ABA‐bound activation states.

Consistent with these findings, our engineered rice ABA receptors also conferred partial ABA‐independent activity. Transgenic rice plants overexpressing OsPYL5L93W N102Y exhibited delayed germination, reduced seedling growth, and elevated expression of ABA‐responsive genes with or without ABA. Although this double‐point mutation did not confer full constitutive activity, the mutated receptor inhibited OsPP2C51 more effectively than the wild‐type receptor, even in the absence of ABA, suggesting enhanced basal signaling. In field trials, OsPYL5L93W N102Y‐overexpressing lines displayed reduced plant height and grain yield compared to wild‐type and OsPYL5‐overexpressing plants, while panicle number remained unchanged. These results indicate that elevated ABA signaling, even in the absence of stress stimuli, can negatively affect plant growth and yield. However, the same lines also exhibited significantly improved tolerance to drought and salinity stress, as reflected by increased survival and biomass accumulation under stress conditions.

Taken together, we identified novel amino acid residues which contribute ABA independent interaction between OsPYL5 and OsPP2C51 and our results demonstrate that engineering ABA receptor through random mutagenesis is an efficient strategy for improving stress‐tolerant traits in crops.

Supporting information

Figure S1: The ABA‐dependent interaction between OsPYL5 and OsPP2CAs. Figure S2: Multiple amino acid sequence alignment of OsPYL5. Figure S3: The ABA‐independent interaction between OsPYL5L93W N102Y and OsPP2CAs. Figure S4: No interaction was detected between GST and a His‐tagged ABA receptor protein in a GST pull‐down assay. Figure S5: Homology‐based structural model of OsPYL5 based on the structures of AtPYR1 and OsPYL3. Figure S6: Determination of transgene copy number in T0 and T1, T4 transgenic lines and expression levels of OsPYL5 in T1 plants. Table S1: Summary of Y2H screening using OsPYL5 mutant library as a prey and OsPP2C51 as a bait. Table S2: Primer sequences for qPCR analysis.

PCE-49-4688-s001.docx (1.8MB, docx)

Acknowledgements

This work was supported by research program (PJ017209) of the Rural Development Administration, Republic of Korea. S.L. was supported by the National Research Foundation of Korea (NRF) grants (RS‐2025‐00513994 and RS‐2025‐16069622).

Data Availability Statement

The data that supports the findings of this study are available in the supporting material of this article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: The ABA‐dependent interaction between OsPYL5 and OsPP2CAs. Figure S2: Multiple amino acid sequence alignment of OsPYL5. Figure S3: The ABA‐independent interaction between OsPYL5L93W N102Y and OsPP2CAs. Figure S4: No interaction was detected between GST and a His‐tagged ABA receptor protein in a GST pull‐down assay. Figure S5: Homology‐based structural model of OsPYL5 based on the structures of AtPYR1 and OsPYL3. Figure S6: Determination of transgene copy number in T0 and T1, T4 transgenic lines and expression levels of OsPYL5 in T1 plants. Table S1: Summary of Y2H screening using OsPYL5 mutant library as a prey and OsPP2C51 as a bait. Table S2: Primer sequences for qPCR analysis.

PCE-49-4688-s001.docx (1.8MB, docx)

Data Availability Statement

The data that supports the findings of this study are available in the supporting material of this article.

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