ABA-independent PP2C-binding in PYLs traces to bacterial origins and persists in land plants

Domain: biology
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
Land plants have evolved strategies to survive water deficiency. Among these adaptations, the “drying without dying” strategy evolved in early land plants and is maintained in the desiccated seeds of angiosperms. This process is regulated by a family of ABA receptors known as the PYR/PYL/RCAR (PYL) family, which can bind to clade A protein phosphatases 2Cs (PP2Cs) and suppress their inhibition of water stress responses. ABA-independent PYLs first emerged in an aquatic alga lineage; however, their evolutionary origins and the mechanistic basis of ABA-independent PYL variants in land plants remain poorly characterized. Here, we characterize ABA-independent PYL-like proteins from bacteria, algae, and land plants that retain constitutive PP2C binding but lack ABA-enhanced inhibitory activity, supporting their putative bacterial origin via horizontal gene transfer. We identified a bacterial PYL homolog (PrPYL) in Paraburkholderia rhynchosiae with PP2C-binding ability, three PP2C-inhibiting PYL homologs in Zygnematales algae, and ABA-independent PYL-like proteins in seed plants (e.g., AtPYL13 and AtPYL13-like proteins). AtPYL13-like genes in dicots exhibited high expression during seed maturation and in desiccated seeds, suggesting a functional shift from canonical ABA receptors to ABA-independent PYL-like proteins following gene family expansion. Two invariant residues underlie ABA dependence in canonical PYL receptors. Transcription factor ABI3 mediates AtPYL13 expression during the mature seed stage, thereby locally restricting constitutively activated stress signaling. Our findings indicate that ABA-independent PYL-like genes likely originated via horizontal transfer from bacteria and function in basal stress signaling in seed plants.

Introduction
Terrestrialization represents a pivotal event in green plant evolution, requiring adaptive mechanisms to overcome multiple environmental challenges, particularly fluctuating water availability. Plants employ two principal strategies to cope with water deficiency: “drying without dying (desiccation tolerance)” or “avoiding drying (drought avoidance)”1. Early land plants are mainly poikilohydric and adopt the “drying without dying” strategy to survive extreme conditions2. Vascular land plants retain the “drying without dying” strategy in specific organs, such as seeds and pollen, but mainly utilize the “avoiding drying” strategy for their vegetative stages. These processes both depend on the activation of stress responses by the phytohormone ABA3,4.

The activation of ABA signaling occurs through a protein phosphorylation cascade, in which SNF1-related protein kinase 2 (SnRK2) kinases have a central role. Upon ABA binding, the PYR/PYL/RCAR (PYL) ABA receptors interact with and inhibit clade A protein phosphatase 2Cs (PP2Cs), releasing SnRK2s from PP2C inhibition and activating downstream stress responses5,6. The ABA-binding properties of PYLs vary significantly in seed plants; likewise, their PP2C inhibition mechanisms show distinct ABA-dependence patterns: (i) strictly ABA-dependent (no basal inhibition activity without ABA, e.g., the dimeric subfamily III PYLs in angiosperms)7; (ii) ABA-enhanced (basal inhibition activity potentiated by ABA)8; or (iii) ABA-independent (not modulated by ABA). The “ABA-independent PYL-like proteins” include: 1) constitutively active PYLs that activate stress responses regardless of ABA9; and 2) inactive PYLs incapable of activating stress responses. Overall, ABA signaling plays a central role in the “avoiding drying” strategy via stress-induced ABA accumulation4,8,10,11 and also regulates seed development and germination via tightly regulated dynamic ABA levels4,12. Although it is predictable that ABA-independent PYL-like proteins constitutively activate stress signaling, it remains unclear which plants encode such proteins and whether they contribute to the “drying without dying” strategy in vascular plants.

PYLs existed in the last common ancestor of Zygnematophyceae (green algae) and Embryophyta (land plants)13,14. The PYL-homolog ZcPYL8 from the alga Zygnema circumcarinatum shows ABA-independent PP2C inhibition, enabling constitutive activation of stress signaling and desiccation tolerance9. In the nonvascular liverwort Marchantia, MpPYL1 shows ABA-enhanced activity to inhibit PP2Cs, although with high basal activity9, which triggers high basal stress signaling and actively represses plant growth15. Strictly ABA-dependent PYLs with minimal basal activity emerged in Bryophytina mosses and angiosperms8,16, potentially enabling a broader range of ABA responses and improved balance between growth and stress responses. However, it remains unclear how ABA-independent PYL-like proteins emerged and were subsequently incorporated into land plant lineages.

One hypothesis is that the ancestral PYLs were transferred horizontally from soil bacteria to charophytes during their evolution14. Here, we analyzed the activity and ABA-dependence of PYLs from bacteria, charophytes, bryophytes, and angiosperms, identifying ABA-independent PYL-like proteins across these groups. The bacterial PYL-homolog from Paraburkholderia rhynchosiae possesses PP2C-binding ability, and PYLs from Zygnematales exhibit PP2C inhibitory ability, supporting that PYLs in plants might have originated horizontally from soil bacteria. The ABA-independent PYL-like proteins exist in multiple genera in seed plants, including PYL13 and PYL13-like proteins in dicots and monocots. Most dicot AtPYL13-like proteins are highly expressed in mature and desiccated seeds and confer constant activation of stress signaling. The seed-specific expression of AtPYL13 is controlled by the transcription factor ABI3. These ABA-independent PYL-like proteins differ from the ABA-receptor PYLs in the highly conserved ABA-binding pocket by two amino acid residues − the invariant lysine and leucine residues that are crucial for ABA receptors. Our discoveries suggest that the ABA-independent PYL-like genes might have originated horizontally from bacteria and function in basal stress signaling in seed plants.

Results
The bacterial PYL-homolog from Paraburkholderia rhynchosiae possesses PP2C-binding ability

Phylogenetic analyses propose that ancestral PYLs in plants were acquired through horizontal gene transfer from soil bacteria14. The PYL-homolog genes from plants form a monophyletic group that nests within the diversity of twenty bacterial genes from two phyla: Proteobacteria and Actinobacteria14. We selected four bacterial PYLs showing high similarity to PYLs from green algae, bryophytes, and angiosperms for further analyses (Fig. 1a; Supplementary Fig. 1a). However, these bacterial PYLs (from Paraburkholderia, Pseudomonas, Frankia, and Sphingomonas) have apparent differences with plant PYLs in the CL2 region, which is critical for interactions with ABA and PP2Cs17–26.

To test whether PYL homologs in bacteria function as ABA receptors, we evaluated their ABA dependence in the Arabidopsis ABA-insensitive pyl duodecuple mutant, in which all AtPYLs except AtPYL6 and AtPYL13 are mutated4. As expected, transfection of the pyl duodecuple mutant protoplasts with SnRK2.6 and ABF2 did not induce expression of the ABA-responsive reporter RD29B-LUC, even in the presence of ABA; however, co-transfection of SnRK2.6, ABF2 with an ABA receptor (AtPYR1, MpPYL1, or PpPYL1) resulted in the induction of RD29B-LUC in the presence of ABA (Fig. 1b; Supplementary Fig. 1b). PYL homologs from bacteria, including Proteobacteria (Sphingomonas, Pseudomonas, and Paraburkholderia) and Actinobacteria (Frankia), failed to induce RD29B-LUC expression with or without ABA treatment (Fig. 1b), indicating these bacterial PYLs are ABA-independent PYLs.

Despite lacking ABA dependence, ancestral PYLs should, first and foremost, interact with PP2Cs. Yeast two-hybrid (Y2H) assays were used to evaluate the PP2C-binding ability of these bacterial PYLs with Arabidopsis clade A PP2Cs. The Paraburkholderia rhynchosiae PYL (PrPYL), but not the bacterial PYL homologs from Sphingomonas and Frankia, interacted with several PP2Cs, including ABI1, HAB2, HAI3, and PP2CA, in an ABA-independent manner (Fig. 1c, d; Supplementary Fig. 1c, d). We further confirmed physical interactions between PrPYL and AtABI1 using split luciferase (LUC) complementation (LCI) assays. Transient co-expression of PrPYL-nLUC with either cLUC-AtABI1 or cLUC-AtHAB2 generated weak reconstituted LUC signals in Nicotiana benthamiana (Nb) leaves (Supplementary Fig. 1e). The weak PrPYL-PP2C interactions may be outcompeted by endogenous proteins (e.g., plant PYLs and SnRK2s), thus generating very weak reconstituted LUC signals in planta. These results suggest that PrPYL represents a prototype ABA receptor.

The PP2C inhibitory activity of PrPYL was evaluated using in vitro SnRK2 kinase assays in the presence and absence of ABA (Fig. 1e, f). Recombinant GST-tagged ABI1 and PP2CA inhibited the kinase activity of MBP-SnRK2.6. As a positive control, recombinant His-PYR1 released SnRK2.6 from PP2C-mediated inhibition in an ABA-dependent manner. In contrast, recombinant PrPYL could not release PP2C-mediated inhibition with or without external ABA (Fig. 1e, f). We further assessed PP2C inhibitory activity using the phosphatase substrate p-nitrophenyl phosphate (pNPP), and found that PrPYL did not inhibit PP2CA, ABI1, or HAB1 with or without external ABA (Fig. 1g, h; Supplementary Fig. 1f). These results indicated that bacterial PrPYL binds to PP2Cs but lacks PP2C-inhibitory activity.

The structure of PrPYL was predicted using AlphaFold3 and then aligned to the reported ABA-PYL1-ABI1 complex (PDB: 3KDJ)19. The PYL1-ABI1 interaction is mainly mediated by the CL loops, especially CL2 in AtPYL119. Ser112 in the CL2 loop of AtPYL1 plays an essential role in the PYL1-ABI1 interaction, with its hydroxyl group and backbone carbonyl oxygen forming hydrogen bonds with the Glu142 and Gly180 residues of ABI1. The predicted structure of PrPYL resembled that of AtPYL1 (Fig. 1i). However, the CL2 loop of AtPYL1 (gold) was closer to ABI1 (cyan) compared to that in the predicted PrPYL structure (purple). While Ser52 in PrPYL occupies a position structurally equivalent to Ser112 in AtPYL1 within the “S/TGLPA” motif (CL2 loop) and is predicted to form a hydrogen bond with Gly180 in ABI1, the distance of 4.5 Å (instead of 3.0 Å and less) (Fig. 1j) could partially explain the weak interaction between plant PP2Cs and PrPYL. In addition, the ligand-binding pocket of AtPYL1 (gold) accommodates the ABA molecule (green and red sticks) (Fig. 1k), but the ligand-binding pocket of PrPYL was too small to hold ABA (Fig. 1l), explaining its inability to respond to ABA. We also analyzed the binding affinity between PrPYL and ABA using isothermal titration calorimetry (ITC) and found that PrPYL has no binding affinity for ABA (Supplementary Fig. 1g). Our results support the hypothesis that PYLs originated via horizontal gene transfer from bacteria, but acquired their PP2C-inhibitory and ABA-binding functions in plants.

Zygnematales PYLs exhibit PP2C inhibitory activity in an ABA-independent manner

PYLs in plant lineages appear to have emerged before the last common ancestor of Zygnematophyceae and land plants13,14. The algal PYL-homolog ZcPYL8 in Zygnema circumcarinatum exhibited an ABA-independent PP2C inhibitory activity in vitro9. We mined transcriptome resources from representative land plants and algae and identified PYL homologs in five species of Zygnematophyceae: Zygnema circumcarinatum, Zygnemopsis sp., Mesotaenium endlicherianum, Mesotaenium kramstae, and Spirogyra sp., of which the first four are classified in Zygnematales and the last is classified in Spirogyrales (Fig. 2a and Supplementary Data 1). However, it remains unclear whether PYL homologs from Mesotaenium, Zygnemopsis and Spirogyra have ABA-dependent PP2C inhibitory activity. To test whether PYL homologs in green algae function as ABA receptors, we evaluated the ABA dependence of PYLs in the ABA-insensitive pyl duodecuple mutant. Among PYL homologs from Zygnematales, MeenPYL3 from Mesotaenium endlicherianum, MekrPYL1 from Mesotaenium kramstae, and ZcPYL8 from Zygnema circumcarinatum constitutively activated expression of RD29B-LUC in an ABA-independent manner, while ZyspPYL1 from Zygnemopsis, SpprPYL1 from Spirogyra, and MeenPYL1 and MeenPYL2 failed to induce RD29B-LUC expression (Figs. 1a and 2b). A previous study showed that ZcPYL8 inhibits ZcABI1 in an ABA-independent manner and cannot bind ABA9. These results indicated that PYL homologs gained ABA-independent PP2C inhibitory activity before the last common ancestor of Zygnematophyceae and land plants.

Consistent with their ability to constitutively activate expression of RD29B-LUC, MeenPYL3 and ZcPYL8 strongly interacted with multiple PP2Cs in an ABA-independent manner, as assessed by Y2H assays (Fig. 2c and Supplementary Fig. 2a). Although MeenPYL1 failed to induce RD29B-LUC expression (Fig. 2b), it did strongly interact with all clade A PP2Cs except HAI1 and AHG1 (Supplementary Fig. 2a), suggesting that MeenPYL1 has gained PP2C binding ability but not the strong PP2C inhibitory activity. It should be noted that AHG1 is resistant to PYL-mediated inhibition since it lacks the conserved tryptophan for ABA-PYL-PP2C and PP2C-SnRK2 interactions19,20,22,27–29. We further confirmed physical interactions between MeenPYL3 and PP2Cs using LCI assays. The transient co-expression of MeenPYL3-nLUC and cLUC-PP2Cs generated LUC signals in Nb leaves (Fig. 2d), suggesting interactions between MeenPYL3 and Arabidopsis PP2Cs, including ABI1, HAB1, PP2CA and HAI1.

We further evaluated the PP2C inhibitory activity of ZcPYL8 and MeenPYL3 using in vitro SnRK2 kinase assays (Fig. 2e, f). The recombinant AtHAB1 and AtPP2CA phosphatases repressed the kinase activity of the recombinant SnRK2.6. The recombinant ZcPYL8 partially released SnRK2.6 from HAB1-mediated inhibition, while the recombinant MeenPYL3 partially released SnRK2.6 from PP2CA-mediated inhibition in an ABA-independent manner. We further assessed PP2C inhibitory activity using pNPP as substrate. The recombinant MeenPYL3 partially repressed phosphatase activity of HAB1 and ABI1 on pNPP, while the recombinant ZcPYL8 slightly repressed phosphatase activity of PP2CA on pNPP but not HAB1, in an ABA-independent manner (Fig. 2g–j and Supplementary Fig. 2b). The differential results between the two PP2C activity assays could be due to differences in substrates, since pNPP is not a physiological substrate, and PP2Cs inhibit SnRK2s not only by dephosphorylation but also by physical interactions28. The AlphaFold3-predicted structure of MeenPYL3 was aligned to the ABA-PYL1-ABI1 complex (PDB: 3KDJ)19 as a template, showing that R63 in MeenPYL (corresponds to K86 in AtPYL1) leads to a clear steric hindrance (<2 Å) that blocks binding of ABA (in green; Fig. 2k). These results further confirm that PYL homologs obtained PP2C inhibition ability before the last common ancestor of Zygnematophyceae and land plants (Fig. 2l).

Structural analysis of PYLs identified four highly conserved loops (CL1–CL4) critical for ABA binding, among which the CL2 region, with a “S/TGLPA” motif, is crucial for the interactions between PYLs and PP2Cs19. Interestingly, these algal PYLs have a similar “S/TGL/IPG” motif, while the bacterial PYL homologs are distinguished from PYLs from algae and land plants in the CL2 region (Fig. 2a and Supplementary Fig. 1a). This partially explains the weak interaction of PrPYL-ABI1 and strong interactions between algal PYLs and Arabidopsis PP2Cs as well as the constitutive activation of RD29B-LUC expression in pyl duodecuple mutant protoplasts by algal PYLs (Figs. 1d and 2, and Supplementary Fig. 2a). In ABA-dependent or enhanced PYLs, including AtPYR1, AtPYL1/2/3/9/10, and OsPYL2, the ABA carboxylate forms charge-stabilized hydrogen bonds with the amino group of a conserved lysine in CL1, while the aliphatic portion of ABA interacts with the amino group of a conserved leucine in CL217–26. The key lysine residue that binds to the carboxyl group of ABA is replaced by another basic residue, arginine (R), in the three MeenPYL homologs (Fig. 2a). We also analyzed the binding affinity between MeenPYL3 and ABA using ITC and found that MeenPYL3 has no binding affinity for ABA (Supplementary Fig. 2c). To test whether these sequence variations may explain the ABA insensitivity of MeenPYL3, we co-transfected SnRK2.6, ABF2 with wild-type and mutated MeenPYL3 in pyl duodecuple mutant protoplasts. Neither MeenPYL3-R63K nor MeenPYL3-R63K/I93L altered the ABA-independent phenotype in the pyl duodecuple mutant protoplasts under ABA treatment, and MeenPYL3-R63K even failed to induce RD29B-LUC expression (Supplementary Fig. 2d). To our surprise, the AtPYL4-K81R mutant lost its high basal activity in elevating RD29B-LUC expression (Supplementary Fig. 2d), suggesting that arginine is a better choice than lysine in CL1 to generate PYL ABA receptors with no basal activity. Since high basal stress signaling would have favored the terrestrialization of land plant progenitors, we propose that this critical arginine was not conserved during evolution to maintain high basal activity of PYLs.

An ABA-independent PYL-like protein exists in Arabidopsis

The ABA-independent PYL-like proteins evolved before the last common ancestor of Zygnematophyceae and land plants, with some lineages subsequently gaining PP2C-inhibition capability. PYLs then acquired ABA-binding and -responsive functions in the last common ancestor of land plants9,16. The dimeric subfamily III PYLs in angiosperms exhibit strict ABA dependency7, and some moss-specific PYLs also display strict ABA dependency despite being monomeric16. However, it is unclear whether ABA-independent PYL-like proteins exist and function in angiosperms. Therefore, we first evaluated the ABA dependence of PYLs from the model species Arabidopsis thaliana in the ABA-insensitive pyl duodecuple mutant (Fig. 3a). Among these ABA-responsive PYLs, the ABA-enhanced AtPYL5-6 and 8–12 exhibited basal activities, leading to the induction of RD29B-LUC expression, while the ABA-dependent AtPYR1, AtPYL1 and AtPYL7 could not induce RD29B-LUC expression without ABA treatment. In contrast, AtPYL13 failed to induce RD29B-LUC expression with or without ABA treatment. These results indicated that ABA-independent PYL-like proteins exist in angiosperms.

AtPYL13 differs from the other AtPYLs by two amino acid residues, which correspond to Q38 in CL1 and F71 in CL2 in AtPYL13 (Fig. 3b). The key lysine residue that binds to the carboxyl group of ABA is replaced by glutamine in AtPYL13, and the key leucine residue that binds to the aliphatic portion of ABA is replaced by phenylalanine in AtPYL13 (Fig. 3b). To test whether these sequence variations may explain the ABA insensitivity of AtPYL13, we co-transfected SnRK2.6, ABF2 with wild-type and mutated AtPYL13 in pyl duodecuple mutant protoplasts. The wild-type AtPYL13 could not activate RD29B-LUC under ABA treatment, while the expression of AtPYL13-Q38K/F71L restored the ABA-dependent activation of RD29B-LUC in the pyl duodecuple mutant protoplasts (Fig. 3c), indicating that AtPYL13 is otherwise functional and that the two natural sequence variations in the CL1/CL2 domains confer ABA independence to AtPYL13. Phylogenetic analysis of Brassicaceae PYLs revealed a stepwise transition from ABA-enhanced receptors to ABA-independent paralogs after gene family expansion (Supplementary Fig. 3a; steps i–iii). This progression is evidenced by: (i) initial divergence of the AtPYL11/12/13 clade30 (node marked with a purple asterisk), which has only been identified in eudicots; (ii) secondary divergence of AtPYL13 (gold-highlighted branch) from the AtPYL11/12 subclade; (iii) subsequent acquisition of lineage-specific variations within the AtPYL13 subclade, including one in Camelina sativa and two in the Arabidopsis lineage (A. thaliana, A. lyrata, and A. halleri; variation sites labeled in red). These changes support the hypothesis that ABA-independent PYL-like proteins reemerged in the Brassicaceae family.

There are nine members of clade A PP2Cs in Arabidopsis, and the protoplast transient expression assays suggested that AtPYL13 cannot inhibit most, if not all, PP2Cs in the presence of ABA. We thus reconstituted the ABA signaling pathway in vitro, using the recombinant MBP-tagged SnRK2.6, GST-tagged ABI1 and PP2CA, and His-tagged AtPYL13. The SnRK2 activity was evaluated by in vitro kinase assay with (γ-32P)ATP (Fig. 3d, e), anti-phospho-S175-SnRK2s antibody (Supplementary Fig. 3b) that recognizes activated SnRK2s4, and PP2C enzymatic assay using pNPP as substrate (Supplementary Fig. 3c). The incubation of ABI1 or PP2CA with SnRK2.6 repressed SnRK2.6 activity. The repression of SnRK2.6 by PP2CA was released by wild-type AtPYL13 in the absence or presence of ABA, suggesting AtPYL13 constitutively represses PP2CA in an ABA-independent manner (Fig. 3e). In contrast, AtPYL13-Q38K/F71L repressed PP2C phosphatase activity and released PP2C-mediated inhibition of SnRK2 activity in an ABA-enhanced manner (Fig. 3d, e, and Supplementary Fig. 3b, c).

Due to the very weak phenotype of pyl13 mutants31, we then evaluated the properties of AtPYL13 in the pyr1pyl1pyl2pyl4 (pyr1-pyl124) quadruple mutant background. We generated transgenic Arabidopsis overexpressing either AtPYL13 or AtPYL13-Q38K/F71L driven by the constitutive 35S cauliflower mosaic virus (CaMV) promoter (Supplementary Fig. 3d). We found that ectopic expression of AtPYL13-Q38K/F71L complemented the ABA-hypersensitive phenotype of the pyl quadruple mutant during seedling establishment and subsequent growth (Fig. 3f, g). In contrast, ectopic expression of AtPYL13 did not complement the ABA-hypersensitive phenotype of the pyl quadruple mutant plants. Together with our previous data31,32, these results indicate that AtPYL13 has ABA-independent PP2C inhibitory activity and that the two natural amino acid variations in AtPYL13 confer ABA insensitivity.

Two invariant residues are crucial for PYL ABA receptors

To further address the effects of the noncanonical amino acid residues in AtPYLs on ABA dependence, mutated AtPYLs were generated and used in the transient activation assay in pyl duodecuple mutant protoplasts. The expression of 12 mutated AtPYLs, except AtPYL8-K61Q/L87F, did not activate RD29B-LUC expression (Fig. 3h). Expression of AtPYL8-K61Q/L87F enabled weak ABA-enhanced activation of RD29B-LUC expression, likely due to residual ABA dependence. While co-transfection of wild-type AtPYL8 with ABI1 enabled ABA-induced RD29B-LUC expression, co-transfection of AtPYL8-K61Q/L87F with ABI1 failed to induce expression even with ABA treatment. This suggests AtPYL8-K61Q/L87F lacks strong ABI1-inhibitory capacity. We then analyzed the ABA-binding properties of wild-type PYL9 and its K63Q/L89F mutant using ITC and found that this mutation abolished the ABA-binding affinity of PYL9 (Supplementary Fig. 3e, f). We further evaluated the ABA dependence of wild-type and mutated AtPYL9 with the reconstituted ABA signaling pathway in vitro. The incubation of ABI1 with SnRK2.6 repressed SnRK2.6 activity. The wild-type AtPYL9 blocked the phosphatase activity of ABI1 and HAB1 and released the repression of SnRK2 by PP2Cs in an ABA-dependent manner (Fig. 3i and Supplementary Fig. 3g–i). In contrast, AtPYL9-K63Q/L89F exhibited limited or no inhibition of ABI1 or HAB1 in an ABA-independent manner (Fig. 3i and Supplementary Fig. 3g–i). Moreover, the expression of native promoter-driven AtPYL1-K86Q/L114F did not complement the ABA-hyposensitive phenotype of the pyl quadruple mutant plants (Fig. 3j, k, Supplementary Fig. 3j). As a positive control, the expression of native promoter-driven AtPYL1 partially complemented the ABA-hyposensitive phenotype of the pyl quadruple mutant during seedling establishment (Fig. 3j, k). These results indicate that the invariant lysine and leucine residues are crucial for ABA dependence of plant PYL ABA receptors.

ABA-independent PYL-like proteins exist in seed plants

We further identified putative ABA-independent PYL-like proteins in seed plants according to natural variations in the key lysine and leucine residues (Fig. 4a and Supplementary Data 1). In addition to AtPYL13, several AtPYL13-like proteins differ from the ABA-dependent PYLs by one or two amino acid residues, which correspond to Q38 in CL1 and/or F71 in CL2 in AtPYL13 (Fig. 4b). The key lysine residue that binds to the carboxyl group of ABA is replaced by either glutamine in the Brassicaceae family (Arabidopsis lyrata, Camelina sativa, Capsella rubella) and in the genus Oryza, or by threonine in the genus Hordeum (Fig. 4b). Moreover, phenylalanine replaces the key leucine residue that binds to the aliphatic portion of ABA in PYLs in the genera Arabidopsis, Capsicum, Nicotiana, and Oryza (Fig. 4b). We evaluated the ABA dependence of these AtPYL13-like proteins in pyl duodecuple mutant protoplasts. Overexpression of PYLs from the genera Camelina (CsPYL13L1), Capsicum (CaPYL13L2), Nicotiana (NsPYL13L and NaPYL13L), and Hordeum (HvPYL13L1/2) could not induce expression of RD29B-LUC, while overexpression of PYLs from the genera Oryza (OsPYL12) and Capsella (CrPYL13L) constitutively activated expression of RD29B-LUC in an ABA-independent manner (Fig. 4c). These results indicated that these alterations are associated with ABA dependence of PYL proteins and suggested that the transformation from ABA receptors to ABA-independent PYL-like proteins occurred in seed plants after expansion of the gene family (Supplementary Fig. 3a and Supplementary Data 1).

According to the Klepikova Arabidopsis Atlas eFP browser33, AtPYL13 is specifically expressed in dry seeds but not in other tissues (Supplementary Fig. 4a). Next, we investigated the expression pattern of ABA-independent PYL-like genes in seed plants. According to the eFP browser on the Nicotiana attenuata Data Hub34, NaPYL13L (NIATv7_g33692) is highly expressed in Nicotiana attenuata seeds but not in other tissues (Supplementary Fig. 4b). The expression of NaPYL13L was dramatically reduced after seed imbibition and was undetectable after seedling establishment (Fig. 4d). In contrast, the expression of NaPYL4L (NIATv7_g37793) was undetectable in dry seeds but was elevated after seed imbibition, while NaPYL8L (NIATv7_g17046) was expressed in dry and imbibed seeds (Fig. 4d). In Camelina sativa, CsPYL13L2 was also highly expressed in seeds but not in other tissues, according to ePlant Camelina (Fig. 4e). Moreover, HvPYL13L (HORVU.MOREX.r3.3HG0247520) had the highest expression level in barley seeds compared to other tissues, according to RNA-Seq of 16 developmental stages of barley (PRJEB14349) (Fig. 4f). In rice, OsPYL12 was expressed in imbibed rice seeds and seedlings but not in dry seeds, while OsPYL13 was highly expressed in dry seeds with reduced expression after seed imbibition (Fig. 4g). The F76 variation in OsPYL12 (lavender) causes steric clash (atom distance <2 Å) with ABA, explaining its ABA independence (Fig. 4h). ITC analysis confirmed OsPYL12 cannot bind ABA (Supplementary Fig. 4c). These results indicated that most ABA-independent PYL-like genes are expressed in dry seeds, which differs from the expression pattern of ABA-dependent or ABA-enhanced PYLs (Fig. 4i).

ABI3 mediates seed-specific expression of AtPYL13

We evaluated the expression of the 14 Arabidopsis PYLs during seed development (Fig. 5a). Expression of AtPYL11, 12, and 13 was drastically elevated from 12 days after flowering and peaked at 15-18 days after flowering (Fig. 5a and Supplementary Fig. 5). On the other hand, the expression of PYR1 and PYL2/5/7/9 gradually elevated during seed maturation, although at levels that were orders of magnitude lower than that of AtPYL11/12/13, while expression of PYL1/3/4/6/8/10 gradually decreased during seed maturation (Fig. 5a). We further confirmed the seed-specific expression of AtPYL13 using transgenic lines expressing a β-glucuronidase-encoding gene driven by the native AtPYL13 promoter. GUS staining results confirmed that the AtPYL13 promoter was highly active in Arabidopsis seeds 15 days after flowering (Fig. 5b). These results indicate that AtPYL13 is a seed-specific ABA-independent PYL-like gene.

Analysis of the promoters of AtPYL11/12/13 and AtPYL13-like genes in Brassicaceae identified the presence of RY motifs and ABRE core motifs (Fig. 5c and Supplementary Fig. 5d). The transcription factor ABI3 directly binds to RY motifs via its B3 domain and may also indirectly interact with ABRE motifs35. We next investigated whether ABI3 controls the seed-specific expression of AtPYL13. The drastic elevation of AtPYL11/12/13 expression during seed maturation was nearly blocked in the abi3-6 mutant, and the gradual increases of AtPYL2/5/9 were also defective in the abi3-6 mutant (Fig. 5a and Supplementary Fig. 5). Moreover, the strong GUS staining of PYL13pro:GUS seen in dry seeds of the Col-0 WT background was dramatically reduced in the abi3-6 mutant background (Fig. 5d). To investigate binding between ABI3 and the AtPYL13 promoter, we performed yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) to test whether ABI3 could bind the RY motif-containing region in vitro. The GAL4 AD fusion of full-length ABI3 did not support yeast growth on synthetic dropout (SD)-Ura/Leu media, and the GAL4 AD fusion of ABI3 or ABI3-B3 did not support yeast growth on SD-Ura/Leu media supplemented with Aureobasidin A (AbA) (Fig. 5e). The R64K/R66K/P69S (KKS) substitutions in ABI3-B3 allow yeast growth in the Y1H system and enhance DNA binding without compromising sequence specificity36. These mutations (ABI3-B3-KKS) promoted yeast growth only with the PYL13 promoter fragment containing a wild-type RY motif but not with a mutated RY motif (Fig. 5e). Recombinant His-tagged full-length ABI3 bound to the Cyanine5 (Cy5)-labeled PYL13 promoter fragment containing the RY motif (Fig. 5f). Addition of wild-type unlabeled probes, but not unlabeled probes carrying a mutated RY motif, repressed the interaction between His-ABI3 and the Cy5-labeled PYL13 promoter fragment (Fig. 5f). Chromatin immunoprecipitation (ChIP)-qPCR assays using mature green seeds (about 15 days after flowering) of OE-ABI3-Flag plants revealed significant enrichment of fragments with the RY motif (Fig. 5g). These results indicated that the seed-specific expression of AtPYL13 is transcriptionally regulated by ABI3 (Fig. 5h).

Methods
Plant materials
The pyr1pyl124 quadruple and pyr1pyl12458379101112 duodecuple mutants used in the present study have been described previously (Park et al., 2009; Zhao et al., 2018). To generate 35Spro:AtPYL13 transgenic plants in the pyr1pyl124 quadruple mutant background, the coding sequences of AtPYL13 and AtPYL13-Q38K/F71L were cloned into a pGWB517 vector under the control of the 35S promoter, after which the construct was transformed into the pyr1pyl124 quadruple mutant.
To generate AtPYL1pro:AtPYL1 transgenic plants in pyr1pyl124 quadruple mutant background, the coding sequences of AtPYL1 and AtPYL1-K86Q/L114F were fused to the AtPYL1 promoter and cloned into pGWB513, which was then transformed into the pyr1pyl124 quadruple mutant. To generate PYL13pro:GUS transgenic plants, the GUS gene was driven by a −2000 to −1 bp fragment of PYL13 promoter containing RY motif. The PYL13pro:GUS transgenic plant in the abi3-6 mutant background was generated by a genetic cross and subsequent genotyping. Two-way ANOVA, *p < 0.05, **p < 0.01, ****p < 0.0001.

Phenotypic analysis
Arabidopsis seeds were collected from wild type Col-0, the pyr1pyl124 quadruple mutant, and T2 or T3 generation transgenic Arabidopsis plants grown under the same well-watered conditions, after which they were allowed to dry for at least 2 weeks, surface sterilized and then sown onto 1/2 MS plates supplemented with or without 3 µM ABA. After stratification in the dark at 4°C for 3 days, the seeds were grown horizontally in a Percival CU36L4 growth chamber. Seedling establishment was scored as the percentage of seeds with green expanded cotyledons on the 12th day. Seedling establishment images were collected on the 15th day.

Multiple sequence alignment and phylogenetic analysis
Multiple sequence alignment and phylogenetic analysis were conducted using MEGA12 software, with the ClustalW and maximum likelihood method and the Jones-Taylor-Thornton model of amino acid substitutions.

Transient expression assay in Arabidopsis protoplasts
The putative ABA-irresponsive PYL homologs from bacteria, algae, and land plants were synthesized and cloned into the pHBT95 vector. All plasmids were confirmed by sequencing and purified with the QIAGEN Plasmid Maxi Kit.
The pyr1pyl12458379101112 duodecuple mutant was grown under short-day conditions and high humidity (10 h of light/14 h of darkness, 80% humidity). Rosette leaves of 4-week-old plants were used to generate protoplasts, as previously reported (Fujii et al., 2009; Zhao et al., 2013). Protoplast expression constructs, including pHBT95-PYL, -ABI1, -SnRK2.6, -ABF2, RD29B-LUC, and ZmUBQ-GUS constructs, were the same as previously reported (Fujii et al., 2009; Zhao et al., 2013). All mutants were generated via two-step PCR and verified by plasmid sequencing. Assays for transient expression in protoplasts were performed as described previously (Fujii et al., 2009; Zhao et al., 2013), and all steps were performed at room temperature. For each transform, 6 µg RD29B-LUC, 3 µg ABF2, 3 µg SnRK2.6 and 0.5 µg ZmUBQ-GUS were added as essential control. Besides, none or 3 µg of different candidate PYL was added as an effector to test ABA-receptor identities. After transfection, the protoplasts were washed twice using W5 buffer and incubated in WI solution without ABA or with 5 µM ABA under light for 16 h.
Introduction of PYL receptors is sufficient to recover ABA response in the pyl duodecuple protoplast transient assay, while addition of SnRK2 and ABF supports robust activation of luciferase signals in the presence of PYL receptors. Therefore, we co-expressed SnRK2 and ABF to evaluate the ABA-responsiveness of PYLs in the pyl duodecuple protoplast transient assay.

Yeast two-hybrid (Y2H) assay
Yeast two-hybrid assays were performed using the Matchmaker GAL4 Two-Hybrid System from Clontech, following the manufacturer’s instructions. The yeast strain Y2H Gold was co-transformed with paired constructs and then plated on selective dropout nutrition-deficient media containing or lacking 10 µM ABA.

Firefly luciferase complementation imaging (LCI) assay
The full-length coding sequences of PrPYL and MeenPYL3 were cloned into the pCAMBIA-35S-nLUC, while the coding sequences of ABI1, HAI1, HAI3, HAB2, and PP2CA were amplified by PCR and cloned into the pCAMBIA-35S-cLUC. LCI assay was performed by transient expression of paired combinations of constructs in Nicotiana benthamiana leaves through Agrobacterium-mediated infiltration. After infiltration, tobacco plants were grown in the dark for one day and under white light for another 2 days. Luciferase activity was detected with a CCD Tanon-5200 by applying firefly D-luciferin (NanoLight).

Protein expression and purification from E. coli
The coding sequences of PrPYL, ZcPYL8, MeenPYL3, AtPYR1, AtPYL9 and AtPYL13 were amplified and cloned into the pET28a vector to obtain His-PrPYL, His-ZcPYL8, His-MeenPYL3, His-AtPYR1, His-AtPYL9, and His-AtPYL13 constructs. All mutants were generated via two-step PCR and verified by plasmid sequencing. The ABI1, PP2CA, and HAB1 coding sequences were amplified and cloned into the pGEX-6P-1 vector to obtain the GST-ABI1, GST-PP2CA, and GST-HAB1 constructs. MBP-SnRK2.6 was the same as before46.
The wild-type or mutated His-PYLs, GST-PP2Cs and MBP-SnRK2.6 constructs were transformed into the competent cells of E. coli BL21(DE3). Expression of proteins was induced by 1 mM isopropyl β-D-thiogalactoside (IPTG) at 18°C for 16 h. To purify His-tag-fusion proteins, cells were harvested and sonicated in lysis buffer containing 50 mM NaHPO4, 300 mM NaCl, pH 8.0 on ice using a Branson SFX250 Sonifier (50% duty, output 5, 2 min, three times). After centrifuging the lysate at 20,000 × g at 4°C for 40 min, the recombinant proteins were purified from the supernatant with the corresponding agarose beads according to the protocols. After washing three times with the washing buffer (50 mM NaHPO4, 300 mM NaCl, 50 mM imidazole, pH 8.0), the recombinant proteins were eluted with elution buffer containing 50 mM NaHPO4, 300 mM NaCl, 250 mM imidazole pH 8.0, and frozen at −80°C. The lysis buffer, wash buffer and elution buffer were altered according to the suggested protocols for MBP- and GST-tagged proteins. It is worth noting that after purification, wild-type or mutated AtPYL13, ZcPYL8, and MeenPYL3 proteins must be used fresh immediately for subsequent assays due to their extreme instability.

In vitro kinase assays
The purified recombinant wild-type and mutated PYL proteins were incubated with GST-tagged PP2Cs and MBP-SnRK2.6 protein kinase at 30°C for 2 h in 25 μL of reaction buffer containing 20 mM Tris-HCl, pH 8.0, 1 mM DTT, 5 mM MgCl2, 1 μM ATP and 5 μCi [γ-32P]ATP, with or without 100 μM ABA. The reaction was stopped by adding 4× SDS loading buffer, and proteins were separated by a 12.5% SDS-PAGE gel. The phosphorylation signals were detected by a Personal Molecular Imager (Bio-Rad). Coomassie Brilliant Blue (CBB) staining was used as a loading control.
SnRK2 kinase activity was also evaluated using the anti-phospho-S175-SnRK2s antibody4. Different combinations of His-PYLs, GST-PP2Cs, and MBP-SnRK2.6 were incubated in 25 μl of reaction buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 0.25 mM DTT, 1 μM ATP) for 2 hours. Afterward, samples were boiled with protein SDS loading buffer and separated by a 10%/12.5% gradient SDS-PAGE gel. The anti-phospho-S175-SnRK2s antibody (ABclonal, AP1481, 1:3000 dilution) was used to detect SnRK2 kinase activity. Anti-His antibody (ABclonal, AE086, 1:5000 dilution) was used to detect His-tagged PYL, while anti-MBP antibody (Abmart, M20051M, 1:5000 dilution) was used to quantify MBP-tagged SnRK2.6.

PYL-mediated PP2C enzyme activity assay
The coding sequences of PYLs (PrPYL, ZcPYL8, MeenPYL3, PYR1, and PYL9) were cloned into pET28/32a vectors to generate 6× His-tagged PYLs. The coding sequences of PP2Cs (ABI1 and PP2CA) were cloned into pGEX-6P-1 vector to generate GST-tagged ABI1 and PP2CA. The coding sequence of HAB1 171-end was cloned into pMAL vector to generate MBP-tagged HAB1 171-end.
Initial reaction velocities for GST-PP2Cs were measured using the synthetic phosphatase substrate pNPP. Reactions contained 0.5 μM PP2Cs (GST-ABI1, GST-PP2CA, or MBP-HAB1 171-end), with 0, 0.5, 1.0, or 2.0 μM 6× His-PYLs (PrPYL, ZcPYL8, MeenPYL3, PYR1, or PYL9). Reaction buffer consists of 33 mM Tris-OAc (pH 7.9), 66 mM KOAc, 0.1% BSA, 25 mM Mg(OAc)2, and 10 μM (+)-ABA. After 20 minutes, 50 mM pNPP was added to initiate reactions. Hydrolysis of pNPP was monitored at A405 using a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices). PP2C activity in the absence of receptors was defined as 100% activity.

Structure prediction and alignment
The structures of PrPYL, MeenPYL3, and OsPYL13 with ABA were predicted using alphafold3 (https://alphafoldserver.com/). The ligand (ABA) binding pocket was determined by presenting surrounding residues within a 5 Å radius. The molecular visualization, alignment, and ligand-protein interaction analysis were performed using Pymol (https://pymol.org/2/).

RNA extraction and quantitative RT-PCR
The total RNA of seeds or seedlings was extracted using FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics-rich, Vazyme, RC401-01) according to the manufacturer’s protocols. Reverse transcription was carried out using HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper, Vazyme, R233-01). Expression levels were quantified using 2× Universal SYBR Green Fast qPCR Mix (ABclonal, RK21203). The constitutively expressed AtPP2AA3 or NaIF5a was used as an internal inference in Arabidopsis thaliana or Nicotiana attenuate, respectively.

Histochemical GUS staining for seeds
GUS reporter transgenic seeds were soaked in water for 15 minutes. The seed coats and endosperm were carefully removed with fine forceps under a stereomicroscope. The embryos were extracted and incubated in a GUS substrate staining solution (O’BioLab, S102) at 37°C for ~4 hours. Following incubation, the samples were decolorized with 75% and 95% ethanol and water, subsequently at 37°C and finally imaged by a Stemi 305 stereomicroscope (Zeiss).

Yeast one-hybrid (Y1H) assay
Yeast one-hybrid assays were conducted using the Yeast One-Hybrid Library Screening System (Clontech), following the manufacturer’s instructions. Three tandem copies of the 16 nt fragment containing RY or mutated RY motif from the PYL13 promoter were synthesized and ligated into the pAbAi vector. The RY and mRY reporter yeast strains were generated by homologous integration to Y1H Gold and selected by growth on SD/-Ura plates. Subsequently, the minimal inhibitory concentration of Aureobasidin A (AbA) for each bait strain was determined, showing 1 µg/mL for RY or 0.2 µg/mL for mRY reporter yeast strain. The pGADT7-empty, ABI3-B3, and ABI3-B3-KKS plasmids were transformed into the corresponding generated bait-strain competent cells. Dilutions (10−1 and 10−2) of the same OD liquid suspension of yeast cells were cultured onto the plates and incubated at 30°C for 2–4 days. The interaction was determined by growth on SD/-Trp/-Leu solid medium with indicated concentrations of AbA.

Electrophoretic mobility shift assay
The full-length ABI3 coding sequence with stop codon was amplified and cloned into pET28a, then introduced into E. coli BL21(DE3). His-ABI3 protein was expressed and purified following the manufacturer’s manual for Ni-NTA Agarose (QIAGEN). The synthetic complementary oligonucleotides of PYL13 (or mPYL13 with mutated RY motif) were annealed and cloned into the T-vector to generate the probes. The labeled probes were amplified using Cy5-labeled T7 primer and M13R, while the unlabeled probes were amplified using the normal T7 primer and M13R. The purified proteins were then incubated with the Cy5-labeled probe at room temperature for 20 min in cold EMSA buffer (20 mM Tris-acetate, pH 8.3, 5% glycerol, 0.5 mM EDTA, and 0.05 mg/mL BSA). After incubation, the reaction mixture was electrophoresed on an 8% native polyacrylamide gel with running buffer (40 mM Tris-acetate, pH 8.3) and then imaged with an Amersham Typhoon imager (GE Healthcare). Note that the mutated variants used in the EMSA and Y1H assay differ. Relevant details of oligonucleotides can be checked in the Primer list.

ChIP-qPCR assays
ChIP assays were performed using 2 g seeds (about 15 days after flowering) from Col-0 and OE-ABI3-Flag. Samples were fixed with 1% formaldehyde. Nuclei were isolated using isolation buffer (1 M sucrose, 10 mM HEPES pH 8.0, 5 mM MgCl2, 5 mM KCl, 0.6% Triton X-100, 0.4 mM PMSF, protease inhibitor cocktail), filtered through Miracloth, washed with CHIP buffer (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1 mM EDTA, 5 mM β-mercaptoethanol, protease inhibitor cocktail), and pelleted by centrifugation. Nuclei were resuspended in 300 μl lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, protease inhibitor cocktail) and sonicated with a Bioruptor (Diagenode). The nuclei lysates were incubated overnight with anti-Flag antibody (Sigma-Aldrich, #F1804, 1:100 dilution), followed by 2-h incubation with Protein G beads (Invitrogen). The precipitated protein–DNA mixtures were washed with low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% SDS, 1% Triton X-100, 2 mM EDTA), high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% SDS, 1% Triton X-100, 2 mM EDTA), LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% NP-40, 1% Sodium Deoxycholate, 1 mM EDTA) and TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). DNA was eluted from beads with elution buffer (1% SDS, 0.1 M NaHCO3) at 65°C. The DNA was recovered after reverse cross-linking and proteinase K treatment. Enriched DNA fragments were analyzed by qPCR using the indicated primers (Table S1), with TUB8 serving as a negative control.

ITC assays
ITC experiments were performed with an ITC200 Microcalorimeter (MicroCal). All the ligands and proteins were in the same reaction buffer consisting of 20 mM HEPES (pH 7.5) and 150 mM NaCl. The ligand 1 mM (+)-ABA was titrated against 50 µM PrPYL, MeenPYL3, PYL9 or PYL9-K63Q/L89F proteins. The ligand 2 mM (+)-ABA was titrated against 10 µM OsPYL12 proteins. The stoichiometry between the ligands and proteins was set to 1 for all analyses. Data fitting was performed by using the ORIGIN™ software supplied with the instrument. Thermodynamic constants were determined by Origin software.

Statistical analysis
The ImageJ software was used to analyze the relative band intensity during in vitro kinase assays. Statistical analysis was examined using Student’s t test or two-way ANOVA with Tukey’s multiple comparisons test using the Graph Pad Prism 9 software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study presents an intriguing hypothesis regarding the evolutionary origins of PYL genes, proposing to provide experimental evidence that suggests a bacterial PYL homolog that was transmitted to the plant lineage. However, the central claims of the paper, particularly as stated in the title, are not adequately supported by the presented data. Most of the experimental work essentially reproduces findings from previous studies rather than providing novel insights. Furthermore, the manuscript suffers from substantial clarity issues, with unsupported or misinterpreted claims in the introduction and a presentation of results that obfuscates key conclusions (see specific examples below).\n\nMajor Concerns\nQuery #1:\nFunctional Characterization of a Bacterial PYL Homolog\nA central premise of this study is the identification of a bacterial PYL homolog with ABA-independent activity. However, the assays used fail to assess this function rigorously. The experimental systems either contain endogenous ABA (as in plant-based systems) or rely on overexpression, which can artificially drive protein-protein interactions independent of ligand binding. Notably, even well-characterized ABA-dependent receptors, such as PYL4, exhibit ABA-independent PP2C binding under similar conditions. A more appropriate in vitro approach, where protein concentrations are strictly controlled and ABA is entirely absent, is required to definitively assess ligand dependency.\n\nQuery #2:\nHorizontal Gene Transfer Hypothesis Lacks Supporting Evidence\nThe claim of horizontal gene transfer (HGT) is presented without substantive support. Establishing HGT in a system that diverged over 600 million years ago is inherently difficult. Yet, the authors fail to explore/roll out alternative evolutionary scenarios, such as convergent evolution, which remain equally plausible. A robust phylogenetic analysis should provide statistical confidence measures and exclude other evolutionary possibilities. If the phylogenetic tree showed plant sequences embedded within bacterial lineages, it could suggest recent gene transfer, but the current analysis lacks the necessary rigor. The arbitrary nature of the sequence relationships as presented does not substantiate the proposed HGT event. While the reviewer is not a bioinformatician, the absence of statistical validation and failure to test alternative hypotheses significantly weaken this claim.\n\nQuery #3:\nSpecific examples:\nIn the title “….in the seeds of land plants”\nThe relations described in the study extend beyond seed plants, as the PYL gene family is conserved across the entire lineage of land plants, not just in seed-bearing species. Also the title should explicitly state which functional aspect of PYL receptors has been retained throughout evolution—specifically, their ability to bind PP2C phosphatases.\n\nQuery #4:\nFurthermore, the claim of horizontal gene transfer (HGT) remains a hypothesis rather than a demonstrated/conclusion. As such, it should either be removed from the title or clearly framed as a hypothesis rather than a definitive statement. The current phrasing in the title risks misleading readers by presenting an unverified assumption as an established finding.\n\nQuery #5:\n-Abstract: Has inaccuracies\nExamples: Authors claim: “which requires constitutive activation of stress signaling, was mainly adopted by early land plants and persists in seeds.”\nThis statement is incorrect—ABA levels are crucial for seed dormancy and decrease to allow germination. During dormancy, ABA levels remain high to prevent premature germination. However, for germination to occur, ABA levels must decline (while gibberellins increase).\n\nQuery #6:\nAuthors claim: “Through evolution, the PYR/PYL/RCAR family (PYL) of ABA receptors gained several functions over the course of evolution, namely inhibition of protein phosphatases (PP2Cs), ABA-enhanced inhibition of PP2Cs, and ABA dependent inhibition of PP2Cs”\nThe only well-established physiological function of PYL receptors is their ability to inhibit PP2C phosphatases, which are central to ABA signaling. If the authors intended to suggest an additional function, they should clarify it and provide supporting evidence.\n\nQuery #7:\nAuthors claim: “However, it remains unclear how the ABA-irresponsive PYLs originated and whether they exist in land plant. “\nThis statement is incorrect—it has been shown that PYL receptors in land plants likely originated from algal ancestors, and homologous proteins are present across all land plant lineages. This suggests that the PYL family is not exclusive to land plants but instead evolved from pre-existing proteins in algae before being co-opted into the ABA signaling pathway in terrestrial plants. See doi.org/10.1073/pnas.1914480116\n\nQuery #8:\nAuthors claim: “AtPYL13 and AtPYL13-like genes were highly expressed in dicot seeds, suggesting the transformation from ABA receptors to ABA-irresponsive PYLs after expansion of the gene family”.\nThis statement misrepresents the actual expression profile of PYL in Arabidopsis seeds. While PYL13 expression can be detected, public datasets (e.g., BAR ePlant) indicate that its expression is approximately 100-fold lower than PYR1, suggesting it plays a minor role in seed germination. Additionally, experimental evidence challenges the significance of PYL13 in this context. For example, activating PYR1 alone, using a specific agonist, was sufficient to inhibit germination. Dimeric multiple mutants exhibit an early germination phenotype, further suggesting that the primary regulators of germination are other PYL receptors. See Park 2009 DOI: 10.1126/science.1173041 and Okamoto 2011 https://doi.org/10.1073/pnas.1305919110\nGiven these findings, if PYL13 has any role in germination, it is likely minimal or redundant, and its functional relevance in this process remains questionable.\n\nQuery #9:\n-Introduction- Has inaccuracies\nSome Examples:\nAuthors claim: “Terrestrialization is a pivotal event in the evolution of green plants, which requires mechanisms to survive under drought stress.”\nThe term \"drought stress\" is not appropriate to describe the primary challenges of terrestrialization (refers to the evolutionary transition from an aquatic to a terrestrial environment). Terrestrialization involves adaptation to a gradual shift from consistently wet conditions to environments with fluctuating water availability rather than exposure to extreme drought. Using \"drought stress\" oversimplifies the complexity of this transition, which encompasses not only water limitation but also factors such as desiccation tolerance, gas exchange regulation, light intensity, and structural adaptations to terrestrial life.\n\nQuery #10:\nAuthors claim: “Plants mainly utilize two strategies to survive under water deficiency: “drying without dying” or “without drying”. “\nThese terms are not clear.\n\nQuery #11:\nAuthors claim: “The early land plants are mainly poikilohydric and adopt the “drying without dying” strategy, which requires constitutive activation of stress signaling and enables plants to get through extreme conditions” –\nEarly land plants possess mechanisms for water regulation and stress adaptation that are independent of vascular systems, as described in the cited paper. A more relevant and evolutionarily conserved mechanism is osmoregulation, which is shared between mosses and seed plants.\nRegarding the statement “PP2Cs can be ABA-dependent or ABA-enhanced”, this terminology is unclear. PP2C inhibition occurs through both ABA-dependent and ABA-independent mechanisms, and the distinction between \"ABA-enhanced\" and \"ABA-dependent\" needs to be explicitly defined to avoid confusion.\n\nQuery #12:\nAuthors claim: “The entirely ABA dependent dimeric PYLs emerged in angiosperms and have nearly no basal activity”-\nThis applies to other PYLs as well— not just dimeric forms. This should be considered as it's not the only mechanism of low basal activity (in Arabidopsis and other species), as indicated in DOI: 10.1016/j.cub.2024.12.043.\"\n\nQuery #13:\nAuthors claim: “which enables angiosperms to have a wider range of ABA responses and improved balance between growth and stress responses”-\nThis is an assumption and should be stated as such.\n\nQuery #14:\nAuthors claim: “However, it remains unclear how the ABA-irresponsive PYLs originated.”\nThe focus should be more specific—there is evidence that land plant lineages and the common ancestor of Zygnema circumcarinatum possessed a nonresponsive PYL. What remains unclear is how this protein was ‘adopted’ into the plant lineage.\"\n\nQuery #15:\n-Specific points in the result section:\nThe section titled: “The bacterial PYL-homolog from Paraburkholderia rhynchosiae possesses PP2C-binding ability”\nAuthors claim: “It is proposed that the ancestral PYLs in plants were gained through horizontal gene transfer from soil bacteria based on phylogenetic analyses (Fig. 1a).” –\nThe phylogenetic analysis does not support horizontal gene transfer occurring over 600 million years ago.\n\nQuery #16:\nAuthors claim: “Despite lacking ABA responsiveness”\nThis should be tested in vitro using assays that directly assess the involvement of ABA as a ligand, such as a PP2C inhibition assay, isothermal titration calorimetry, or any equivalent assay.\nThe authors identified the interaction between bacterial PrPYL and PP2C via a yeast two-hybrid assay. However, this interaction was not reproducible in planta nor when SnRK2 competed for the PP2C interface in an in vitro assay. Based on this, the authors concluded that there is an interaction with PP2C but no PP2C inhibition. As stated, 'These results suggested that the bacterial PrPYL possesses PP2C-binding but not PP2C inhibitory activity.' This conclusion is flawed for two reasons: 1. The interaction between PYL and PP2C typically blocks the active site of the PP2C enzyme. Therefore, a 'canonical interaction' should, by default, result also in inhibition. 2. To properly claim this, the interaction and PP2C activity must be evaluated through specific assays, not as a default by a negative result. At this point, it is an unsubstantiated guess.\nAn alternative explanation could be that, in yeast, the protein steady-state concentration is high, allowing for the monitoring of low-affinity interactions. In vitro and in-planta, however, are more challenging, as other proteins could compete for the PP2C interface. Consequently, it is unsurprising that the weak interaction with bacterial PrPYL is outcompeted by SnRK2s (whether from plants or purified protein), resulting in the absence of an interaction signal.\"\n\nQuery #17:\nThe section titled “ABA-irresponsive PYLs exhibit PP2C inhibitory activity in Zygnematales”.\nSun et al. (2019) showed that algal PYL interacts with PP2C in an ABA-independent manner. In that study, Sun also demonstrated that ZcPYL8 cannot bind ABA (by isothermal calorimetry titration). In this current study, the authors aimed to test additional algal PYLs. They used an Arabidopsis PYL mutant with endogenous ABA levels, which were unsuitable for testing ABA-independent activity. The SnRK activity (following release) assay is sensitive to PYLs with basal ABA-independent activity but cannot distinguish if there is a mild ABA effect. These two assays do not support the claim that the 'PYL homologs gained ABA-independent PP2C inhibitory activity during the evolution of Zygnematales.' In vitro assays that directly test ABA binding would be better suited to scratch the surface of this hypothesis. I suggest starting by following the biochemical assays performed by Sun in 2019.\n\nQuery #18:\nThe section titled “An ABA-irresponsive PYL exists in Arabidopsis”\nThe authors claimed that “the wild-type AtPYL13 could not activate RD29B-LUC with ABA treatment, while the expression of AtPYL13- 204 Q38K/F71L restored the ABA-dependent activation of RD29B-LUC in the pyl duodecuple mutant protoplasts (Fig. 3c), indicating that AtPYL13 is otherwise functional and that the two natural sequence variations in the CL1/Cl2 domains of AtPYL13 confer ABA irresponsiveness”\nA paper from the Grill group addresses PYL13 activity and the specific mutation analyzed here: https://doi.org/10.1073/pnas.1322085111. These mutations were thoroughly tested in that study. I failed to see the added value of repeating them here.\"\n\nQuery #19:\nThe sections titled “Two invariant residues are crucial for PYL ABA receptors.”, “ABA-irresponsive PYLs exist in seed plants” and “ABI3 mediates seed-specific expression of AtPYL13”\nI don’t understand the rationale behind the ectopic expression of impaired-active mutated PYLs. What can be learned from these experiments? This entire section seems unrelated to the main focus of the study. Furthermore, it is well-established that dimeric PYLs are key players in seed germination biology. This has been tested through agonist activation of specific PYLs and by examining expression levels (actual reads versus the relative expression tested here). In fact, PYL13 expression is barely detectable.\"", "reviewer_2": "Lu et al. report the evolutionary trajectory of PYL ABA receptors. Based on phylogenetic and functional analyses, they found that an ABA-irresponsive PYL in soil bacteria was horizontally transferred from soil bacteria, gained PP2C interaction in Paraburkholderia rhynchosiae, became capable of inhibiting PP2C in the common ancestor of Zygnematales and land plants, evolved into ABA receptors in liverworts, and then diversified to possess various affinities to ABA, including an irresponsive receptor exclusively expressed in seeds.\n\nThe presented work attracts a wide range of biologists who study protein evolution and hormone signaling. The work is comprehensive and based on solid functional analysis. The following are my comments to improve the manuscript:\n[1] The title needs to be reconsidered:\n(1) \"Horizontally transferred\" typically refers to genes, not proteins.\n(2) I would think that the ABA-irresponsive receptors that was horizontally transferred from soil bacteria and retained functions in seeds are qualitatively distinct. PrPYL is a prototype of ABA receptor, while AtPYL13 is a variant of ABA receptor. The current title sounds continuous ABA-irresponsive receptors.\n(3) Related to this, the overall context appears to have two distinct parts. I recommend the authors revise the text to create a cohesive narrative.\n[2] Co-evolution of PYL and PP2C:\nThe authors examined the function of ancient PYLs and their interaction with Arabidopsis PP2Cs. The evolution of PYLs during this period heavily depends on the nature of PP2C. The evolution/conservation of required amino acid residues in PP2Cs should be discussed. Ideally, PP2C residues required for PYL interaction or inhibition can be separately discussed. If possible. This is a core part of the story.\n[3] ABA-irresponsive PYLs:\nThe functionality of ABA receptors or PYLs can be assigned by their ability to inhibit PP2C and their ABA dependency, regardless of whether they act as monomers or dimers. ABA receptors evolved as PYLs with ABA-enhanced PP2C inhibition. The present work demonstrated that ABA-irresponsive PYLs constitutively inhibit some PP2Cs, but their PP2C inhibition activity is not quantitatively compared with other receptors. The ability to induce downstream events (or inhibit PP2C activity) among PYL members is a critical aspect of their functionality, as they function in the same cells with other ABA-responsive receptors in vivo. This makes it difficult for readers to assess their role in vivo. The phenotype was primarily assessed by overexpressors. In vitro SnRK2 activation was quantified, but this quantification does not provide the degree of strength among family members. The authors discussed, “Although the pyl duodecuple mutant is viable and fertile, we cannot get any seeds for the pyl quattuordecuple mutant that has further mutated AtPYL6 and AtPYL13, supporting the critical role of AtPYL13 in fertility and seed maturation.” The multigenic mutant phenotypes are the most critical data to assess the role of AtPYL13 among family members, especially in seeds where it is exclusively expressed. I encourage the authors to describe the multigenic mutant phenotypes in more detail (I suggest that the overexpressor phenotype be included in the supplemental materials).\nRelated to the above, I am curious if the two critical residues (Q and F) in ABA-irresponsive PYLs are only characteristic conservations of PYL13-like proteins.", "reviewer_3": "The manuscript by Lu et al. reported an evolutionary model of ABA-irresponsive PYL-like proteins and proposed their roles in desiccation tolerance of angiosperms seeds. They demonstrated in vitro that a bacterial PYL-like protein can interact with but not inhibit the activity of Arabidopsis ABI1 in an ABA-independent manner, supporting a previous idea that plant PYL family is originated from soil bacteria. They also performed the same experiment set against PYL-like genes from Zygnematophyceae, showing that algal PYLs have the binding and inhibitory function against Arabidopsis PP2Cs in an ABA-independent manner. They focused on two important amino acids located at conserved loops 1 and 2 which are not conserved in ABA-irresponsive PYLs. The two amino acids were also not conserved in seed-specific ABA-irresponsive AtPYL13 and substitution of these amino acids to that of ABA-responsive PYLs were sufficient to confer ABA-responsiveness. They further proposed that AtPYL13 was under the regulation of seed-specific ABI3 transcription factor, which is essential for the acquisition of seed desiccation tolerance.\n\nIt is a potentially interesting research paper to give insights into the evolution of PYLs and plant desiccation tolerance; however, the experimental evidence was not sufficient support their claims. Moreover, I recommend the authors should discuss with an expert of evolutionarily research since the manuscript contains many flaws in evolutionary aspect. For example, “PYLs then gained ABA-binding and -responsive activities in liverworts,”. Liverworts are extant plant group and not ancestor of land plants. My major concern is that there is no in vivo experimental data showing that PYL13 is directly regulated by ABI3 and plays an essential role like ABI3 in seed desiccation tolerance. I was also confused about the “ABA-irresponsive” PYLs. What are the criteria for this word? For example, MeenPYL1/2 have no capacity but MeenPYL3 does has the capacity to activate RD29B-LUC in pyl protoplasts with or without ABA. These are all ABA-irresponsive PYLs? Probably it should be, since the authors describe AtPYL13 as ABA-irresponsive, which cannot activate RD29B-LUC in pyl protoplasts like MeenPYL1/2. Is ABA-irresponsive PYL re-evolved in angiosperms? What are the differences between ABA-irresponsive algal PYLs and ABA-irresponsive angiosperms PYLs.\n\nComments\nL88- and Fig.1a. What is the reason to add SnRK2 and ABF in the pyl protoplast transient assay? Was introduction of AtPYL(s) not sufficient to recover ABA response?\nL96- This experiment should include a positive control of ABA-dependent PYL.\nL121- and Fig. 1k. This is also an extant bacterium and not a doner of PYL, therefor, misleading. The authors did not show that the target of bacterial PYLs is PP2Cs, and Fig. 1k should be deleted or revised.\nL122- The Figure layout should be arranged according to the order in the main text. There is no mention about Fig. 2a before Fig.2b.\nL155- Fig. 2e,f need quantification.\nL162- Is this Ser the same with Ser112 described in L115? The numbers do not match each other.\nL178- Reduction of high basal activity should be supported by an appropriate statistical analysis.\nL180-182 The description is not clear.\nL181- What does “original land plants” mean?\nL184- “Zygnematales” are extant plants.\nL185- I mentioned above.\nL186 and L196 The authors should describe the PYLs subfamilies with their function in the introduction.\nL191- There is no data for AtPYL7. What is the criterion of “basal activity”? Is there any significant difference between AtPYR1 and AtPYL9 autoactivation?\nL193- Please describe more about the “stepwise transformation”. For me, it is not evident from the figures. It will be helpful to indicate where transformation occurred on the phylogenic tree.\nL213- Which is not evident from Fig. 3c, as the authors described. Please explain why AtPYL13 failed to activate RD29B promoter. Is PP2CA seed-specific factor?\nL217- No PYR1 data in supplementary Fig.3b.\nL234-236 Please explain what does this result indicate or suggest.\nL249-250 Please clearly explain which are the putative ABA-irresponsive PYLs in Supplementary Data 1.\nL280- Why the authors used OsPYL13 for structure prediction? There is no functional data for OsPYL13 (Fig. 4c).\nL309-310 Please explain why are these mutations required in this experiment briefly.", "reviewer_4": "Abscisic Acid (ABA) is arguably one of the most important biomolecules on the planet, as a consequence of its central importance in promoting plant dehydration tolerance: a state essential to the initial plant colonization of land ca. 500Ma that had planetary consequences: the photosynthetic activity of earliest land plants resulted in elevation of the oxygen content of the atmosphere to a level enabling the evolution of large land animals.\n\nABA, a small molecule derived from the breakdown of carotenoids and ubiquitous in Nature, acts as a ligand to the multimember family of ABA receptors (“PYLs”). These receptors are START-domain proteins that are structurally modified by ABA-binding so that they form molecular complexes with a sub-family of PP2C protein phosphatases blocking their dephosphorylation activity. This activates SnRK2 protein kinases that direct transcription factors to drive the expression of genes encoding a plethora of protective gene products that enables plants to survive dehydration.\n\nThis manuscript describes the functional evolution of the PYL receptor family that is thought to have been acquired by horizontal gene transfer from microbe(s) to an ancestral alga from which all land plants subsequently diverged and proliferated.\n\nThe paper comprises:\n1. Examination of receptor-like functions of bacterial PYL homologs:\n(i) Does a bacterial PYL-like gene complement the Arabidopsis 12x pyl- line?\n- No: there is no activation of the Rd29b reporter gene either with or without added ABA\n(ii) Does a bacterial PYL-like gene bind to an Arabidopsis PP2C\n- Yes, but only one of the bacterial proteins tested showed binding in Yeast-2-Hybrid and split luciferase assays.\n(Paraburkholderia rhynchosiae “Pr”)\n(iii) Does the PrPYL activate SnRK2 phosphorylation in vitro?\n- No. The bacteria tested were those identified in Reference 11 (which focused on algal progenitors in land plant evolution, but indicated these species to be taxonomically related potential HGT progenitors of plant PYL genes). Of these\nParaburkholderia remains the leading contender.\n(iv) Can structural modeling indicate whether PrPYL could interact with a plant PP2C protein.\n- Up to a point: there are similarities between the modeled PrPYL structure and that of the crystallographically determined\nArabidopsis PYL-ABA-PP2C co-complex, but the predictions indicate structural inconsistencies that would preclude ABA-interaction (not least, the lack of a “gate” motif in the bacterial sequence)\n\nI think that these essentially negative findings are not unexpected, and one would not expect PYL homolog within an extant bacterium that has been independently evolving for the last 500 million years to necessarily be similar to whichever microbial sequence actually might have been passed into the genome of the algal ancestor of today’s land plants. But it is important that this has now been experimentally tested. While comparative genomics has been a powerful tool in developing our understanding of land plant evolution, there will always be some some uncertainty in making accurate predictions.\n\n2. Do algal PYL homologs within the Zygnematales have properties similar to angiosperm PYLs in activating ABA-mediated gene expression?\n- Yes, but not all the properties of PYLs in modern land plants. PYL function has now been analysed extensively in angiosperms, and also to a lesser extent in early-diverging land plants (Bryophytes: Ref 12 and in a very recently published paper (Zimran et al. 2025. Current Biology 24: 818-830).\n\nThe results reported here showing ABA-independent “basal” activity in PYL-PP2C interactions are consistent with these recent papers. Whereas these two papers use direct analysis of PP2C inhibition in vitro, this manuscript nicely reinforces the evidence by showing PYL-binding-specific activation of SnRK2 kinase activity being promoted in an ABA-independent manner.\n\nI suggest that the title of this section be altered to “Zygnematales PYLs exhibit PP2C inhibitory activity in an ABA-independent manner” as the current wording implies that the experiments had all been carried out in algae, rather than analysing the activity of PYLs from algae.\n\n3. Characterizing ABA-independent PYL activity in Arabidopsis\n- ABA-independent basal activity has been associated with PYL family members that are monomeric, with the dimeric angiosperm subfamily 3 PYLs being highly ABA-dependent. The authors have carried out complementation of the 12x pyl- mutant to identify Arabidopsis PYLs with basal activity and those without, confirming this.\n\nThey also state in Lines 192-194: “Phylogenetic analysis of PYLs from the Brassicaceae family exhibited the stepwise transformation from ABA receptors to ABA-irresponsive PYLs after expansion of the gene family”.\n\nI do not follow this argument. The support for this is supposedly in Supplementary Figure 3a and Supplementary Data 1 but I cannot follow the logic (This may be my fault). I should have expected basal activity alone (ABA-independence) to be the ancestral state with ABA-dependence with some residual ABA-independence to follow and complete ABA dependence to be the latest acquisition. (Essentially this is what is described in the abstract in lines 17-19). I think the authors need to clarify this.\n\n4. Do specific mutations identify residues required for ABA-dependence?\n- The residues Q38 and F71 in PYL13 are identified as variant and required for ABA binding, and the PYL13 gene is characterized by basal activity rather than ABA-dependence using site-specific mutations to correct this, and to examine the requirement for Lysine and Leucine at the corresponding positions in other PYL genes.\n\nThis then leads on to analysis of PYLs in other plants that carry similar mutations, and that also exhibit principally basal activity in their regulation of SnRK2 phosphorylation.\n\n5. Seed specificity of PYLs\n- A major outcome of PYL activation of the ABA response is in the regulation of seed development. The control of ABA-dependent acquisition of desiccation tolerance is one of the most important functions for plant reproduction, enabling the development of resilient reproductive propagules that can be dispersed in space, and as a result of ABA-induced dormancy, dispersed also in time. This property is also of fundamental importance for human social evolution, as the ability to store dry sees from one year to the next is the basis of agriculture and transformed human societies from hunter-gathering tribes to settled communities leading to the advent of civilisation.\n- The authors note that a number of PYLs are specifically expressed during seed development and are thus direct activators of the genetic programme that leads to desiccation tolerance in seeds. They additionally note that AtPYL13 is one of these seed specific PYLs, and that it (and likely the others) is expressed exclusively during this time, the transcription of the gene being directed by the ABI3 transcription factor – itself generally described as being seed-specific in its functions (actually, this is not quite correct: it is also active in bud dormancy).\n- However, it is not clear to what extent the AtPYL13 is essential for this process, since seed desiccation tolerance is certainly principally ABA-dependent, and thus necessarily principally reliant on those other seed-expressed PYLs named in this section of the manuscript. I do not imagine that the basal activity of AtPYL13 contributes significantly, by comparison.\n- This last section, while interesting, seems somewhat out of place by comparison with the foregoing information, and it might be better published separately in another journal.\n\nRecommendations\nThis is clearly a well-executed piece of work, but the authors should consider my comments above. I also have a few specific recommendations:\n\n1. The title et seq.\nFirst, I do not think that the term “ABA-irresponsive” is appropriate as a description of the PYL receptors. First, no PYL is either “responsive” or “not responsive” to ABA. PYLs are receptors that become active through interacting with ABA as a ligand in order to enable ABA responsive gene expression.\n\nIt is the phosphatase activity and the subsequent pathway of gene activation that is responsive – not the PYL. In the case of PYL homologs that bind to PP2CA phosphatases without binding ABA, - defined as “basal activity” of these PYLs, it remains to be determined how much of a response is elicited.\n\nFor example:\nLines 82-83: “we evaluated the ABA responsiveness of PYLs in the Arabidopsis ABA-insensitive pyl duodecuple mutant”\n- Actually, this isn’t testing the “ABA responsiveness” of the PYL, it’s measuring the ABA responsiveness of the Rd29b-Luc reporter when the 12x mutant is complemented by wild-type PYLs from Arabidopsis, Marchantia and Physcomitrella, and so-called bacterial PYLs (or more accurately, genes identified as having some similarity to bona fide PYLs\n\nI recommend the use of ABA-independent, rather than-ABA irresponsive, in the title:\n“ABA-independent PYL-like proteins…”\n…and throughout the manuscript.\n\n2. Minor corrections\nLines 46-7 and 57-8: Use a standard font, not bold text. The use of bold text to emphasise a point is acceptable in a grant proposal, but not in a paper (unless it is a section heading).\nLine 49: “encoded by the algae Zygnema…” to: “encoded by the alga Zygnema…”\nLines 192-194: “Phylogenetic analysis of PYLs from the Brassicaceae family exhibited the stepwise transformation from ABA receptors to ABA-irresponsive PYLs after expansion of the gene family”\nThis implies that an ABA receptor with low basal activity arose first, and subsequently PYLs evolved with high basal activity.\nI’d have thought that basal activity was the ancestral state. Of course, this is being described in a relatively late-evolving plant group, in which there could have been prior loss of members with basal activity prior to the re-emergence of PYLs with high basal activity.\n\nSupplementary dataset 1: Phylogenetic analysis of PYLs.\n- The authors have fallen in to the trap of failing to determine whether genes annotated as ABA receptors are indeed bona fide receptors. Thus the set of PYL homologs identified for P. patens identifies three gene models that were identified as encoding ABA receptors in a report by Sussmilch et al. (2017) Plant Signaling & Behavior 12: e1365210, but that are actually non-functional pseudogenes. These are Pp3c3_660, Pp3c7_4410 and Pp3c15_19950. The first two of these are predicted to contain a short intron that is unsupported by RNAseq data. In each case the predicted intron (automatically assigned by the software used to generate the models) contains an in-frame termination codon that would render an unspliced transcript non-functional. In 3_660, the available RNAseq data for this locus do not indicate the presence of an intron. For 7_4410 the gene model is unsupported by any RNAseq reads, it lies in an intergenic region and there is no evidence of a start codon. For 15_19950, the predicted polypeptide sequence shows the N-terminus to lie only 14 residues before the SGIPAT “gate” sequence: however, a BLASTX analysis of the predicted 5’UTR sequence immediately prior to the candidate N-terminal methionine residue shows a more likely N-terminal coding region with significant similarity to that of PpPYL1 (Pp3c26_15240). Readthrough shows that these two sequences are out of frame, with two consecutive termination codons between the “UTR” and “coding” sequences. These are likely degenerated pseudogenes and should be excluded.\nSimilarly, the M. polymorpha model Mapoly0145s0004 is speculative, as there is very little RNAseq support for this locus.\nFor Sphagnum fallax, The sequence assembly used (based on scaffolds, rather than chromosomes) is not the most recent: the V1.1 locus identifiers are as follows:\nV0.5 Sphfalx0046s0087 = V1.1 Sphfalx16G013800\nV0.5 Sphfalx0031s0007 = V1.1 Sphfalx16G062700\nV0.5 Sphfalx0002s0105 = V1.1 Sphfalx19G066800\nV0.5 Sphfalx0024s0170 = V1.1 Sphfalx11G055600\nV0.5 Sphfalx0310s0087 = V1.1 Sphfalx04G53400"}, "author_response": "Responses: We thank the reviewer for raising critical methodological considerations regarding ligand dependency assessment. To address these points, we conducted additional in vitro biochemical characterization under rigorously controlled conditions: 1) PP2C phosphatase activity assays using pNPP substrate (Fig. 1g, h; Supplementary Fig. 1f); 2) Isothermal Titration Calorimetry (ITC) (Supplementary Fig. 1g). Together with the SnRK2.6 kinase activity assays at previous version (Fig. 1e, f), these experiments employed purified recombinant proteins in systems with precise control of protein concentrations/ratios and ABA levels. Collectively, our data demonstrate that PrPYL exhibits ligand-independent activity, validating its functional characterization as a bacterial ABA-independent PYL homolog.\nConcerning in vivo validation systems, we are actively developing ABA-deficient plant material through CRISPR/Cas9-mediated knockout of ABA2 in the pyl duodecuple mutant background. This approach aims to generate plants with combined defects in ABA biosynthesis and perception. While the developmental phenotypes associated with the parental mutant complicate line establishment, we are optimizing growth conditions to overcome these challenges.\n\nQuery #2:\nHorizontal Gene Transfer Hypothesis Lacks Supporting Evidence\nThe claim of horizontal gene transfer (HGT) is presented without substantive support. Establishing HGT in a system that diverged over 600 million years ago is inherently difficult. Yet, the authors fail to explore/roll out alternative evolutionary scenarios, such as convergent evolution, which remain equally plausible. A robust phylogenetic analysis should provide statistical confidence measures and exclude other evolutionary possibilities. If the phylogenetic tree showed plant sequences embedded within bacterial lineages, it could suggest recent gene transfer, but the current analysis lacks the necessary rigor. The arbitrary nature of the sequence relationships as presented does not substantiate the proposed HGT event. While the reviewer is not a bioinformatician, the absence of statistical validation and failure to test alternative hypotheses significantly weaken this claim.\n\nResponses: We appreciate the reviewer’s engagement with the evolutionary context of this work. As our study fundamentally constitutes an experimental investigation of bacterial PYL functionality but not a phylogenetic analysis, the Cheng et al. (2019) reference primarily provides the evolutionary premise motivating our functional inquiry. Accordingly, we have revised the title and manuscript to frame HGT strictly as a hypothesis.\nOur core contribution demonstrates cross-kingdom functional conservation: PrPYL exhibits PP2C-binding capabilities mirroring plant PYLs (Fig. 1d) and shows structural convergence with AtPYL1 (Fig. 1i-l). These results establish mechanistic conservation of PYL function, though definitive determination of evolutionary origins requires specialized genomic analyses beyond this study’s scope.\n\nQuery #3:\nSpecific examples:\nIn the title “….in the seeds of land plants”\nThe relations described in the study extend beyond seed plants, as the PYL gene family is conserved across the entire lineage of land plants, not just in seed-bearing species. Also the title should explicitly state which functional aspect of PYL receptors has been retained throughout evolution—specifically, their ability to bind PP2C phosphatases.\n\nResponses: We thank the reviewer for this constructive suggestion. Accordingly, we have revised the title to: “ABA-independent PP2C-binding in PYLs traces to bacterial origins and persists in land plants”.\n\nQuery #4:\nFurthermore, the claim of horizontal gene transfer (HGT) remains a hypothesis rather than a demonstrated/conclusion. As such, it should either be removed from the title or clearly framed as a hypothesis rather than a definitive statement. The current phrasing in the title risks misleading readers by presenting an unverified assumption as an established finding.\n\nResponses: We sincerely thank the reviewer for pointing this out. We agree that horizontal gene transfer (HGT) represents one plausible evolutionary scenario rather than a definitively proven mechanism for the origin of bacterial-derived PYL-like sequences in plants. To address this, we have revised the title to: “ABA-independent PP2C-binding in PYLs traces to bacterial origins and persists in land plants”. We have also clearly framed horizontal gene transfer of PYL genes as a hypothesis throughout the main text.\n\nQuery #5:\n-Abstract: Has inaccuracies\nExamples: Authors claim: “which requires constitutive activation of stress signaling, was mainly adopted by early land plants and persists in seeds.”\nThis statement is incorrect—ABA levels are crucial for seed dormancy and decrease to allow germination. During dormancy, ABA levels remain high to prevent premature germination. However, for germination to occur, ABA levels must decline (while gibberellins increase).\n\nResponses: We sincerely thank the reviewer for identifying this critical inaccuracy regarding stress signaling in seeds. We have revised the abstract to: “Among these adaptations, ‘drying without dying’ strategy, which requires constitutive stress signaling activation, evolved in early land plants and is maintained in desiccated seeds.” The constitutive stress signaling activation in desiccated seeds is maintained primarily through ABA-dependent mechanism during dormancy, with ABA-independent mechanisms1,2.\n\nQuery #6:\nAuthors claim: “Through evolution, the PYR/PYL/RCAR family (PYL) of ABA receptors gained several functions over the course of evolution, namely inhibition of protein phosphatases (PP2Cs), ABA-enhanced inhibition of PP2Cs, and ABA dependent inhibition of PP2Cs”\nThe only well-established physiological function of PYL receptors is their ability to inhibit PP2C phosphatases, which are central to ABA signaling. If the authors intended to suggest an additional function, they should clarify it and provide supporting evidence.\n\nResponses: We sincerely thank the reviewer for pointing this out. Accordingly, we have revised the abstract to: “During evolution, the PYR/PYL/RCAR (PYL) family of ABA receptors acquired PP2C binding and inhibitory activities.”\n\nQuery #7:\nAuthors claim: “However, it remains unclear how the ABA-irresponsive PYLs originated and whether they exist in land plant. “\nThis statement is incorrect—it has been shown that PYL receptors in land plants likely originated from algal ancestors, and homologous proteins are present across all land plant lineages. This suggests that the PYL family is not exclusive to land plants but instead evolved from pre-existing proteins in algae before being co-opted into the ABA signaling pathway in terrestrial plants. See doi.org/10.1073/pnas.1914480116\n\nResponses: We sincerely thank the reviewer for prompting critical clarification regarding PYL evolution. As requested by one reviewer, we changed “ABA-irresponsive” PYLs to “ABA-independent PYL-like proteins”. We have accordingly revised the abstract: “However, the origin of algal PYLs and the mechanistic basis of ABA-independent PYL variants in land plants remain uncharacterized.” We define these ABA-independent PYL-like proteins in the subsequent sentence as those “retain constitutive PP2C binding but lack ABA-enhanced inhibitory activity”.\n\nQuery #8:\nAuthors claim: “AtPYL13 and AtPYL13-like genes were highly expressed in dicot seeds, suggesting the transformation from ABA receptors to ABA-irresponsive PYLs after expansion of the gene family”.\nThis statement misrepresents the actual expression profile of PYL in Arabidopsis seeds. While PYL13 expression can be detected, public datasets (e.g., BAR ePlant) indicate that its expression is approximately 100-fold lower than PYR1, suggesting it plays a minor role in seed germination. Additionally, experimental evidence challenges the significance of PYL13 in this context. For example, activating PYR1 alone, using a specific agonist, was sufficient to inhibit germination. Dimeric multiple mutants exhibit an early germination phenotype, further suggesting that the primary regulators of germination are other PYL receptors. See Park 2009 DOI: 10.1126/science.1173041 and Okamoto 2011 https://doi.org/10.1073/pnas.1305919110\nGiven these findings, if PYL13 has any role in germination, it is likely minimal or redundant, and its functional relevance in this process remains questionable.\n\nResponses: We appreciate the reviewer’s insightful comments regarding the expression and functional relevance of AtPYL13. We agree that AtPYL13 exhibits very low expression levels in most tissues and does not play a central role in seed germination. To clarify this point, we have revised the abstract as follows: “AtPYL13-like genes in dicots exhibited high expression during seed maturation and in desiccated seeds, suggesting a functional shift from canonical ABA receptors to ABA-independent PYL-like proteins following gene family expansion.” This statement aligns with the transcriptional analyses presented in our manuscript (Fig. 4d, e; Fig. 5a, b). Based on their distinct expression patterns, we propose that AtPYL13-like genes primarily contribute to seed vigor maintenance rather than seed germination regulation. We have also clarified this point in the Discussion.\n\nQuery #9:\n-Introduction- Has inaccuracies\nSome Examples:\nAuthors claim: “Terrestrialization is a pivotal event in the evolution of green plants, which requires mechanisms to survive under drought stress.”\nThe term \"drought stress\" is not appropriate to describe the primary challenges of terrestrialization (refers to the evolutionary transition from an aquatic to a terrestrial environment). Terrestrialization involves adaptation to a gradual shift from consistently wet conditions to environments with fluctuating water availability rather than exposure to extreme drought. Using \"drought stress\" oversimplifies the complexity of this transition, which encompasses not only water limitation but also factors such as desiccation tolerance, gas exchange regulation, light intensity, and structural adaptations to terrestrial life.\n\nResponse: We sincerely thank the reviewer for this insightful observation. We agree that “drought stress” does not fully capture the multifaceted challenges of terrestrialization. As suggested, we have revised the Introduction to more accurately reflect the complexity of this evolutionary transition: “Terrestrialization represents a pivotal event in green plant evolution, requiring adaptive mechanisms to overcome multiple environmental challenges, particularly fluctuating water availability.”\n\nQuery #10:\nAuthors claim: “Plants mainly utilize two strategies to survive under water deficiency: “drying without dying” or “without drying”.\nThese terms are not clear.\n\nResponses: We appreciate the reviewer’s valuable comment regarding the clarity of these terms. In the revised manuscript, we have now explicitly defined these survival strategies as: “Plants employ two principal strategies to cope with water deficiency: “drying without dying (desiccation tolerance)” or “avoiding drying (drought avoidance)”3.\n\nQuery #11:\nAuthors claim: “The early land plants are mainly poikilohydric and adopt the “drying without dying” strategy, which requires constitutive activation of stress signaling and enables plants to get through extreme conditions” –Early land plants possess mechanisms for water regulation and stress adaptation that are independent of vascular systems, as described in the cited paper. A more relevant and evolutionarily conserved mechanism is osmoregulation, which is shared between mosses and seed plants.\nRegarding the statement “PP2Cs can be ABA-dependent or ABA-enhanced”, this terminology is unclear. PP2C inhibition occurs through both ABA-dependent and ABA-independent mechanisms, and the distinction between \"ABA-enhanced\" and \"ABA-dependent\" needs to be explicitly defined to avoid confusion.\n\nResponses: We sincerely appreciate the reviewer’s insightful comments regarding these important mechanistic distinctions. We have carefully revised the text to incorporate these points.\nFor the poikilohydric strategy discussion: “Early land plants are mainly poikilohydric and adopt the “drying without dying” strategy, which relies on fundamental stress adaptation mechanisms including osmoregulation and enables plants to survive extreme conditions.”\nFor the PP2C terminology clarification: “The ABA-binding properties of PYLs vary significantly in seed plants; likewise, their PP2C inhibition mechanisms show distinct ABA-dependence patterns: (i) strictly ABA-dependent (no basal inhibition activity without ABA, e.g., the dimeric subfamily III PYLs in angiosperms)4, (ii) ABA-enhanced (basal inhibition activity potentiated by ABA)5, or (iii) ABA-independent (not modulated by ABA). The “ABA-independent PYL-like proteins” include: 1) constitutively active PYLs that activate stress responses regardless of ABA6; and 2) inactive PYLs incapable of activating stress responses.”\n\nQuery #12:\nAuthors claim: “The entirely ABA dependent dimeric PYLs emerged in angiosperms and have nearly no basal activity”-This applies to other PYLs as well— not just dimeric forms. This should be considered as it's not the only mechanism of low basal activity (in Arabidopsis and other species), as indicated in DOI: 10.1016/j.cub.2024.12.043.\"\nAuthors claim: “which enables angiosperms to have a wider range of ABA responses and improved balance between growth and stress responses”-This is an assumption and should be stated as such.\n\nResponses: We sincerely thank the reviewer for these insightful comments. We have revised the text as follows: “Strictly ABA-dependent PYLs with minimal basal activity emerged in Bryophytina mosses and angiosperms5,7, potentially enabling broader range of ABA responses and improved balance between growth and stress responses.”\n\nQuery #13:\nAuthors claim: “However, it remains unclear how the ABA-irresponsive PYLs originated.”\nThe focus should be more specific—there is evidence that land plant lineages and the common ancestor of Zygnema circumcarinatum possessed a nonresponsive PYL. What remains unclear is how this protein was ‘adopted’ into the plant lineage.\"\n\nResponses: We sincerely thank the reviewer for this insightful comment. We have revised the text as follows: “However, it remains unclear how ABA-independent PYL-like proteins emerged and were subsequently incorporated into plant lineage.”\n\nQuery #14:\n-Specific points in the result section:\nThe section titled: “The bacterial PYL-homolog from Paraburkholderia rhynchosiae possesses PP2C-binding ability”\nAuthors claim: “It is proposed that the ancestral PYLs in plants were gained through horizontal gene transfer from soil bacteria based on phylogenetic analyses (Fig. 1a).” –The phylogenetic analysis does not support horizontal gene transfer occurring over 600 million years ago.\n\nResponses: We sincerely thank the reviewer for this important observation. Our phylogenetic analysis indeed does not provide definitive support for horizontal gene transfer events occurring over 600 million years ago. Accordingly, we have revised the manuscript by properly citing Cheng et al. (2019)8 and repositioning Fig. 1a.\n\nQuery #15:\nAuthors claim: “Despite lacking ABA responsiveness”\nThis should be tested in vitro using assays that directly assess the involvement of ABA as a ligand, such as a PP2C inhibition assay, isothermal titration calorimetry, or any equivalent assay.\n\nResponses: We thank the reviewer for this important methodological suggestion. We have now performed: 1) PP2C inhibition assays using pNPP substrate (Fig. 1g, h; Supplementary Fig. 1f); 2) Isothermal Titration Calorimetry (ITC) (Supplementary Fig. 1g). Combined with our previous SnRK2.6 kinase activity assays (Fig. 1e, f), these results confirm that PrPYL lacks ABA binding and responsiveness.\n\nQuery #16:\nThe authors identified the interaction between bacterial PrPYL and PP2C via a yeast two-hybrid assay. However, this interaction was not reproducible in planta nor when SnRK2 competed for the PP2C interface in an in vitro assay. Based on this, the authors concluded that there is an interaction with PP2C but no PP2C inhibition. As stated, 'These results suggested that the bacterial PrPYL possesses PP2C-binding but not PP2C inhibitory activity.' This conclusion is flawed for two reasons: 1. The interaction between PYL and PP2C typically blocks the active site of the PP2C enzyme. Therefore, a 'canonical interaction' should, by default, result also in inhibition. 2. To properly claim this, the interaction and PP2C activity must be evaluated through specific assays, not as a default by a negative result. At this point, it is an unsubstantiated guess.\nAn alternative explanation could be that, in yeast, the protein steady-state concentration is high, allowing for the monitoring of low-affinity interactions. In vitro and in-planta, however, are more challenging, as other proteins could compete for the PP2C interface. Consequently, it is unsurprising that the weak interaction with bacterial PrPYL is outcompeted by SnRK2s (whether from plants or purified protein), resulting in the absence of an interaction signal.\"\n\nResponses: We sincerely thank the reviewer for this insightful critique and valuable hypothesis.\nOur experimental data demonstrate that: (1) PrPYL exhibits weak PP2C binding without inhibitory activity (Fig. 1); (2) PrPYL lacks the conserved Gate loop (S/TGLPA) (Supplementary Fig. 1a) that mimics SnRK2 activation loop in SnRK2-PP2C complex9; and (3) Molecular docking confirms PrPYL’s inability to properly occupy ABI1’s catalytic site (Response Fig. 1). These results indicate a non-canonical binding mechanism distinct from angiosperm PYLs. As suggested, we have incorporated the possibility that weak PrPYL-PP2C interactions may be outcompeted by endogenous proteins (e.g., plant PYLs and SnRK2s) into our main text. We have revised the text as follows: “The weak PrPYL-PP2C interactions may be outcompeted by endogenous proteins (e.g., plant PYLs and SnRK2s), thus generating very weak reconstituted LUC signals in planta. These results suggest that PrPYL represents a prototype ABA receptor.”\n\nResponse Fig. 1. Molecular docking of AtPYL1 and PrPYL into catalytic active site of AtABI1.\n\nQuery #17:\nThe section titled “ABA-irresponsive PYLs exhibit PP2C inhibitory activity in Zygnematales”.\nSun et al. (2019) showed that algal PYL interacts with PP2C in an ABA-independent manner. In that study, Sun also demonstrated that ZcPYL8 cannot bind ABA (by isothermal calorimetry titration). In this current study, the authors aimed to test additional algal PYLs. They used an Arabidopsis PYL mutant with endogenous ABA levels, which were unsuitable for testing ABA-independent activity. The SnRK activity (following release) assay is sensitive to PYLs with basal ABA-independent activity but cannot distinguish if there is a mild ABA effect. These two assays do not support the claim that the 'PYL homologs gained ABA-independent PP2C inhibitory activity during the evolution of Zygnematales.' In vitro assays that directly test ABA binding would be better suited to scratch the surface of this hypothesis. I suggest starting by following the biochemical assays performed by Sun in 2019.\n\nResponse: We thank the reviewer for this critical methodological suggestion. To address these concerns, we have now conducted: 1) PP2C inhibition assays using pNPP substrate (Fig. 2g-j; Supplementary Fig. 2b); and 2) Isothermal Titration Calorimetry (ITC) to directly assess ABA binding (Supplementary Fig. 2c). Together with our SnRK2.6 kinase activity assays (Fig. 2e, f), these experiments confirm that MeenPYL3 functions in an ABA-independent manner.\n\nQuery #18:\nThe section titled “An ABA-irresponsive PYL exists in Arabidopsis”\nThe authors claimed that “the wild-type AtPYL13 could not activate RD29B-LUC with ABA treatment, while the expression of AtPYL13-Q38K/F71L restored the ABA-dependent activation of RD29B-LUC in the pyl duodecuple mutant protoplasts (Fig. 3c), indicating that AtPYL13 is otherwise functional and that the two natural sequence variations in the CL1/Cl2 domains of AtPYL13 confer ABA irresponsiveness”\nA paper from the Grill group addresses PYL13 activity and the specific mutation analyzed here: https://doi.org/10.1073/pnas.1322085111. These mutations were thoroughly tested in that study. I failed to see the added value of repeating them here.\"\n\nResponse: We sincerely appreciate the reviewer’s guidance in highlighting the foundational work by Grill lab. Our intent is not to challenge but to complement existing knowledge. While previous studies established AtPYL13’s ABA responsiveness using transient expression in ABA-deficient backgrounds (aba2-1), our study extends this observation by demonstrating in a genetic null background lacking endogenous ABA receptors (pyl duodecuple mutant) that: (i) wild-type AtPYL13 fails to activate ABA-responsive reporters (Fig. 3a), and (ii) AtPYL13-Q38K/F71L restores ABA-dependent signaling (Fig. 3c). This independent validation underscores the functional plasticity of PYL13’s ligand-binding domains under physiologically relevant receptor-free conditions.\n\nQuery #19:\nThe sections titled “Two invariant residues are crucial for PYL ABA receptors.”, “ABA-irresponsive PYLs exist in seed plants” and “ABI3 mediates seed-specific expression of AtPYL13”\nI don’t understand the rationale behind the ectopic expression of impaired-active mutated PYLs. What can be learned from these experiments? This entire section seems unrelated to the main focus of the study. Furthermore, it is well-established that dimeric PYLs are key players in seed germination biology. This has been tested through agonist activation of specific PYLs and by examining expression levels (actual reads versus the relative expression tested here). In fact, PYL13 expression is barely detectable.\"\n\nResponse: We thank the reviewer for raising these critical points. Our ectopic expression experiments serve two key purposes—mechanistic validation and functional essentiality. By introducing Q38K/F71L mutations into ABA-independent PYL13 (Fig. 3c-f), we demonstrate these ancestral residues sufficiently restore ABA responsiveness. Reciprocal mutagenesis in canonical ABA receptors (Fig. 3h-k) confirms these invariant residues are strictly required for ABA perception.\nWhile dimeric PYLs dominate germination control, our transcriptional analysis (Fig. 5) reveals AtPYL13’s selective upregulation specifically during seed maturation—not germination—suggesting its ABA-insensitive activity may enable distinct developmental programming at this critical stage."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have revised their manuscript appropriately, and the revised version is much clearer in terms of wording and the description of the evolution of ABA receptors.\n\nOne point I would like the authors to further improve concerns the function of AtPYL13. Based on the reduced seed production of the quattuordecuple mutant (generated by introducing the pyl13 and pyl6 mutations into the duodecuple pyl mutant), the authors argue for a role of AtPYL13 in fertility and seed maturation. However, I find this evidence insufficient to support their claim for the following reasons. First, this observation does not provide direct insight into the specific role of AtPYL13 alone. Second, it does not clarify the function of AtPYL13 in seed maturation. Since the central argument of this work is that AtPYL13 plays a role in seed maturation (particularly in the desiccation stage), it is critical to discuss AtPYL13 function in this specific developmental context.\n\nI note that mutations in seed maturation–specific genes result in seeds with desiccation intolerance or distinct morphological defects. In contrast, phenotypes related to fertility usually leads to the absence of embryonic/zygotic structures in seeds, or as aberrant segregation ratios. If the reduced seed phenotype observed here is due to sterility (either male or female), it does not support the authors’ main claim regarding a role for AtPYL13 in seed maturation.\n\nI encourage the authors to provide clear phenotypic evidence supporting the function of AtPYL13 in seed maturation. Otherwise, I suggest removing this ambiguous observation and tone down their claim accordingly.", "reviewer_3": "In the revised manuscript, the authors appropriately answer my comments to the previous manuscript, and I have no further question or comment.", "reviewer_4": "First, my apologies for my tardiness in returning this review: I received the request to consider the revised manuscript immediately before taking a vacation, and it is only in the last week that I’ve had time to examine the revision.\n\nI’m happy to say that so far as my own comments of the original manuscript are concerned, I think that the authors have addressed all of the comments I raised satisfactorily.\n\nMy comments below are principally the correction of some phrases to conform with standard English usage. However I have also made some suggestions where some alterations in the text add clarity: for example an alternative opening to the Abstract (immediately below)\n\nI found the Abstract to lack clarity: in order to rectify this I have rewritten the opening 6 lines in a way that I think make it easier for the reader to understand (and hopefully, then proceed to read the entire paper!):\n\"Land plants have evolved strategies to survive water deficiency. Among these adaptations, a “drying without dying” strategy, regulated by the hormone Abscisic Acid (ABA), evolved in early land plants and is maintained in the desiccated seeds of angiosperms. This is regulated by a family of ABA receptors (the PYR/PYL/RCAR (PYL) family) able to bind protein phosphatases of the PP2CA family and suppress their inhibitory action on water stress responses. ABA-independent PYLs first appear in an aquatic algal lineage, however their origins and the mechanistic basis of ABA-independent PYL variants in land plants remain uncharacterized. Here we characterize……etc.\"\n\nThe Introduction begins: Early land plants are mainly poikilohydric and adopt the “drying without dying” strategy, which relies on fundamental stress adaptation mechanisms including osmoregulation and enables plants to survive extreme conditions2.\nVascular land plants retain the “drying without dying” strategy in specific organs, such as seeds or pollen, but utilize the “avoiding drying” strategy for their vegetative stages, which depends on the activation of stress responses by the phytohormone ABA3,4.\n- It is important to realise that this is not a binary choice, and that both acquisition of dehydration tolerance and dehydration avoidance involve activation of multiple stress responses by ABA (for example promotion of expression of genes encoding LEA proteins as a prerequisite for achieving desiccation tolerance, and the implementation of stomatal closure as a means of drought avoidance).\n\nI suggest rewriting the opening lines of the paragraph commencing at Line 41 as follows:\n\"ABA activation occurs through a protein phosphorylation cascade, in which Snf1-related protein kinase 2 (SnRK2) kinases have a central role. Upon ABA binding, the PYR/PYL/RCAR (PYL) ABA receptors…..etc.\"\n\nL58-60: \"In the nonvascular liverwort Marchantia, MpPYL1 shows ABA-enhanced activity to inhibit PP2Cs, although with high basal activity9 , which triggers high basal stress signaling and actively represses plant growth15\"\n- Reference 15, whilst being a comprehensive review of the regulation of plant growth responses, does not make any reference to Marchantia or the basal activity of MpPYL1 (the focus is primarily on stress and growth regulation in angiosperms)\n\nL61: potentially enabling broader range of ABA responses\nTo: potentially enabling a broader range of ABA responses\n\nL63: were subsequently incorporated into plant lineage.\nTo: were subsequently incorporated into land plant lineages.\n\nL81: The PYL genes from plants form monophyletic group\nTo: The PYL genes from plants form a monophyletic group\n\nResults section\nL81: The PYL genes from plants form monophyletic group\nTo: The PYL genes from plants form a monophyletic group\n\nL182: This partially explains the weak interaction of PrPYL-ABI1 and strong interactions between algal PYLs and Arabidopsis PP2Cs as well as the constitutive activation of RD29B-LUC expression in pyl duodecuple mutant protoplasts by algal PYLs (Fig. 1, 2, and Supplementary Fig. 2a)\n- I can see the interactions between algal PYLs and Arabidopsis PP2Cs, and the activation of RD29B-LUC in Figures 2 and Supplementary 2, but not in Figure 1.\n\nL198-200: Since high basal stress signaling favored the terrestrialization of land plant progenitors, this critical arginine was not conserved during evolution to maintain high basal activity of PYLs.\nTo: Since high basal stress signaling would have favored the terrestrialization of land plant progenitors, we propose that this critical arginine was not conserved during evolution to maintain high basal activity of PYLs.\n\nL280: several AtPYL13-like proteins differ from the ABA-responsive PYLs by one or two amino...\nTo: several AtPYL13-like proteins differ from the ABA-dependent PYLs by one or two amino...\n\nL286: We evaluated the ABA responsiveness of these AtPYL13-like proteins\nTo: We evaluated the ABA dependence of these AtPYL13-like proteins\n\nDiscussion\nL355: algae, the ABA receptor PYLs are not present until the last common ancestor of lant plants\nTo: algae, the ABA receptor PYLs are not present until the last common ancestor of land plants\n\nL357-8: Our results showed that PYL-like proteins are present in bacteria, might have gained PP2C binding ability in some soil bacteria. Plants acquired PYL genes horizontally...\nTo: Our results showed that PYL-like proteins are present in bacteria, and might have gained PP2C binding ability in some soil bacteria. Plants likely acquired PYL genes horizontally...\n\nL365-6: Besides, the monomeric PYLs also evolved the strict ABA dependency in mosses\nTo: Independently, the monomeric PYLs also evolved strict ABA dependency in mosses\n\nL366-7: The ABA-binding and -responsive properties of PYLs confer ABA-dependent or ABA-enhanced PP2C-inhibition activity, which enables land plants to change...\nTo: The ABA-binding properties of PYLs confer ABA-dependent or ABA-enhanced PP2C-inhibition activity, which enabled land plants to change…."}, "author_response": "Response: We thank the reviewer for pointing out the overinterpretation in this point.\nWe agree that fertility, seed maturation, and desiccation tolerance represent distinct physiological processes. In an independent unpublished study, we assessed transcriptional reprogramming during seed maturation and observed that the vast majority of transcriptional changes are completed prior to the desiccation phase. Thus, the ABI3-mediated seed-specific expression of AtPYL11, AtPYL12, and AtPYL13 suggests their potential involvement in seed maturation and desiccation tolerance. Both ABA-dependent and ABA-independent PYLs employ SnRK2s and ABFs to mediate transcriptional reprogramming and act redundantly in maintaining seed viability1-3. Plants accumulate high levels of ABA during seed maturation, and ABA concentrations gradually decline during vernalization4. Therefore, elevated basal stress signaling mediated by ABA-enhanced and ABA-independent PYLs is likely necessary for seed viability at stages characterized by low ABA levels, such as following vernalization.\nAs we were unable to obtain a pyl tredecuple mutant harboring the PYL13 mutation, we have removed the contested observations related to the quattuordecuple mutant and revised our conclusions accordingly to reflect a more cautious interpretation. The text now focuses on correlative evidence from ABI3-mediated seed-specific expression (Fig. 5) to propose a possible, rather than definitive role for AtPYL13 during seed maturation.\n\nQuery #2:\nAdditional comments regarding the response to Reviewer #1:\nReviewer 1 provided the most extensive and detailed comments throughout the manuscript. Thus, the authors focused their revision efforts primarily on addressing Reviewer 1’s concerns. I believe the authors did a good job in responding to Query #1. The two in vitro experiments and one in vivo experiment they conducted are considered gold-standard experiments, and the results support their claims convincingly.\nRegarding Query #2, I find the authors’ explanation reasonable. They clarified that the focus of this work is on the functional aspects of evolutionary divergence, distinguishing it from Cheng et al., 2019. Compared to previous studies that discussed the evolution of PYL proteins from algae to land plants, this manuscript covers a much broader evolutionary timeline. Overall, Reviewer 1’s comments were constructive, and the authors responded appropriately. As I am not an evolutionary biologist, I do not have further comments beyond this.\n\nResponse: We sincerely appreciate the reviewer’s supportive feedback and valuable perspective. To further strengthen the evolutionary analysis in our study, we have engaged in extensive discussion with Professor Guanzhu Han, an expert in plant evolutionary biology, which has helped us to better clarify the evolutionary trajectory of the PYL gene family in the previous revised version.\n\nReviewer #4 (Remarks to the Author):\nFirst, my apologies for my tardiness in returning this review: I received the request to consider the revised manuscript immediately before taking a vacation, and it is only in the last week that I’ve had time to examine the revision.\nI’m happy to say that so far as my own comments of the original manuscript are concerned, I think that the authors have addressed all of the comments I raised satisfactorily.\n\nQuery #1:\nMy comments below are principally the correction of some phrases to conform with standard English usage. However I have also made some suggestions where some alterations in the text add clarity: for example an alternative opening to the Abstract (immediately below)\nI found the Abstract to lack clarity: in order to rectify this I have rewritten the opening 6 lines in a way that I think make it easier for the reader to understand (and hopefully, then proceed to read the entire paper!):\n\"Land plants have evolved strategies to survive water deficiency. Among these adaptations, a “drying without dying” strategy, regulated by the hormone Abscisic Acid (ABA), evolved in early land plants and is maintained in the desiccated seeds of angiosperms. This is regulated by a family of ABA receptors (the PYR/PYL/RCAR (PYL) family) able to bind protein phosphatases of the PP2CA family and suppress their inhibitory action on water stress responses. ABA-independent PYLs first appear in an aquatic algal lineage, however their origins and the mechanistic basis of ABA-independent PYL variants in land plants remain uncharacterized. Here we characterize……etc.\"\n\nResponse: We are grateful to the reviewer for the constructive feedback and for the time taken to refine the wording of our abstract. We have adopted the proposed opening lines and agree that they have greatly improved the clarity of our introduction.\n\nQuery #2:\nThe Introduction begins: Early land plants are mainly poikilohydric and adopt the “drying without dying” strategy, which relies on fundamental stress adaptation mechanisms including osmoregulation and enables plants to survive extreme conditions2. Vascular land plants retain the “drying without dying” strategy in specific organs, such as seeds or pollen, but utilize the “avoiding drying” strategy for their vegetative stages, which depends on the activation of stress responses by the phytohormone ABA3,4.\n- It is important to realise that this is not a binary choice, and that both acquisition of dehydration tolerance and dehydration avoidance involve activation of multiple stress responses by ABA (for example promotion of expression of genes encoding LEA proteins as a prerequisite for achieving desiccation tolerance, and the implementation of stomatal closure as a means of drought avoidance).\n\nResponse: We are grateful to the reviewer for this constructive suggestion regarding the non-binary nature of these adaptive strategies. We have revised the manuscript to reflect that both dehydration tolerance and avoidance involve ABA-mediated stress responses.\n\nQuery #3:\nI suggest rewriting the opening lines of the paragraph commencing at Line 41 as follows:\n\"ABA activation occurs through a protein phosphorylation cascade, in which Snf1-related protein kinase 2 (SnRK2) kinases have a central role. Upon ABA binding, the PYR/PYL/RCAR (PYL) ABA receptors…..etc.\"\n\nResponse: We thank the reviewer for the suggested improvement and have revised the text at the beginning of the paragraph on Line 41 as indicated.\n\nQuery #4:\nL58-60: \"In the nonvascular liverwort Marchantia, MpPYL1 shows ABA-enhanced activity to inhibit PP2Cs, although with high basal activity9 , which triggers high basal stress signaling and actively represses plant growth15\"\n- Reference 15, whilst being a comprehensive review of the regulation of plant growth responses, does not make any reference to Marchantia or the basal activity of MpPYL1 (the focus is primarily on stress and growth regulation in angiosperms)\n\nResponse: We thank the reviewer for pointing this out. We have corrected the citation by replacing Reference 15 with the primary research article by Jahan et al. (2019), which specifically documents the growth repression by MpPYL1 in Marchantia.\n\nQuery #5:\nL61: potentially enabling broader range of ABA responses\nTo: potentially enabling a broader range of ABA responses\nL63: were subsequently incorporated into plant lineage.\nTo: were subsequently incorporated into land plant lineages.\nL81: The PYL genes from plants form monophyletic group\nTo: The PYL genes from plants form a monophyletic group\n\nResponse: We thank the reviewer for these helpful corrections. The manuscript has been revised as suggested.\n\nQuery #6:\nResults section\nL182: This partially explains the weak interaction of PrPYL-ABI1 and strong interactions between algal PYLs and Arabidopsis PP2Cs as well as the constitutive activation of RD29B-LUC expression in pyl duodecuple mutant protoplasts by algal PYLs (Fig. 1, 2, and Supplementary Fig. 2a)\n- I can see the interactions between algal PYLs and Arabidopsis PP2Cs, and the activation of RD29B-LUC in Figures 2 and Supplementary 2, but not in Figure 1.\n\nResponse: We are grateful to the reviewer for the careful reading and for identifying this citation error. We have revised the text to accurately reference the figures. The Y2H assay in Figure 1d shows the interaction of PrPYL with PP2Cs, and when contrasted with the stronger interaction of MeenPYL3 in Figure 2c, it supports our conclusion regarding the differential activities of these PYLs.\n\nQuery #7:\nL198-200: Since high basal stress signaling favored the terrestrialization of land plant progenitors, this critical arginine was not conserved during evolution to maintain high basal activity of PYLs.\nTo: Since high basal stress signaling would have favored the terrestrialization of land plant progenitors, we propose that this critical arginine was not conserved during evolution to maintain high basal activity of PYLs.\nL280: several AtPYL13-like proteins differ from the ABA-responsive PYLs by one or two amino...\nTo: several AtPYL13-like proteins differ from the ABA-dependent PYLs by one or two amino...\nL286: We evaluated the ABA responsiveness of these AtPYL13-like proteins\nTo: We evaluated the ABA dependence of these AtPYL13-like proteins\nDiscussion\nL355: algae, the ABA receptor PYLs are not present until the last common ancestor of lant plants\nTo: algae, the ABA receptor PYLs are not present until the last common ancestor of land plants\nL357-8: Our results showed that PYL-like proteins are present in bacteria, might have gained PP2C binding ability in some soil bacteria. Plants acquired PYL genes horizontally...\nTo: Our results showed that PYL-like proteins are present in bacteria, and might have gained PP2C binding ability in some soil bacteria. Plants likely acquired PYL genes horizontally...\nL365-6: Besides, the monomeric PYLs also evolved the strict ABA dependency in mosses\nTo: Independently, the monomeric PYLs also evolved strict ABA dependency in mosses\nL366-7: The ABA-binding and -responsive properties of PYLs confer ABA-dependent or ABA-enhanced PP2C-inhibition activity, which enables land plants to change…\nTo: The ABA-binding properties of PYLs confer ABA-dependent or ABA-enhanced PP2C-inhibition activity, which enabled land plants to change….\n\nResponse: We thank the reviewer for these helpful corrections. The manuscript has been revised as suggested."}]

Decision letter