FL7 is an ancient ABA-independent inhibitor of PP2C-As regulating plant stress responses

Domain: biology
Published: 2025-12-15
Editorial outcome: major_revision

Abstract
Clade A protein phosphatase 2Cs (PP2C-As) play crucial roles in plant stress responses. Although the ABA receptors PYLs inhibit PP2C-As in an ABA-dependent manner, other modulators of these phosphatases remain largely unknown. Here, we identify the FORKED-LIKE 7 (FL7) protein as a broad PP2C-A interactor that effectively suppresses PP2C activity through an ABA-independent, noncompetitive mechanism. By inhibiting PP2C-A activity, FL7 positively regulates osmotic tolerance and plant immunity in an ABA-independent manner. The N-terminal auxin canalisation (AC) domain of FL7 is required for its PP2C-As inhibitory activity. Further evolutionary analyses reveal that FL7 homologues containing an AC domain belong to an ancient family that emerged in a common ancestor of Klebsormidiophyceae algae and land plants. Genetic analyses indicate that algal FL7 homologues have a conserved function as PP2C-A inhibitors. Our study reveals an ABA-independent layer of PP2C-A modulation that regulates biotic and abiotic stress responses, which is likely conserved across a billion years of streptophyte evolution and predated the PYL-ABA regulation established in the common ancestor of land plants.

Introduction
Owing to the climate crisis, plants face ever-increasing soil salinization and periods of drought, imposing osmotic stresses that adversely affect crop growth, yield, and resistance to disease1,2. High soil salinity, in particular, heightens plant vulnerability to soil-born pathogens. For instance, irrigation with saline water significantly compromises onion resistance to Fusarium oxysporum3, while tomato and chrysanthemum grown in high-salinity nutrient solutions exhibit increased disease severity caused by Phytophthora parasitica or P. cryptogea4. This phenomenon is partially attributed to the accumulation of the phytohormone abscisic acid (ABA), which antagonizes salicylic acid (SA), a vital defence hormone, under osmotic stress conditions5,6. Consistently, ABA application induces disease susceptibility in different plants, such as Arabidopsis thaliana, tomato, and soybean, independent of the pathogen’s lifestyle7,8. These findings highlight the crosstalk between plant osmotic stress and defence responses; however, the underlying mechanisms remain largely elusive, posing a considerable challenge to the development of climate-resilient crop varieties.

During evolution, intricate signalling mechanisms have emerged through which plants exhibit appropriate responses to withstand osmotic and pathogenic stress9. Central to these stress response pathways is the reversible phosphorylation of proteins, facilitated by the coordinated actions of protein kinases and protein phosphatases10. In particular, the clade A protein phosphatase 2C (PP2C-A) family plays a pivotal role in regulating plant stress signalling networks11 and might act as a hub linking plant osmotic and defence responses. Arabidopsis encodes 9 PP2C-As, including ABA INSENSITIVE 1/2 (ABI1/2), HYPERSENSITIVE TO ABA 1/2 (HAB1/2), HYPERSENSITIVE GERMINATION 1/3 (AHG1/3), and HIGHLY ABA-INDUCED 1/2/3 (HAI1/2/3). All of the enzymes are functional phosphatases and negative regulators of the plant osmotic stress response12,13. For example, HAI1/2/3 single mutants exhibit increased proline and osmoregulatory solute accumulation at low water potential14, whereas AHG1/3 or ABI1/2 mutants exhibit increased drought tolerance15. PP2C-As also negatively regulate plant defence responses. HAI1 interacts with mitogen-activated protein kinases 3/6 (MPK3/6) and dephosphorylates them to suppress MPK3/6 activation, leading to weakened plant immunity16. ABI1 can also interact with and dephosphorylate MPK617.

PP2C-As lack regulatory subunits18,19; thus, their activities are precisely fine-tuned by those of other proteins. PYR/PYL/RCAR proteins (PYLs), which are important components of the ABA signalling pathway, are well known regulators of PP2C-As. In the absence of ABA, PP2C-As are associated with Snf1-related kinases 2 (SnRKs), including SnRK2.2/2.3/2.6, keeping them inactive by dephosphorylating their activation loops and blocking their catalytic clefts20,21. PYLs are ABA receptors: in the presence of ABA, PP2C-As form complexes with PYL-ABA, and their activities are thus inhibited22. Such inhibition releases SnRK2s from suppression by PP2C-As, leading to the induced expression of many downstream effectors, such as RD29B23. In the absence of ABA, several PYLs, such as PYL5, PYL6, PYL8, PYL9, and PYL10, can still interact with and inhibit PP2C-As, but the inhibition is weaker than that caused by ABA-bound PYLs24.

In addition to PYLs, Enhancer of ABA Co-receptor 1 (EAR1) and the ROP GTPases ROP11 can enhance the activities of a subset of PP2C-As25–27. Direct suppressors of PP2C-A phosphatase activity have also been identified. The RNA helicase RH8 and the haem-binding protein DOG1 physically associate with and suppress the activity of AHG3 and AHG1, respectively28,29. Despite the numerous enzyme activity regulators identified for PP2C-As, these regulators merely function on several specific PP2C-As. Apart from PYLs, it remains unknown whether other broad-spectrum regulators target multiple PP2C-As.

The FORKED-LIKE (FL) protein family is characterised by an N-terminal auxin canalisation (AC) domain and a C-terminal pleckstrin homology-like (PH) domain30,31. These proteins were initially discovered to be involved in leaf vein development by mediating asymmetric localisation of the auxin efflux protein PINFORMED1 (PIN1)30. Recent studies have highlighted additional functions of FL proteins in plant biology. FL1 and FL3 are interaction partners of the blue light receptor cryptochrome (CRY2). CRY2 physically interacts with FL1 and FL3 to inhibit their ability to promote root elongation32. Our group found that FL7 transcripts are present across multiple plant tissues, including leaves, roots, stems, flowers, and siliques, with the protein localising in both nuclear and cytoplasmic compartments33. Furthermore, we found that FL7 directly interacts with HAI1/2/3 and inhibits their phosphatase activity to prevent the dephosphorylation of MPK3/6, thereby promoting plant immunity33. This discovery prompted us to investigate whether FL7, similar to PYLs, functions as a general enzyme inhibitor of PP2C-As.

In this study, we provide evidence that FL7 functions as a broad-spectrum inhibitor of PP2C-As in Arabidopsis and is thus a positive regulator of the plant ABA response and osmotic tolerance. Importantly, salt stress often impairs plant resistance to pathogens, but we found that FL7-overexpressing lines presented increased resistance to salt stress. In contrast to PYLs, FL7 inhibits PP2C-A activity in an ABA-independent and noncompetitive manner. Consistent with these findings, FL7 positively regulates the osmotic response independent of ABA. FL7 potently suppresses PP2C-A phosphatase activity by directly binding to its C-terminal phosphatase domain via its conserved N-terminal AC domain. Evolutionary analyses revealed that FL7 homologues emerged in Klebsormidiophyceae green algae and expanded across streptophyte lineages. The algal FL7 homologues, KnFL7 from Klebsormidium nitens and CbFL7 from Chara braunii, had PP2C-A inhibitory activity and exhibited functions that were similar to those of AtFL7. These results indicated that FL7 represents an ancient family of PP2C-A inhibitors that are conserved throughout green plants, predating PYLs, and might integrate plant biotic and abiotic stress responses.

Results
FL7 broadly interacts with PP2C-As and inhibits their phosphatase activity
We previously found that FL7 is an inhibitor of HAI1/2/333, prompting us to test the effect of FL7 on other PP2C-A members. First, we found that FL7 could interact with all of the other six PP2C-As in both yeast two-hybrid and coimmunoprecipitation assays, using HAIs as positive controls and a clade D PP2C (At5g02760) as a negative control (Fig. 1a,b). To confirm the natural interaction between FL7 and PP2C-As in planta, we immunoprecipitated the FL7 proteins from pFL7::gFL7-FLAG fl7 complementation lines (hereafter gFL7). ABI1 and ABI2 proteins could be detected in the precipitates using α-ABI1/ABI2 antibodies (Fig. 1c). Furthermore, we conducted pull-down assays to detect the direct interaction between FL7 and PP2C-As. We successfully purified the ABI1, ABI2, HAB2, and AHG3 proteins. Unlike the clade D PP2C control, all the purified PP2C-As displayed an affinity for FL7 in the pull-down assays (Fig. 1d). These results demonstrate that FL7 is associated with PP2C-As both in vivo and in vitro. Notably, PYL10 is a broad interactor of PP2C-As24 and was also used as a positive control in the Y2H assay. We found that PYL10 interacts with all the PP2C-As except AHG1, whereas FL7 can interact with all of them (Fig. 1a).

We then tested whether FL7 can inhibit the activity of PP2C-As other than HAIs. The results revealed that FL7 inhibited between 23 and 56% of the activity of ABI1, ABI2, HAB2, and AHG3 but not that of the clade D enzyme (Fig. 1e), indicating that FL7 broadly inhibits PP2C-A activity. Given that the N-terminus of some PP2C-As, including ABI1 and AHG3, encompasses an autoinhibitory domain25, we examined whether this region is required for FL7-mediated suppression. N-terminal truncated ABI1 (131–434) and AHG3 (106–398) mutants were generated, and their phosphatase activities were higher than those of the wild-type PP2C-As, indicating successful removal of the autoinhibitory domain (Supplementary Fig. 1a). FL7 still inhibited both the PP2C-A N-terminal truncation mutants (Supplementary Fig. 1a), demonstrating that FL7 directly suppresses PP2C-A activity independent of its autoinhibitory domain.

We then measured PP2C phosphatase activity among the wild-type, fl7 and OE-FL7 lines33 in vivo (Fig. 1f). Our results demonstrated that compared with the wild-type plants, the FL7 overexpression lines presented significantly reduced PP2C activity, whereas the fl7 mutant presented the highest activity level (Fig. 1f). Collectively, these biochemical data confirm that FL7 acts as a broad-spectrum enzyme inhibitor of PP2C-As both in vivo and in vitro.

FL7 suppresses the dephosphorylation of MPK6 and SnRK2.6 mediated by PP2C-As
We further tested whether FL7 could suppress the PP2C-A-mediated dephosphorylation of its substrates. Phosphorylated MPK6 can be dephosphorylated by ABI1 (Supplementary Fig. 1b), which is consistent with a previous report17. In the presence of FL7, but not the GST control, MPK6 dephosphorylation by ABI1 was significantly restored (Fig. 1g). As a control, FL7 alone did not affect MPK6 phosphorylation (Supplementary Fig. 1c).

SnRK2s are well-known substrates of PP2C-As13. The phosphorylation level of SnRK2.6 greatly decreased in the presence of ABI1 (Fig. 1h, Supplementary Fig. 1d). However, this phenomenon was reversed by the addition of FL7 (Fig. 1h, Supplementary Fig. 1d), indicating that FL7 inhibited SnRK2.6 dephosphorylation by ABI1 in vitro. To test whether FL7 inhibits the PP2C-A-mediated dephosphorylation of SnRK2.6 in vivo, we conducted dual-luciferase reporter assays in snrk2.2/3/6 triple-mutant protoplasts (Supplementary Fig. 1e) because phosphorylated SnRK2.6 activates ABF2, which in turn induces RD29B expression23. The results revealed that the expression of SnRK2.6 increased the pRD29B-LUC output, whereas the addition of ABI1 and AHG3 suppressed the chemiluminescence signal (Supplementary Fig. 1f). PYL10 was used as a positive control in this assay, and the addition of PYL10 restored the signals inhibited by ABI1 and AHG3 (Supplementary Fig. 1f).

FL7 positively regulates the plant osmotic stress response
To investigate the function of FL7 as an inhibitor of PP2C-As, we tested the ABA sensitivity of FL7-related genotypes on the basis of the antagonistic effect between total PP2C-A activity and the ABA response25. In the absence of ABA, compared with the wild-type plants, the fl7 mutants and FL7 overexpression lines (hereafter OE-FL7) presented no obvious differences in terms of the germination rate or the primary root length (Fig. 2a, Supplementary Fig. 2a). However, compared with the wild-type plants, the ABA treatment increased the fl7 mutant germination rate and primary root growth, whereas the OE-FL7 seedlings displayed ABA hypersensitivity (Fig. 2a, Supplementary Fig. 2a). Similarly, ABA-induced stomatal closure was impaired in the fl7 mutants but enhanced in the OE-FL7 lines (Fig. 2b). These results indicated that FL7 positively regulates plant ABA responses, probably through inhibition of total PP2C-A activity.

PP2C-As are vital negative regulators of the plant osmotic response34. We thus explored the role of FL7 in plant osmotic tolerance. Under normal conditions, all the Arabidopsis lines exhibited similar growth. After 20 days of drought, the fl7 mutant displayed the lowest survival rate, whereas the OE-FL7 lines showed significantly increased survival (Fig. 2c), demonstrating that FL7 positively regulates drought tolerance. Similarly, water loss from detached leaves was also more rapid in the fl7 mutant and weakened in the OE-FL7 lines (Supplementary Fig. 2b). To obtain a genome-wide view of gene expression changes in the fl7 mutant and OE-FL7 lines in response to osmotic stress, we conducted RNA sequencing (RNA-seq) analysis on Col-0, fl7 and OE-FL7 lines treated with 300 mM mannitol. After 12 h of treatment, 2035, 2823 and 3314 differentially expressed genes (DEGs) (|Log2(fold change)| >= 1, P < 0.05) were detected in Col-0, fl7 and OE-FL7 seedlings, respectively (Supplementary Data 1). The overlapping pattern of DEGs between Col-0, fl7, and OE-FL7 seedlings suggested a common osmotic response mechanism among these lines (Fig. 2d). The k-means clustering of all DEGs between Col-0, fl7, and OE-FL7 seedlings resulted in six clusters with distinct expression patterns (Supplementary Fig. 3). Cluster 3 included genes whose expression was strongly induced by mannitol in the OE-FL7 line but weakly induced in both the Col-0 and fl7 lines. The genes in Cluster 6 exhibited the strongest mannitol-induced expression in the OE-FL7 line, moderate induction in Col-0, and the weakest induction in the fl7 mutant (Supplementary Fig. 3). Gene Ontology (GO) enrichment analysis of the DEGs in Clusters 3 and 6 revealed that these genes were highly enriched in potential abiotic stress response-related GO terms such as “response to abiotic stimulus” and “response to stress” (Supplementary Fig. 3). Moreover, these clusters are enriched in many plant osmotic response marker genes, such as ABI1/2, RD29B and RAB1835 (Supplementary Fig. 3). These marker genes generally exhibited the strongest mannitol-induced expression in the OE-FL7 line, moderate induction in Col-0, and the weakest induction in the fl7 mutant (Fig. 2e). These results indicated that FL7 positively regulates the plant osmotic response.

Enhanced pathogenic resistance of FL7-overexpressing lines is not impaired by salt stress
We previously reported that FL7 is a positive regulator of plant immunity33. Combined with the data presented here, these findings reveal that FL7 is a positive hub regulator of both the osmotic response and the defence response, indicating that FL7 might be a promising genetic resource for the development of resistant plants suitable for saline soils. We first tested the role of FL7 in plant salt tolerance and found that FL7 promotes plant tolerance to salinity stress caused by NaCl through assays of electrolyte leakage (Fig. 2f). Activation of SnRK2s is a central step in the plant response to salt stress, and we found that NaCl-induced SnRK2.2, SnRK2.3, and SnRK2.6 phosphorylation was reduced in the fl7 mutant but increased in the OE-FL7 lines (Supplementary Fig. 4a). To test whether FL7 responded to salt stress, we analysed its protein accumulation level in OE-FL7 and gFL7 plants after NaCl treatment. The FL7 protein level increased with treatment (Supplementary Fig. 4b). These results indicated that FL7 responds to and positively regulates plant salt tolerance.

We next tested whether FL7 could promote plant immunity under salt stress. Four-week-old Arabidopsis plants were irrigated with water that contained 100 mM NaCl to simulate soil salinization, and the leaves were detached for the inoculation assay at 24 h post-treatment (Supplementary Fig. 4c). In the absence of NaCl treatment, the fl7 mutant was more susceptible, whereas the OE-FL7 line was more resistant to Phytophthora capsici (Fig. 2g), supporting FL7 as a positive plant immune regulator. After NaCl treatment, the susceptibility of the wild-type, fl7 mutant and gFL7 complementation lines to P. capsici significantly increased (Fig. 2g), which is consistent with previous findings that soil salinization increased disease severity caused by Phytophthora parasitica and P. cryptogea4. Interestingly, no obvious lesion area changes could be detected in the OE-FL7 lines after mock or NaCl treatment, indicating that the degree of immune weakness caused by NaCl treatment greatly decreased in the OE-FL7 plants (Fig. 2g).

Genetic analysis of fl7 with the pp2c-As mutants
To further analyse the genetic relationship between FL7 and PP2C-As, we knocked out FL7 in the abi1 hab1 pp2ca triple mutant using CRISPR-Cas9, generating an abi1 hab1 pp2ca fl7 quadruple mutant (Supplementary Fig. 4d). The abi1 hab1 pp2ca triple mutant is sensitive to ABA during seed germination and root growth (Fig. 3a,b), which is consistent with previous reports15. Although fl7 showed reduced ABA sensitivity compared with wild-type, the abi1 hab1 pp2ca fl7 quadruple mutant exhibited a phenotype comparable to that of the abi1 hab1 pp2ca triple mutant (Fig. 3a,b). Similarly, a drought tolerance analysis revealed that the abi1 hab1 pp2ca fl7 quadruple mutant displayed a phenotype comparable to that of the abi1 hab1 pp2ca triple mutant, with both showing enhanced drought tolerance (Fig. 3c). These results indicate that FL7 functioning is dependent mainly on PP2C-As.

FL7 inhibits PP2C-As in an ABA-independent and noncompetitive manner
ABA promotes the inhibition of PP2C-As by PYLs22. However, our phosphatase activity indicated that the inhibitory function of FL7 is independent of ABA (Fig. 1e)—in contrast to that of canonical PYLs. To further test whether ABA is required for FL7 function, a microscale thermophoresis binding assay was conducted by using PYL10 as a positive control. The results revealed an interaction between ABA and PYL10 but no binding affinity for ABA for the FL7 and GST control (Fig. 4a). We then tested whether ABA influences the interaction between FL7 and PP2C-As. Consistent with previous findings22, ABA stimulated PYL10-ABI1 complex formation (Fig. 4b). In contrast, ABA did not affect FL7-ABI1 associations (Fig. 4b). We next investigated whether ABA regulates FL7 inhibition of PP2C-A activity. Enzymatic analyses demonstrated that ABA bolstered PYL10-mediated but not FL7-mediated inhibition of ABI1 (Fig. 4c). Similar ABA-independent inhibition activity was observed for FL7 when ABI2 and AHG3 were used (Supplementary Fig. 5a,b). Furthermore, ABA did not enhance the ability of FL7 to suppress ABI1-mediated dephosphorylation of SnRK2.6 (Fig. 1h, Supplementary Fig. 1d). These results indicated that the inhibitory activity of FL7 on PP2C-As is independent of ABA.

To elucidate the biochemical mechanisms underlying the inhibitory activities of FL7 and PYL10, we performed enzyme kinetic analyses (Fig. 4d). Competitive inhibitors compete with substrate(s) for the active site of the enzyme, and the inhibition is reversible upon substrate saturation. In contrast, noncompetitive inhibitors alter active site conformation or chemistry to reduce substrate binding/catalysis independently of substrate levels (Supplementary Fig. 5c)36. Consistently, ABA did not increase FL7 inhibition in this assay (Fig. 4d). In contrast to the reversible inhibition of ABI1 by PYL10 upon substrate saturation despite the presence of ABA, FL7-mediated ABI1 suppression was unresponsive to both increased substrate availability and increased ABA treatment (Fig. 4d). Identical patterns also occurred for AHG3 (Supplementary Fig. 5d), corroborating a noncompetitive inhibition mechanism for FL7.

FL7 enhances plant osmotic stress tolerance through an ABA-independent pathway
To investigate whether FL7 regulates the plant osmotic response independently of ABA, we crossed the ABA-deficient aba1-1 mutant with fl7 to generate the fl7 aba1-1 double mutant (Supplementary Fig. 5e,f). We found that, in the absence of ABA, the stomata of aba1-1 were more open than those of fl7 and Col-0 were, while the stomatal aperture of the fl7 aba1-1 double mutant was the largest (Fig. 4e). Drought tolerance assay revealed that compared with the aba1-1 single mutant, the fl7 aba1-1 double mutant had lower survival rates (Fig. 4f, Supplementary Fig. 5g). These results further support that FL7 suppresses PP2C-A activity and positively regulates the plant osmotic response in an ABA-independent manner.

PYLs function as ABA receptors and broad suppressors of PP2C-As22. To examine the relationship between FL7 and PYLs, we employed ABA ANTAGONIST 1 (AA1), a broad-spectrum antagonist that blocks all PYR/PYL-PP2C interactions37 (Fig. 4g). We analysed the germination rates of Col-0 and fl7 mutant seeds grown on 1/2 MS plates supplemented with either ABA alone or ABA plus AA1. The addition of AA1 significantly increased the germination rate of Col-0, indicating successful inhibition of PYL activity (Fig. 4h). Notably, the germination rate of fl7+AA1 was higher than that of Col-0+AA1 (Fig. 4h), suggesting that FL7 continues to regulate the PP2C pathway even when PYL activity is blocked. In addition, we found that the presence of both PYL and FL7 led to a stronger inhibition of ABI1 phosphatase activity compared with either component alone, indicating an additive effect (Supplementary Fig. 5h). Collectively, these data support that FL7 and PYLs function in parallel pathways.

FL7 inhibits PP2C activity through an N-terminal auxin canalisation domain
The FL7 protein contained an N-terminal auxin canalisation domain (AC) and a C-terminal pleckstrin homology-like domain (PH) (Fig. 5a). To investigate which domain of FL7 is critical for the inhibitory activity on PP2C-As, we generated FL7 truncation mutants (FL7N and FL7C) (Fig. 5a). ABI1 and AHG3 were selected as representatives of PP2C-As to test their interactions with FL7 truncation mutants. The results showed that the AC domain, but not the PH domain, sufficiently interacted with the phosphatase domain of the tested PP2C-As in a Y2H assay (ABI1N for the autoinhibition domain; ABI1C for the phosphatase domain) (Fig. 5a, Supplementary Fig. 6a). Similarly, the AC domain was sufficient to suppress the activities of ABI1 and AHG3 (Fig. 5b, Supplementary Fig. 6b), and this inhibitory activity was not enhanced by ABA (Fig. 5c, Supplementary Fig. 6c). Furthermore, we added the AC domain or the PH domain of FL7 under the control of the native promoter of FL7 to the fl7 mutant line (Supplementary Fig. 6d). The AC domain, but not the PH domain, completely restored the fl7 mutant phenotype, while overexpression of the AC domain mimicked the phenotype of the OE-FL7 lines (Fig. 5d–f, Supplementary Fig. 6e–g). Compared with the OE-FL7 lines, the protein extracts of OE-FL7N lines also exhibited impaired PP2C activity (Fig. 1f). These results indicated that the AC domain is sufficient for the suppression of PP2C activity.

FL7 emerged in an ancient streptophyte
When did FL7 evolve? To investigate the conservation of FL7, we performed comparative genomic and phylogenetic analyses of the AC domain in the genomes of 17 representative plant species that cover a broad diversity of plants (Fig. 6a; Supplementary Data 2). Using a Markov model of the AC domain generated by the Pfam database, we identified a total of 32 FL7 homologues with the AC domain in the genomes of 9 plants (Fig. 6a; Supplementary Data 3 and 4). The number of FL7 homologues we identified in Arabidopsis aligns with previous reports30, bolstering the identification stability of our approach. In addition to the land plant clade, homologues of FL7 were found in the streptophyte algal families Klebsormidiophyceae and Charophyceae but not in the phragmoplastophytic Coleochaetophyceae and Zygnematophyceae or the clade of Mesostigmatophyceae and Chlorokybophyceae (Fig. 6a). We performed a large-scale phylogenetic analysis of these proteins with the AC domain. We found that plants formed a monophyletic group with high support values (Fig. 6a). The copy number of FL7 homologues is low in algal and bryophyte species but is expanded in vascular plants (Fig. 6a, b), indicating that FL7 homologues are monophyletic.

PYLs originated in the common ancestor of Zygnematophyceae and land plants38 and were likely gained by horizontal gene transfer from soil bacteria39. Interestingly, FL7 homologues were identified in Klebsormidiophyceae, suggesting that they emerged in a last common ancestor (LCA) of Klebsormidiophyceae and Phragmoplastophyta, which likely lived more than a billion years ago40. These findings indicate that FL7 homologues might be more ancient PP2C inhibitors than PYLs are. To further study FL7 homologues in streptophyte algae, we performed similarity searches and phylogenetic analyses within six charophyte transcriptomes41,42. We found that FL7 homologues are present in representatives of two other charophyte genera: Nitella, another Charophyceae, and, importantly, Coleochaete, a Coleochaetophyceae (Supplementary Data 5). These findings further underscore the emergence of FL7 homologues in the LCAs of Klebsormidiophyceae and Phragmoplastophyta. Since the absence of one gene in the transcriptome of one species does not guarantee its absence in that species43, it might be that FL7 homologues are even more widespread in streptophytes. Our results further demonstrate that the AC domain is widely present in streptophyte algae. Although some plant FL7 homologues lack a PH domain (Fig. 6a; Supplementary Data 4), these proteins cluster with those with PH domains in the phylogenetic tree (Fig. 6a), indicating that they might have arisen from AC-PH proteins by deleting PH. Taken together, our comparative genomic analyses provide evidence for the deep evolutionary origin of FL7 homologues in a streptophyte predating PYLs (Fig. 6b).

FL7 function is conserved across a billion years of streptophyte evolution
PYL was an evolutionary innovation of a common ancestor of Zygnematophyceae and land plants39,44. Interestingly, our evolutionary analysis revealed that compared with PYLs, FL7 homologues are more ancient PP2C inhibitors. To investigate their conserved function across streptophytes, we synthesised FL7s from the two streptophyte algae Klebsormidium nitens and Chara braunii. Y2H and pull-down assays revealed that, like AtFL7, algal FL7s also interact with AtABI1 (Fig. 7a,b). Furthermore, algal FL7 inhibited the activity of AtABI1 (Fig. 7c), indicating that the function of algal FL7 is conserved with AtFL7. We constructed transgenic plants in which the algal FL7s were under the control of the AtFL7 promoter and introduced them into the fl7 mutant (Supplementary Fig. 7a,b). Compared with the wild-type plants, the complemental lines presented a similar osmotic response (Fig. 7d,e). Thus, our results indicated that FL7 is a functionally conserved PP2C-A inhibitor across a billion years of streptophyte evolution.

Methods
Plant materials and growth conditions
Arabidopsis plants were grown and maintained in plant growth chambers at an ambient temperature of 23°C under a 16-h light/8-h dark photoperiod. Light was provided by white fluorescent bulbs with an intensity of ~120 μmol/m2/s. All Arabidopsis lines used in this study were from a Col-0 accession. T-DNA insertion mutants were obtained from Arashare (https://www.arashare.cn/index/).

We previously obtained fl7, OE-FL7, gFL7, abi1 hab1 pp2ca and snrk2.2/3/6 triple mutants33,52,53. The 1000 bp FL7 promoter (pFL7) was amplified from Col-0 genomic DNA. The sequences of KnFL7 and CbFL7 were synthesised by a biological company. The coding sequences of FL7 and PYL10 were subsequently cloned via RT‒PCR. To generate complementation transgenic lines expressing FLAG-tagged pFL7:FL7N, pFL7:FL7C, pFL7:KnFL7 and pFL7:CbFL7, DNA fragments encoding these sequences were fused to a C-terminal 3×FLAG tag and cloned and inserted into the pCambia1300 vector. To generate transgenic lines overexpressing FLAG-tagged FL7N, the sequence encoding FL7N was fused to a C-terminal 3×FLAG tag and cloned and inserted into the pCambia1300 vector. All the constructs were subsequently introduced into Agrobacterium (Agrobacterium tumefaciens) strain GV3101 for plant transformation using the floral dip method. Positive transformants were recovered by antibiotic selection and confirmed by genomic PCR. The abi1 hab1 pp2ca fl7 quadruple mutant was generated by knocking out FL7 in the abi1 hab1 pp2ca triple mutant using CRISPR-Cas9. To generate the fl7 aba1-1 double mutant, an fl7 T-DNA insertion mutant (SALK_07717C) was crossed with an aba1-1 T-DNA insertion mutant (SALK_027326C). The primers used in this study are listed in Supplementary Data 6.

For the root growth assay, the corresponding seeds were germinated on 1/2 MS medium. After incubation at 4°C for 3 days, the seeds were transferred to a greenhouse with a 16-h light/8-h dark cycle for an additional 3 days. Seedlings with similar root lengths were then transferred to 1/2 MS medium supplemented with various concentrations of ABA and vertically grown for an additional 5 days. The root lengths were measured using ImageJ software.

For the germination assays, seeds were sown on 1/2 MS medium supplemented with ABA and incubated at 4°C for 3 days. Unless otherwise stated, a concentration of 0.2 μM ABA was used for all germination assays. The plates were then transferred to a greenhouse and grown for an additional 7 days before determining the seed germination ratio. For the experiment shown in Fig. 4h, a concentration of 0.3 μM ABA was employed, as this assay was conducted following the relocation of our laboratory, during which it was determined that 0.2 μM ABA was insufficient to effectively suppress seed germination.

Drought-stress tolerance
To assess drought tolerance, the seedlings were transferred to plastic pots, grown for 3 weeks under short-day conditions, and then subjected to drought conditions by withholding water from the plants for 20 days. The drought-induced phenotypes were photographed with a camera. The survival rates of each genotype were analysed at 2 days after rewatering.

Stomatal aperture measurements
For the stomatal aperture assay, leaves were collected from 5-week-old plants and incubated in MES buffer (10 mM MES-KOH (pH 6.15), 10 mM KCl, and 50 μM CaCl2) under light for 3 h at 23°C to open the stomata. To examine ABA-induced stomatal closure, 20 μM ABA was added to the MES buffer, which was subsequently incubated for an additional 2 h under the same conditions. In each individual sample, at least 80 stomata were randomly observed, and images were obtained with a microscope. The stomatal aperture was measured using ImageJ software.

Measurement of water loss
Three-week-old seedlings grown under short-day conditions (16-h light/8-h dark) were used for water loss measurements. The whole rosettes were cut from the plants and weighed at the indicated points.

Ion leakage assay
Ion leakage induced by cell death was analysed as previously described54. In brief, three 9-mm leaf discs were rinsed three times with distilled water and then soaked in 9 mL distilled water for 3 h at room temperature. The conductivity of the solution was measured using a conductivity meter to generate S1. The leaf discs were then boiled for 20 min, after which the conductivity was measured again to determine the value of S2. Electrolyte leakage was calculated as the percentage (%) of “S1”/“S2”. The assay was repeated three times.

Immunoblot assay
To extract proteins from plant materials, leaves were frozen in liquid nitrogen and ground to a fine powder. Extraction buffer (50 mM HEPES, 150 mM KCl, 1 mM EDTA, and 0.1% [v/v] Triton X-100; pH 7.5) containing 1 mM DTT and protease inhibitor cocktail (Sigma) was used for protein extraction. Anti-GFP antibody (1:5000; #M20004; Abmart) and HRP-conjugated anti-mouse IgG (1:10,000, Sigma‒Aldrich) secondary antibody were used for immunoblotting protein with a GFP tag. mAb-HRP-DirecT anti-FLAG antibody (1:5000; #M185-7; MBL) was used for immunoblotting proteins with a FLAG tag. mAb-HRP-DirecT anti-HA antibody (1:5000; #M180-7; MBL) was used for immunoblotting proteins with an HA tag.

Yeast two-hybrid assay
To detect the interaction between FL7 and PP2C-As, the cDNA fragments of FL7 and PP2C-As were amplified by PCR and cloned and inserted into the pGBKT7 and pGADT7 vectors, respectively. The bait and prey constructs were cotransformed into the yeast strain AH109. Successfully transformed colonies were selected on yeast SD medium lacking Trp and Leu. Transformants were subsequently transferred to selective SD medium lacking Trp, Leu, and His for growth analysis. Photographs were taken after 4 days of incubation at 30°C.

Co-IP assay
Arabidopsis protoplasts were transfected with the indicated plasmid combinations and control constructs, and transfection was performed as described above. Immunoprecipitation with anti-FLAG antibodies was carried out as previously described55. Total protein and immunoprecipitates were separated by SDS–PAGE and detected by immunoblotting.

Protein purification
Proteins with different tags were expressed in Rosetta (DE3) Escherichia coli cells growing in LB medium supplemented with 0.5 mM IPTG for 18 h at 18°C. The culture was then collected by centrifugation at 6000×g for 10 min at 4°C. For His-tagged proteins, the pellet was resuspended in lysis buffer (50 mM NaHPO4, 30 mM NaCl, 10 mM imidazole (pH 8.0), 1 mM PMSF, and protease inhibitor cocktail [Abmart, A10004]) and then sonicated for 10 min. After centrifugation for 30 min at 7000g and 4°C, the supernatant was incubated with Ni Sepharose (GE) resin for 3 h at 4°C. The resin was subsequently washed three times with washing buffer (50 mM NaH2PO4, 30 mM NaCl, and 50 mM imidazole; pH 8.0) to remove nonspecifically bound proteins. The His-tagged proteins were eluted from the resin with elution buffer (50 mM NaHPO4, 30 mM NaCl, and 250 mM imidazole, pH 7.4). For the GST-tagged proteins, the pellet was resuspended in phosphate-buffered saline (PBS) (150 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl (pH 7.4), 1 mM PMSF, and inhibitor cocktail) and then sonicated for 10 min. After centrifugation for 30 min at 7000g and 4°C, the supernatant was incubated with glutathione Sepharose 4B (GE) resin for 3 h at 4°C. The resin was subsequently washed three times with PBS to remove nonspecifically bound proteins. The GST-tagged proteins were eluted from the resin with GSH buffer (50 mM Tris-HCl, pH 8.0, and 10 mM GSH). For MBP-tagged proteins, the pellet was resuspended in lysis buffer (20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 1 mM PMSF, and inhibitor cocktail) and then sonicated for 10 min. After centrifugation for 30 min at 7000g and 4°C, the supernatant was incubated with amylose resin (NEB) for 3 h at 4°C. The resin was subsequently washed three times with lysis buffer to remove nonspecifically bound proteins. The MBP-tagged proteins were eluted from the resin with elution buffer (20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, and 10 mM maltose). To measure their concentrations, the purified proteins were mixed with Quick Start Bradford Dye Reagent (Bio-Rad), and the absorbance was measured at 595 nm. Protein concentrations were calculated on the basis of a standard curve with bovine serum albumin (BSA) as a substrate.

Protein pull-down assay
The coding sequences of the genes of interest were subsequently cloned and inserted into the MBP vector or the pGEX-6P vector. Recombinant MBP-PP2C, GST, and GST-FL7 proteins were purified as described above. Purified GST or GST-FL7 (10 mg) in 2 mL of lysis buffer (150 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl (pH 7.4), 1 mM PMSF, and inhibitor cocktail) was incubated with glutathione beads for 2 h at 4°C. After a brief centrifugation at 1000×g for 3 min at 4°C, the buffer was removed, and 2 mg of recombinant MBP-PP2C protein was added to the resin in 2 mL of lysis buffer. The tube was rotated at 4°C for 2 h for protein binding. The resin was then washed five times with PBS to remove nonspecifically bound protein, resuspended in 50 mL of PBS and 10 mL of SDS loading buffer, and boiled for 5 min. After centrifugation at 12,000×g for 1 min at 22°C, the supernatant was subjected to immunoblotting analysis. Immunoblot assays were conducted with anti-GST (1:5000; #M20007; Abmart) or anti-MBP (1:5000; #M20001; Abmart) antibodies and HRP-conjugated anti-mouse IgG (1:10,000; Sigma‒Aldrich) as the secondary antibody.

MST assay
The interactions between ABA and recombinant GST-FL7, GST-PYL10, and GST were measured by MST (microscale thermophoresis). MST assays were performed as previously described56.
First, the purified GST, GST-FL7, and GST-PYL10 sample proteins were exchanged for buffer with labelling buffer (130 mM NaHCO3, 50 mM NaCl, pH 8.2–8.3) and then labelled with dye in assay buffer using the Protein Labelling Kit RED-NHS 2nd Generation (Cat# MO-L011). ABA at a range of concentrations (0.3 μM to 10 mM) was incubated with 2.5 μM labelled protein for 30 min at room temperature, after which the samples were loaded onto NanoTemper glass capillaries (NanoTemper Technologies), and MST was performed. The experimental data were processed, and the Kd values for ABA-FL7, ABA-PYL10, and ABA-GST binding were calculated through MO. Affinity Analysis software.

In vitro dephosphorylation of MAPK
The Escherichia coli strain Rosetta (DE3) was used for the production of recombinant proteins. To prepare phosphorylated MPK6, 5 μg of recombinant purified MBP-tagged kinase-inactive MAPK was incubated with 1 μg of GST-MKK4DD at 30°C for 1 h in kinase buffer (25 mM Tris–HCl (pH 7.5), 1 mM DTT, 10 mM MgCl2, and 200 μM ATP). The kinase reaction was terminated by removing ATP from the reaction using an Amicon Ultra 30K centrifugation unit (Millipore). To test the phosphatase activity of ABI1, phosphorylated MAPK (500 ng) was incubated with MBP or MBP-ABI1 (2 μg) at 30°C for 15 min in phosphatase buffer (25 mM Tris-HCl (pH 7.5), 1 mM DTT, and 10 mM MgCl2). MAPK was detected by immunoblotting as described above. To test whether FL7 inhibits the phosphatase activity of ABI1, phosphorylated MAPK (500 ng) and MBP-ABI1 (2 μg) were incubated with GST or GST-FL7 at 30°C for 15 min in phosphatase buffer (25 mM Tris-HCl (pH 7.5), 1 mM DTT, and 10 mM MgCl2).

Measurement of PP2C-A activity
In vitro PP2C-A activity was measured using a Ser/Thr Phosphatase Assay Kit (Promega, V2460). In brief, the recombinant proteins were purified with buffers that did not contain phosphate. To measure phosphatase activity, different combinations of recombinant purified FL7 and PP2Cs were mixed with 5 mL of 1 mM phosphopeptide and 10 mL of PP2C reaction buffer (250 mM imidazole (pH 7.2), 1 mM EGTA, 25 mM MgCl2, 0.1% [v/v] β-mercaptoethanol, and 0.5 mg/mL BSA) in the wells of a microtiter dish and then incubated at 25°C for 15 min. The reactions were stopped by the addition of 50 mL of molybdate dye/additive mixture, after which the plate was incubated at room temperature for 15 min. The absorbance was measured at 630 nm in a microplate reader (Molecular Devices, SpectraMax M5). Michaelis–Menten plots in GraphPad Prism 9.0 were used to calculate the kinetic parameters.
To measure PP2C activity in vivo, total proteins (0.1 g) from the wild type or fl7, OE-FL7, or OE-FL7N mutants were extracted using phosphatase storage buffer (20 mM Tris–HCl (pH 7.5), 20 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 mM DTT, 0.5% [v/v] Triton X-100, and 50% [v/v] glycerol) with a protease inhibitor cocktail (Abmart, A10004), after which the homogenised lysate was centrifuged at 100,000×g for 1 h at 4°C. The supernatant was filtered through Sephadex G-25 resin to remove endogenous phosphates, after which the lysate was used to measure PP2C activity as described above, but 5 mM okadaic acid was added to inhibit the activity of PP1 and PP2A family Ser/Thr-specific phosphoprotein phosphatases25.

In vitro kinase assays
The recombinant protein was expressed in Rosetta (DE3) and affinity purified according to the instructions described above. Two micrograms of GST or GST-FL7 proteins were incubated with 0.5 μg of MBP-ABI1 with or without 0.5 μM ABA in 20 μL of phosphatase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM EGTA and 0.1% [v/v] β-mercaptoethanol). After 15 min of incubation, 1 μg of SnRK2.6-HIS protein was added to the reaction buffer and incubated for another 15 min. Then, the reaction buffer was mixed with 1 μM ATP and phosphatase inhibitor cocktail 3 to a total volume of 25 μL and incubated for 30 min at 25°C. The reactions were stopped by the addition of SDS loading buffer, and the mixtures were separated by SDS–PAGE. The anti-phospho-S175-SnRK2 antibody was previously reported (1:5000)52. The protein level was analysed by Coomassie Brilliant Blue (CBB) staining.

SnRK2.6 kinase activity in vivo
To assess the kinase activity of snRK2.6 in vivo, total protein was extracted from Col-0, fl7 and OE-FL7 with lysis buffer (100 mM HEPES (pH 7.8), 10 mM Na3VO4, 10 mM NaF, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 1 mM PMSF, and 5% glycerin and protease inhibitor cocktail), separated by SDS–PAGE and detected by immunoblotting with anti-phosphorylation antibodies against Ser175 in SnRK2.635.

Arabidopsis protoplast preparation and transfection
Fully expanded rosette leaves from 4-week-old Col-0 plants were enzymatically digested to release protoplasts55, which were washed and resuspended at ~2×106 cells/ml. For each transfection, 100 μl protoplasts (~2×105 cells) were mixed with 20 μg plasmid DNA and an equal volume of PEG solution (40% PEG 4000, 0.2 M mannitol, 100 mM CaCl2), incubated 10–15 min, then diluted with W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES; pH 5.7) and recovered. Transfected protoplasts were incubated in the dark at 22°C for 12–24 h before downstream assays.

Phylogenetic analysis
Multiple alignments of full-length aa sequences were aligned using MUSCLE. Phylogenetic analysis was performed using full-length sequences with the MEGA X program by the maximum-likelihood method with 100 bootstrap samples and the following parameters: Poisson model, uniform rates, and complete deletion.

RNA-seq analysis
For RNA sequencing, total RNA from nine-day-old seedlings treated with or without 300 mM mannitol on 1/2 MS solid medium for 12 h35 was extracted using an RNA-Simple Total RNA Kit (Zoman Biotech) and prepared for RNA sequencing at Novogene (Beijing, China). The sequencing data were initially filtered, after which the clean reads were mapped to the Arabidopsis genome using HISAT257. The transcripts per million (TPM) value of each gene was quantified using the Stringtie tool57. DESeq2 facilitated the identification of differentially expressed genes (DEGs) across samples, focusing on genes that exhibited a |log2(fold change)| ≥ 1 and a P ≤ 0.05 as DEGs58. Gene expression patterns were clustered using the pheatmap package in R, employing the ward.D2 clustering algorithm. GO enrichment analyses were conducted with the GO.db package in R.

Statistical analysis
All data are shown as the means ± standard deviations (SD) from at least three biological replicates or from three technical replicates in one of three experiments with similar results. Two-tailed Student’s t test was used to compare the means between two samples (* and ** represent P < 0.05 and 0.01, respectively). One- or two-way analysis of variance (ANOVA) was used to test the significance of the difference among different group means (different lowercase letters indicate significant differences; P < 0.05). Detailed statistical reports are provided in the source data for the figures.

Accession numbers
Sequence data from this article can be found in the TAIR (The Arabidopsis Information Resource) databases under the following accession numbers: FL7 (At4g16670), ABI1 (At4g26080), ABI2 (At5g57050), HAB1 (At1g72770), HAB2 (At1g17550), HAI1 (At5g59220), HAI2 (At1g07430), HAI3 (At2g29380), AHG1 (At5g51760), AHG3 (At3g11410), PP2C-D (At5g02760), PYL10 (At4g27920), SnRK2.6 (At4g33950), MPK6 (At2g43790), MKK4 (At1g51660), RD29B (At5g52300), AFP1 (At1g69260), AtNAP (At1g69490), P5CS1 (At2g39800), SAG13 (At2g29350), ABF2 (At1g45249), COR413IM1 (At1g29395), EGY3 (At1g17870), GOLS2 (At1g56600), LEA7 (At1g52690), PNP-A (At2g18660), RAB18 (At5g66400), SINA2 (At3g13672) and ABA1 (At5g67030).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Source data and uncropped western blot membranes are provided with this paper. The RNA-seq raw read data generated in this study have been deposited in the National Genomics Data Center database under accession code CRA020685. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Li et. al reported the function of FL7 as a potent ABA-independent Clade-A PP2Cs predated to ABA-receptor PYLs in the evolution of plants. The experiments were logically carried out and the presented data were scientifically sounds. This report infer that FL7 is a general Clade-A PP2C inhibitor including algae, which contains species without PYL, and explain the ancient regulation mechanisms of Clade-A PP2C before the emergence of ABA receptor PYL, which is now a major inhibitor of Clade-A PP2C in ABA-dependent manner. Interestingly, overexpression of FL7 improved tolerance to pathogen infection under osmotic stress, offering FL7 as a good genetic resource for crop breeding. Nevertheless, I have several questions.\n1) The authors explained in the introduction that salt stress impairs plant resistance to pathogen; however, FL7 is a positive regulator of salt stress response as well as pathogen infection according to OE-FL7 phenotypes. Actually, FL7 accumulates in response to salt stress in WT Col-0; however, salt stress treatment impaired pathogen resistance of Col-0 (Fig. 3h). The reviewer questioned OE-FL7 phenotypes might caused by abnormal accumulation of FL7? How much FL7 accumulates in the OE plants? Authors need to explain this inconsistency.\n2) According to the results shown by authors, FL-7 homologues appeared to repress PP2C activity if they contained AC domain. If so, why single disruptant of FL7 showed phenotypes despite the gene redundancy in Arabidopsis. Does multiple disruptants of FL7 homologs show more severe phenotypes in stress treatment?\n3) Authors concluded that FL7 activity toward Clade-A PP2C is ABA-independent. I agree that FL7 does not bind ABA. But what will happen if PYLs were included in the assay such as Fig. 2d? Is there any synergistic effect between PYL and FL7?\n4) I just wonder why OE-FL7 showed normal germination in the absence of ABA? The Clade-A PP2C activity should be reduced in OE-FL7 plants without ABA treatment. Actually, stomata of OE-FL7 were more closed than WT after H2O2 treatment. This is also the case for SnRK2 activity (Fig. 2f). There was no enhancement of SnRK2 phosphorylation in mock treatment. Did authors ever performed in-gel phosphorylation assay to measure SnRK2 activity?\n5) In the figure legends, Fig. 1g appeared to be a immunoblot analysis, but in M&M, it says ATP assay. Which is true?\n6) Dual LUC assay (Figs. 1h and S1e) requires control experiment without SnRK2.6 to ensure that the promoter activation is mediated by SnRK2.6.\n6) The authors need to explain about FL7 in detail in the introduction, including expression pattern at the tissue level and subcellular localization.\n7) Even acquisition of PYL by plants to regulate Clade-A PP2C in an ABA-dependent way, copy numbers of FL7 homolog has been increased during land plant evolution. What is an authors' idea to explain this?\n8) Transcriptome analysis of fl7 mutants and OE-FL7 plants in response to ABA and/or osomotsress will require to evaluate the role of FL7 in these responses.", "reviewer_2": "In the manuscript titled \"An Ancient Stress-Regulatory Suppressor of PP2C-As Predating PYL-ABA Signaling,\" the authors have uncovered FL7 protein as a versatile PP2C-A interactor that potently inhibits PP2C activity through an ABA-independent, non-competitive mechanism. By suppressing PP2C-A activity, FL7 positively modulates plant ABA responsiveness and enhances osmotic tolerance. Notably, the N-terminal auxin canalization (AC) domain of FL7 is essential for its PP2C-As inhibitory function. Further evolutionary insights reveal that FL7 homologs harboring the AC domain belong to an ancient family that originated in the common ancestor of Klebsormidiophyceae algae and land plants. Genetic analysis corroborates that algal FL7 homologs retain a conserved role as PP2C-A inhibitors. The authors thus unveil an ABA-independent layer of PP2C-A modulation crucial for stress response, which appears to be conserved over a billion years of streptophyte evolution and predates the PYL-ABA regulatory system established in the common ancestor of terrestrial plants. While the data presented are robust and the findings compelling, there are nevertheless some concerns that merit attention.\nMajor points:\n1. The omission of introducing FL7 in the Abstract and Introduction sections is a notable gap. Incorporating a concise yet informative description of FL7's role and significance in the study's context would strengthen the presentation.\n2. To refine the interpretation of soil drying data and ensure uniformity in drought conditions across genotypes, it is imperative that the authors incorporate measurements of soil water potential or water content. This adjustment will fortify the manuscript by providing a more rigorous and comparable framework for evaluating the drought response of different genotypes.\n3. Clarifying the ABA concentration used in the stomatal aperture measurements is crucial. Given the inconsistency between the stated concentrations (50 μM versus 20 μM， both of them are too high), the authors should verify the actual concentration employed and make the necessary corrections throughout the manuscript. Additionally, enhancing the visibility of stomata in the figures by enlarging them and clearly marking the statistical length and width positions would improve the accuracy and interpretability of the data. The authors should also reconcile the discrepancy between the representative stomatal images and statistical results in Figures 4e and 6d, ensuring that the representative pictures accurately reflect the quantitative findings. This consistency is essential for maintaining the integrity of the scientific conclusions drawn from the study.\n4. While the manuscript presents robust molecular and biochemical evidence for the regulation of PP2C-A by FL7, a notable gap remains in the form of genetic evidence directly linking FL7 and PP2C-A within a functional pathway. To strengthen the conclusions, the authors are encouraged to pursue genetic approaches that can definitively establish the interplay between these two factors.\n5. The authors have convincingly demonstrated FL7's inhibition of PP2Cs activity through its N-terminal auxin canalization domain using in vitro assays. However, to fully substantiate this mechanism, in vivo validation is essential. Therefore, it is recommended that the authors conduct additional experiments aimed at assessing phenotypic changes or measuring PP2C phosphatase activity directly within plants. Such evidence would provide a more comprehensive understanding of FL7's mode of action and its physiological relevance."}, "author_response": "Re: Thanks for your positive comments and constructive suggestions! We have carefully studied the following comments, added some experiments, corrected the typos and rephrased many parts of text. Please see our responses which are shown below in bold.\n\n[... Author response text for Round 1 ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors adequately responded to my comments to the previous manuscript, and mostly I satisfied with their responses.\nAdditional comments to the responses.\nResponse 1:\nI still doubt about the role of endogenous FL7 in defense response under salinity condition. Their additional results clearly indicate that the degree of fungal resistance under salinity is not proportional to FL7 protein level, suggesting that endogenous FL7 does not play an important role in immune response under salinity condition. FL7 accumulation level prior to salinity stress might be critical. Nevertheless, I do not argue that FL7 is a good genetic resource to improve plant defense response under salinity condition.\nResponse 2:\nAt least, the authors should mention the possibility of functional redundancy in the text.\nResponse 3:\nNewly added Fig. 1f contains OE-FL7N but no mention in the text. Need revision. Also, the placement is not appropriate. The order of Figure placement should be consistent.\nResponse 5:\nI understand but need the explanation of the nature of pSnRK2 antibody in the method.\nOther comments\n1) There still many typo and/or grammatical errors. Need careful revision.\n2) PYL first appeared in L94 but the explanation was in L99. Please explain where it first appeared. This is also true for FL (L115).\n3) L207-208 is already mentioned in previous text and should be removed.", "reviewer_2": "The manuscript by Li et al. identifies FL7 as a broad-spectrum, ABA-independent inhibitor of clade A PP2C phosphatases and demonstrates its conserved function from streptophyte algae to land plants. The study combines biochemical, genetic, and evolutionary approaches to establish FL7 as an ancient regulator of stress responses that predates the PYL-ABA system.The manuscript is generally well-written, and the authors have conducted thorough revisions in response to previous reviews, significantly strengthening the study. However, several aspects require clarification and additional evidence to fully substantiate the central claims.\nMajor Revisions\n1. Regarding Fig. 1h, as this is an in vitro assay, the purpose of lane 4 is unclear to me—specifically, what control it is intended to represent. Since no ABA receptors are included in this experimental setup, the addition of ABA would not be expected to have a significant effect. Furthermore, comparing the last two lanes suggests that the presence of ABA may attenuate the inhibitory effect of FL7 on ABI1. It would be valuable if the authors could quantitatively analyze these lanes to determine whether the difference is statistically significant.\n2. While the authors show that FL7 protein accumulates in response to NaCl in OE lines (Fig. S4b), a critical piece of data is missing: the quantification of native FL7 protein levels in wild-type plants under the same stress conditions. Demonstrating the endogenous induction level of FL7 is essential to argue that the overexpression phenotypes are not merely artifacts of supraphysiological protein levels but reflect a genuine regulatory mechanism.\n3. The authors' Y2H data (provided in rebuttal) indicate that multiple FL proteins (e.g., FL2, FL5, FL6) can interact with PP2C-As, suggesting possible functional redundancy. However, only fl1, fl2, and fl7 single mutants exhibit clear drought-sensitive phenotypes. The authors' hypothesis—that this is due to FL2 and FL7's broader interaction profiles—remains speculative without direct genetic evidence. To unequivocally demonstrate FL7's dominant role and clarify whether functional redundancy exists, the generation and phenotypic analysis of a higher-order mutant (e.g., fl2 fl7 double mutant) under osmotic stress is strongly recommended. If generating such mutants is not feasible within a reasonable timeframe, this limitation and the possibility of redundancy should be explicitly discussed in the text, as it is important for interpreting the genetic data and understanding the evolutionary maintenance of this gene family.\n4. The authors used a genetic approach (AA1 inhibitor) to show that FL7 and PYLs function in parallel pathways (Fig. 4h). However, the reviewer's original question was biochemical: what happens in a reconstituted system with PYL, FL7, and PP2C-A simultaneously? A direct in vitro phosphatase assay including all three components would provide more mechanistic insight into potential additive, synergistic, or antagonistic effects, strengthening the conclusion about the parallel pathways.\nMinor Revisions\n1. Molecular weight markers should be clearly indicated in Fig. 1g and 1h. The inclusion of markers is essential for assessing the molecular sizes of the proteins and verifying the integrity of the assays. It is recommended that all similar immunoblots and gel-based figures throughout the manuscript be reviewed to ensure molecular weight markers are present and appropriately labeled.\n2. The soil water potential measurements conducted in response to Reviewer #2 (and shown in the rebuttal) should be included in the supplementary information to validate the drought stress assays.\n3. The manuscript would benefit from a final careful proofread by a native English speaker to polish minor grammatical errors and improve phrasing for clarity (e.g., in the Abstract: \"FL7 homologs with AC domain belongs\" should be \"belong\").\n4. In Figure 6a, consider adding bootstrap support values to the key nodes of the phylogenetic tree to reinforce the confidence in the evolutionary relationships depicted.\n5. In the seed germination assays, different concentrations of ABA were applied across experiments. The authors should provide a justification for the variation in ABA concentrations used in different parts of the study to ensure consistency and interpretability of the results."}, "author_response": "Re: Thanks for your positive comments and constructive suggestions! We have carefully studied the following comments, added some experiments, corrected the typos and rephrased many parts of text. Please see our responses which are shown below in bold.\n\n[... Author response text for Round 2 ...]"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors revised the manuscript adequately and I have no further comment.", "reviewer_2": "I have no further comments."}, "author_response": ""}]

Decision letter