Type one protein phosphatases (TOPPs) catalyze EIN2 dephosphorylation to regulate ethylene signaling in Arabidopsis

Meifei Su; Qianqian Qin; Jing Zhang; Yingdong Li; Ailin Ye; Suomin Wang; Suiwen Hou

doi:10.1126/sciadv.aec5937

. 2026 Feb 11;12(7):eaec5937. doi: 10.1126/sciadv.aec5937

Type one protein phosphatases (TOPPs) catalyze EIN2 dephosphorylation to regulate ethylene signaling in Arabidopsis

Meifei Su ^1,^†, Qianqian Qin ^1,^†, Jing Zhang ¹, Yingdong Li ¹, Ailin Ye ¹, Suomin Wang ², Suiwen Hou ^1,^*

PMCID: PMC12893281 PMID: 41671356

Abstract

Type one protein phosphatases (TOPPs) widely modulate phytohormone signaling and stress responses, but their roles in ethylene signaling remain unknown. This study reveals a reciprocal regulatory relationship between TOPPs and ethylene insensitive 2 (EIN2)–mediated ethylene signaling. We identified that ethylene can induce TOPPs’ expression, and topp1/4/5 mutants exhibited partial ethylene insensitivity with reduced EIN3 protein. Mechanistically, TOPPs function upstream of EIN2 and interact with its carboxyl-terminal domain (CEND) to dephosphorylate the S655 residue. This site-specific dephosphorylation promotes EIN2 stability and EIN2 CEND nuclear accumulation, thereby activating ethylene responses. Notably, EIN2^S655A-YFP/ein2-5 plants displayed constitutive ethylene responses and improved salt tolerance. Further investigation showed that EIN3/EIN3 like 1 (EIL1) activates TOPPs’ expression by binding to their promoters, amplifying ethylene signaling accordingly. Together, our finding establishes TOPPs as key regulators in ethylene signaling and reveal a dephosphorylation switch mechanism governing EIN2 function, providing critical insight into how EIN2 posttranslational modifications mediate plant stress adaptation.

TOPPs dephosphorylate EIN2-S655 to reduce ubiquitin-driven degradation and allow CEND nucleus import to trigger ethylene response.

INTRODUCTION

Type one protein phosphatases (TOPPs/PP1) are major serine/threonine phosphatases in eukaryotes that regulate diverse plant cellular processes (1–3). Reversible protein phosphorylation is one of the most common posttranslational modifications (PTMs) mediated by kinases and phosphatases and precisely controls the stability, activity, and subcellular localization of many core components in the signaling cascade (4–6). Arabidopsis contains nine TOPP isoforms (TOPP1 to TOPP9), with conserved homologs identified in major crops including rice, wheat, and soybean (7–9). Studies have demonstrated that TOPPs serve as a molecular switch for typical phytohormone signaling. TOPP4 destabilizes DELLA proteins via dephosphorylation, modulating gibberellin signaling (10), and mediates pin-formed 1 (PIN1) polar localization and endocytic trafficking in pavement cells by regulating the PIN1 phosphorylation status in the auxin signaling pathway (11). In wheat, TdPP1 dephosphorylates BRI1-EMS-suppressor 1 (BES1) to mediate BR-regulated root growth (12). TOPPs also suppress ABA signaling by inhibiting sucrose nonfermenting-1–related protein kinase 2 (SnRK2) activity (13, 14), whereas the Pseudomonas syringae effector AvrE can interact with TOPPs to remove this inhibition, promoting ABA accumulation and stomatal movement (15). In addition, the essential functions of TOPPs in plant stress adaptation and immune response are well established. OsPP1a and TaPP1a mediate salt tolerance in rice and wheat (16, 17), whereas GmTOPP13 confers drought tolerance in soybean (18). Under fixed-carbon starvation, TOPPs promote the formation of the autophagy-related protein 1a (ATG1a)–ATG13a complex by dephosphorylating ATG13a, thereby activating the autophagy pathway (19). TOPPs are important plant immunomodulators, and the accumulation of suppressors of topp4-1, 1 (SUT1) leads to immune activation of the topp4-1 mutant (20, 21). The interaction between TOPPs and kinetochore scaffold 1 (KNL1) is required for the proper localization of TOPPs to kinetochores to silence the spindle assembly checkpoint in Arabidopsis (22).

Ethylene is pivotal in plant growth and development, serving as a key signaling molecule for environmental responses (23–28). Studies elucidating the classical ethylene signaling pathway through Arabidopsis triple-response mutant screens revealed its core regulatory mechanism (29). Without ethylene, the kinase constitutive triple response 1 (CTR1) phosphorylates ethylene insensitive 2 (EIN2) in the endoplasmic reticulum (ER) membrane. This phosphorylation enables recognition by the F-box proteins EIN2 targeting protein 1/2 (ETP1/2), which mediate EIN2 degradation through the 26S proteasome pathway, thereby suppressing ethylene signal transduction (30, 31). When ethylene is present, CTR1 activity is inhibited, thereby reducing EIN2 phosphorylation and enhancing its stability. The stabilized EIN2 is then cleaved to release its C-terminal domain (EIN2 CEND), which functions in the cytoplasm and nucleus. Cytoplasmic EIN2 CEND suppresses translation of E3 ubiquitin ligases EIN3 binding F-box protein 1/2 (EBF1/2) mRNA, thus preventing degradation of the master transcription factor EIN3/EIN3 like 1 (EIL1) (32). Parallelly, nuclear EIN2 CEND interacts with EIN2 target protein 1/2 (ENAP1/2) to enhance EIN3/EIL1 transcriptional activity by regulating histone acetylation. Ultimately, EIN3/EIL1 activates ethylene response factor (ERF) expression and ethylene responses (33–35). Hence, decreasing the EIN2 phosphorylation level is essential for ethylene signal relay from the ER to nucleus. In addition to the inhibition of CTR1 kinase activity, the reduction of EIN2 phosphorylation should also be attributed to the role of protein phosphatase, but the corresponding phosphatase has not been found thus far. Our study demonstrates that TOPPs is the main phosphatase mediating EIN2 dephosphorylation, accordingly regulating ethylene signaling and stress adaptation.

RESULTS

TOPPs are involved in ethylene signaling

To investigate the interplay between TOPPs and ethylene signal, we treated wild-type (WT) plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and found most TOPPs genes were up-regulated (fig. S1). β-Glucuronidase (GUS) staining revealed that ACC treatment notably induced TOPPs promoter activity in hypocotyls, particularly TOPP1, TOPP4, and TOPP5 (Fig. 1A). Next, we analyzed ACC-induced triple responses in TOPP mutants (table S1). All single topp mutants (topp1 to topp9) showed no phenotypic difference from WT (fig. S2, A and B). The functional redundancy among TOPPs family members has necessitated the investigation of multiple mutants (19). Notably, the topp1/4/5 triple mutant uniquely exhibited partial ethylene insensitivity, whereas other double and triple mutant combinations maintained a WT-like response (Fig. 1, B and C). Conversely, overexpression of TOPP4 (TOPP4-OE) or TOPP5 (TOPP5-OE) lines showed ACC hypersensitivity (Fig. 1, D and E). Furthermore, native promoter–driven expression of TOPP1, TOPP4, and TOPP5 fully rescued the topp1/4/5 triple mutant phenotype (Fig. 1, F and G). Molecular analysis demonstrated enhanced EIN3 protein accumulation and ERF1 expression in TOPP4-OE lines after ACC treatment, contrasting with obvious suppression of these responses in topp1/4/5 mutants compared to WT (Fig. 1, H and I). These findings establish that TOPPs are important regulators of ethylene signaling.

Fig. 1. — (A) GUS staining analysis tissue-specific *TOPPs* expression in *TOPPs* promotor-GUS transgenic plant (*pTOPPs-GUS*)*/WT* etiolated seedlings treated with ±10 μM ACC (8 hours). Scale bar, 100 μm. (B, D, and F) Ethylene-responsive phenotype of *topp* mutants *TOPPp:TOPPs-YFP* (*TOPPs-YFP*)*/topp1/4/5* restored lines and overexpressed *TOPPs* (*TOPPs-OE*) lines treated with ±10 μM ACC (4 days). Scale bars, 10 mm. (C, E, and G) Hypocotyl measurements of etiolated seedlings. Each bar is the average length ± SD of at least 15 hypocotyls per line. (H) The EIN3 protein levels were examined in *TOPP4*-OE and *topp1/4/5* etiolated seedlings treated with 100 μM ACC for 0, 8, 16, and 24 hours using anti-EIN3 antibody, with actin serving as a loading control. Relative abundance values shown below blots. h, hours. (I) RT-qPCR analysis of *ERF1* expression was concurrently performed in *topp1/4/5* and *TOPP4*-OE and *TOPP5*-OE etiolated seedlings. Asterisks indicated statistical significance as determined by Student’s t test ( ***P < 0.001; NS, not significant).

Genetic association and physical interactions between TOPPs and EIN2

To elucidate TOPPs function in ethylene signaling, we performed liquid chromatograph–tandem mass spectrometry (LC-MS/MS) to analyze the interacting proteins of TOPP4-GFP. We identified two EIN2 peptide segments containing four phosphoserine residues (S645, S648, S655, and S659) within the intrinsic disordered region (IDR) of EIN2 CEND compared with green fluorescent protein (GFP) controls (fig. S3 and table S1). Notably, TOPP4 emerged as a key EIN2 interactor (data S1), with TOPP1, TOPP4, and TOPP5 colocalized with EIN2 in Nicotiana benthamiana leaf cells, supporting their functional association (fig. S4A). Genetic analyses showed that EIN2 overexpression rescued the topp1/4/5 ethylene insensitive phenotype (Fig. 2, A and B), whereas the ethylene hypersensitivity phenotype of TOPP4-OE and TOPP5-OE lines was abolished in ein2-5 and ein3-1 eil1-1 mutants (Fig. 2, C and D, and fig. S4, B and C). Moreover, the ethylene-triggered EIN3 accumulation was also notably reduced in TOPP4-OE/ein2-5 plants (fig. S4D), confirming that TOPPs are genetically located upstream of EIN2.

Fig. 2. — (A and C) Ethylene-responsive phenotype of the transgenic *35S:EIN2-YFP/topp1/4/5* and *TOPPs-OE/ein2-5* etiolated seedlings treated with ±10 μM ACC (4 days). Scale bars, 10 mm. (B and D) Hypocotyl measurements of etiolated seedlings. Each bar is the average length ± SD of at least 15 hypocotyls per line. Asterisks indicated statistical significance as determined by Student’s t test (***P < 0.001; NS, not significant). (E) Interaction between TOPPs and full-length EIN2/EIN2 CEND were analyzed using mbSUS and Y2H assays. Diploid yeast cells were cultured on selective SD media: −LW, lacking Trp/Leu; −LWHA, lacking Trp/Leu/His/Ade (with 300 μM Met or X-α-Gal). Empty vectors served as negative controls. (F) Pull-down assays demonstrating the direct binding of TOPPs-GST to EIN2 CEND-MBP. (G) BiFC confirmation of the TOPPs-EIN2/EIN2 CEND interaction in *N. benthamiana*. Scale bars, 50 μm. (H) Co-IP validation of TOPPs-EIN2/EIN2C′ interaction in *Arabidopsis*. Total proteins of *TOPPs*-OE etiolated seedlings treated with ±100 μM ACC (16 hours) were immunoprecipitated with GFP magnetic beads. The abundance of EIN2C′ was detected with an EIN2 antibody. WT was used as a negative control.

We further examined the interactions between TOPPs family proteins and EIN2. In vitro analyses using the mating-based split-ubiquitin system (mbSUS), yeast two-hybrid (Y2H), and pull-down experiments revealed direct interactions between TOPP1, TOPP4, or TOPP5 and EIN2, with specific association observed between these TOPP members and EIN2 CEND (Fig. 2, E and F, and fig. S4E). Subsequent in vivo validation through bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation (Co-IP) assays confirmed these interactions (Fig. 2, G and H). These results demonstrate that TOPPs and EIN2 directly interact to regulate ethylene signaling in Arabidopsis.

TOPPs dephosphorylate EIN2 at the S655 residue

Quantitative phosphoproteomics revealed significantly reduced EIN2 phosphorylation levels in TOPP4-OE lines compared to WT (Fig. 3A and data S2). Cell-free kinase and dephosphorylation assays showed that ACC-treated topp1/4/5 mutant extracts obviously enhanced recombinant EIN2 CEND-Maltose-binding protein (MBP) phosphorylation levels versus WT (Fig. 3B and fig. S5A), which was reversed by TOPPs–glutathione S-transferase (GST) supplementation (Fig. 3C and fig. S5B). In addition, in vitro purified CTR1-MBP can phosphorylate EIN2 CEND-MBP, whereas TOPP4-GST can dephosphorylate it (Fig. 3D and fig. S5C). Collectively, our data demonstrate that TOPPs serve as a key phosphatase controlling EIN2 dephosphorylation.

We identified four phosphorylation sites (S645, S648, S655, and S659) of EIN2 by mass spectrometry (table S1). Cell-free dephosphorylation assay revealed that individual alanine substitutions at S645, S648, S655, or S659 in EIN2 CEND-MBP failed to alter phosphorylation levels in ACC-treated topp1/4/5 extracts. Addition of TOPP4-GST obviously reduced EIN2 CEND-MBP phosphorylation, whereas none of the single-site mutants exhibited detectable phosphorylation changes under identical conditions (Fig. 3E and fig. S6, A and B). These phosphorylation sites thus likely mediate TOPPs-regulated EIN2 dephosphorylation.

However, quantitative phosphoproteomics revealed TOPPs-mediated EIN2 dephosphorylation predominantly targets the sLSGEGGsGTGsLSR peptide segments (data S2). To investigate the site specificity of TOPPs for EIN2 dephosphorylation, we used AlphaFold 3 to predict the distinct EIN2-TOPPs interaction complex (Fig. 3, F and G). In the nonphosphorylated EIN2-TOPP1/TOPP4/TOPP5 complex, S655 lies outside the interaction interface (Fig. 3F and fig. S7, A and B), whereas phosphorylated EIN2 specifically anchors S655 to the TOPPs catalytic center (Fig. 3G), comparing to no binding capacity of S645, S648, and S659 sites (fig. S7, C to E). Mutating six catalytic residues of TOPP4 (TOPP4^mA) abolished EIN2 S655 binding to its catalytic center (Fig. 3H). Meanwhile, Y2H assays showed that the S655 phosphorylation state did not affect their interaction (fig. S7F). These data further suggest that phosphorylated EIN2 facilitates S655 site binding to TOPPs’ catalytic center, enabling its dephosphorylation by TOPPs. Furthermore, we synthesized EIN2 phosphopeptides containing pS645 (sequence: APTSNFTVGSDGPPsPR), pS648/pS655 (sequence: sLSGEGGsGTGSLSR), and pS659 (sequence: SLSGEGGSGTGsLSR; Sangon Biotech) (36) and incubated them with TOPP4-GST. Following ultrafiltration removal of the proteins, LC-MS/MS analysis revealed that S645/S648/S659 exhibited equivalent dephosphorylation efficiency with recombinant TOPP4-GST versus control GST. However, the S655 phosphopeptide showed selective dephosphorylation ratio enhancement after TOPP4-GST treatment (Fig. 3I; fig. S6, C and D; and data S3-1, S3-2, and S3-3). Our integrated data demonstrate that S655 is the main site targeted by TOPPs for EIN2 dephosphorylation.

Dephosphorylation at the S655 site is important for EIN2-mediated ethylene signaling

To assess the impact of TOPPs-mediated dephosphorylation on EIN2 functions, we first performed cell-free degradation assays in vitro. Recombinant EIN2 CEND-MBP stability in ACC-treated topp1/4/5 mutant extracts was lower than in WT (fig. S8A), whereas proteasome inhibitor MG132 attenuated this accelerated degradation under the same treatment (fig. S8B). Furthermore, like the effect of alkaline phosphate λpp treatment, TOPP4-GST increased EIN2–yellow fluorescent protein (YFP) and EIN2C′-YFP protein levels in EIN2-YFP/ein2-5 plants, whereas the protein phosphatase inhibitor (Phostop) reduced them (fig. S8, C and D), displaying that TOPPs enhance EIN2 stability in vitro. Meanwhile, in vivo analysis detected reduced EIN2 protein levels in topp1/4/5 mutant versus WT after ACC treatment (Fig. 4A). Consistently, EIN2-YFP and EIN2C′-YFP proteins levels were markedly reduced in EIN2-YFP/topp1/4/5 plants (Fig. 4B and fig. S8E). It should be particularly pointed out that both EIN2-YFP and EIN2C′-YFP protein accumulated to higher levels in EIN2^S655A-YFP/ein2-5 than in EIN2^S655D-YFP/ein2-5 plants (Fig. 4C). These results indicate that TOPPs stabilize EIN2 through dephosphorylation.

Fig. 4. — (A) EIN2 protein levels in WT and *topp1/4/5* mutants treated with ±100 μM ACC (16 hours). Total proteins were subjected to Western blotting with anti-EIN2. (B and C) EIN2-YFP and EIN2C′-YFP levels in *EIN2-YFP/WT*, *EIN2-YFP/topp1/4/5*, and *EIN2p:EIN2^S655A-YFP* (*EIN2^S655A-YFP*)*/ein2-5*, *EIN2p:EIN2^S655D-YFP* (*EIN2^S655D-YFP*)*/ein2-5* plants treated with ±100 μM ACC (16 hours) analyzed with anti-GFP; actin served as a loading control. (D) Nuclear localization of EIN2-YFP (arrows) in roots of *EIN2-YFP/WT* and *EIN2-YFP/topp1/4/5* etiolated seedlings treated with ±100 μM ACC (16 hours). Scale bar, 20 μm. (E) Cytoplasmic (C) and nuclear (N) fractions of EIN2-YFP and EIN2C′-YFP in *EIN2-YFP/WT* and *EIN2-YFP/topp1/4/5* seedlings treated with ±100 μM ACC (16 hours). Tubulin and H2B were used as a cytoplasm and nucleus loading control, respectively. (F) Ethylene-responsive nuclear localization of *EIN2-YFP/ein2-5* and constitutive nuclear localization of *EIN2^S655A-YFP/ein2-5* in *Arabidopsis* root cells. Scale bar, 10 μm. (G and I) Ethylene-responsive phenotype of the transgenic *EIN2^S655A/D-YFP/ein2-5* and *EIN2^S655A-YFP/WT* and *EIN2^S655A-YFP/topp1/4/5* etiolated seedlings treated with ±10 μM ACC (4 days). Scale bars, 10 mm. (H and J) Hypocotyl measurements of etiolated seedlings. Each bar is the average length ± SD of at least 15 hypocotyls per line. Asterisks indicated statistical significance as determined by Student’s t test (***P < 0.001; NS, not significant).

Phosphorylated EIN2 can interact with F-box proteins ETP1/2 and is degraded by 26S proteasome (30). AlphaFold modeling of the EIN2-ETP1/2 complex demonstrated indistinguishable binding configurations between phosphorylated and nonphosphorylated S655 states of EIN2, with consistent interaction scores across prediction models (fig. S9, A to C). Y2H further confirmed stable EIN2-ETP1/2 interaction regardless of the S655 phosphorylation status (fig. S9D). Although EIN2-ETP1/2 interaction remains unaffected by the S655 phosphorylation status, in WT without ACC treatment, EIN2 CEND^S655A-MBP exhibited reduced ubiquitination compared to EIN2 CEND-MBP; following ACC treatment, EIN2 CEND^S655A-MBP ubiquitination remained low, whereas EIN2 CEND^S655D-MBP showed enhanced ubiquitination (fig. S9E). Therefore, EIN2 ubiquitination shows notable phosphorylation-dependent regulation. In planta experiments further showed, under normal conditions, that EIN2^S655A-YFP/ein2-5 exhibited reduced ubiquitination compared to EIN2-YFP/ein2-5 and EIN2^S655D-YFP/ein2-5 (fig. S9F). Notably, ACC treatment further attenuated ubiquitination in EIN2^S655A-YFP/ein2-5 but drastically enhanced ubiquitination in EIN2^S655D-YFP/ein2-5, comparing to EIN2-YFP/ein2-5 (fig. S9F). This regulatory paradigm establishes that S655 dephosphorylation weakens EIN2 ubiquitination, explaining TOPPs-mediated EIN2 stability by dephosphorylating EIN2 and inhibiting its ubiquitination.

ACC treatment could markedly enhance EIN2 fluorescence in nuclear in WT, whereas this phenomenon of increase was not detected in the topp1/4/5 mutant (Fig. 4D and fig. S10A). Nuclear-cytoplasmic fractionation also confirmed that ACC-induced nuclear accumulation of EIN2C′-YFP was remarkedly reduced in the topp1/4/5 background compared to WT (Fig. 4E). Notably, EIN2^S655A-YFP/ein2-5 plants displayed constitutive nuclear localization of EIN2-YFP even without ACC treatment, whereas most of EIN2-YFP in EIN2^S655D-YFP/ein2-5 localized in the cytoplasm (Fig. 4F and fig. S10B). In addition, tobacco leaf cells expressing EIN2 CEND^S655A-YFP showed stronger nuclear fluorescence of EIN2 CEND-YFP than EIN2 CEND^S655D-YFP (fig. S10C). These findings demonstrate that TOPPs promote EIN2 CEND nuclear accumulation via S655 site dephosphorylation.

Because the phosphorylation state is critical for EIN2 function, we investigated the regulatory role of S648 and S655 site dephosphorylation in ethylene signaling. Transgenic EIN2^S648A-YFP/ein2-5 and EIN2^S648D-YFP/ein2-5 lines showed similar ACC sensitivity (fig. S11, A and B). However, EIN2^S655A-YFP/ein2-5 showed obviously accelerated flowering compared to delayed flowering development in both EIN2^S655D-YFP/ein2-5 and ein2-5 plants (fig. S12A). Moreover, EIN2^S655A-YFP/ein2-5 showed constitutive ethylene responses under darkness, and ACC treatment also exacerbated its triple-response phenotype (Fig. 4, G and H, and fig. S12B), with comparable EIN2 expression levels across transgenic lines (fig. S12C). In biochemical analysis, EIN3 accumulation and ERF1 up-regulation were observed in the EIN2^S655A-YFP/ein2-5 (fig. S12, D and E). Crucially, expressing EIN2^S655A-YFP restored the ethylene insensitivity phenotype of the topp1/4/5 mutant (Fig. 4, I and J). These results establish that EIN2 dephosphorylation at the S655 site is important for activating ethylene signaling.

EIN2 dephosphorylation is conducive to salt tolerance in Arabidopsis

Ethylene is critical for plant stress adaptation with the ethylene-constitutive mutant ctr1-1 displaying exceptional salt tolerance, and both ein2-5 and topp multiple mutants exhibits similar salt hypersensitivity (14, 35). Consistently, we observed that TOPP4-OE plants showed enhanced salt tolerance relative to WT, but this advantage was lost in the ein2-5 and ein3-1 eil1-1 background with significantly lower fresh weight under salinity stress (Fig. 5, A and B). We evaluated salt stress responses in EIN2 S655 phosphomutant transgenic lines. Under high-salinity conditions, EIN2^S655A-YFP/ein2-5 displayed higher germination rates than EIN2^S655D-YFP/ein2-5 (Fig. 5, C and D). Notably, salt-stressed EIN2^S655A-YFP/ein2-5 outperformed other transgenic lines in fresh weight accumulation under 200 mM NaCl treatment (Fig. 5E).

Fig. 5. — (A and B) Salt-tolerant phenotype and fresh weight of WT, *TOPP4-OE*, *ein2-5*, *TOPP4-OE/ein2-5*, and *TOPP4-OE/ein3-1 eil1-1* plants treated with 0, 150, and 200 mM NaCl, respectively. (C to G) Seed germination salt-tolerant phenotype (C), seed germination rate (D), fresh weight (E), MDA content (F), and relative ion leakage (G) of WT, *ein2-5*, *ctr1-1*, *EIN2-YFP/ein2-5*, *EIN2^S655A-YFP/ein2-5*, and *EIN2^S655D-YFP/ein2-5* plants treated with 0, 150, and 200 mM NaCl, respectively. (H) NBT staining of O₂⁻ accumulation in unstressed and salt-stressed (12 hours) WT, *ctr1-1*, *EIN2-YFP/ein2-5*, *EIN2^S655A-YFP/ein2-5*, *EIN2^S655D-YFP/ein2-5*, and *ein2-5* plants. Asterisks indicated statistical significance as determined by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant).

To further test the effects of EIN2 dephosphorylation on salt stress response, through comparative analysis of EIN2 S655 phosphomutant transgenic lines, it was found that EIN2^S655A-YFP/ein2-5 seedlings displayed significantly enhanced salt tolerance, with higher survival rates (fig. S13, A and B) and improved cotyledon greening under stress (fig. S13, C and D). In contrast, EIN2^S655D-YFP/ein2-5 showed enhanced sensitivity to 200 mM NaCl, phenotypically resembling the ein2-5 mutant (fig. S13A). Physiological assessments revealed that EIN2^S655A-YFP/ein2-5 maintained membrane integrity through significantly lower malondialdehyde (MDA) content (Fig. 5F) and reduced relative ion leakage (Fig. 5G), coupled with attenuated O₂⁻ accumulation in leaves (Fig. 5H), whereas the enhanced results were detected in EIN2^S655D-YFP/ein2-5 (Fig. 5, F to H). Notably, under normal conditions, no obvious phenotypic and physiologic differences were observed between these transgenic lines and WT (Fig. 5, E to H, and fig. S13, A to D). These results demonstrate that TOPPs-mediated dephosphorylation at the EIN2 S655 site positively regulates plant salt tolerance through mitigation of oxidative stress and membrane damage.

Ethylene-induced TOPPs expression depends on EIN3/EIL1-mediated transcriptional activation

EIN3/EIL1, the master transcriptional regulator of ethylene signaling, activates target genes through canonical EIN3-binding site (EBS) elements (37, 38). To investigate the mechanism of ethylene-induced TOPPs expression, we generated pTOPPs-GUS/ein3-1 eil1-1 plants by crossing pTOPPs-GUS/WT with the ein3-1 eil1-1 mutant. ACC treatment revealed that both TOPP4 and TOPP5 expressions were significantly inhibited in hypocotyls of the ein3-1 eil1-1 mutant (Fig. 6A and fig. S14A), indicating that EIN3 mediates ethylene-responsive TOPPs up-regulation. Promoter analysis identified EBS elements TTGTATCTG in TOPP4 and ATGAAT in TOPP5 (fig. S14B). Both dual luciferase assays and yeast one-hybrid (Y1H) demonstrated that EIN3 interacts with TOPP4 and TOPP5 promoters via EBS elements, which were abolished upon EBS deletion or mutation (Fig. 6, B and C). Electrophoretic mobility shift assay (EMSA) further confirmed direct EIN3 binding to these motifs, with no binding observed when these motifs were mutated (Fig. 6D). In planta validation through chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP-qPCR) showed that the EIN3 protein could immunoprecipitate TOPP4 and TOPP5 promoter regions containing intact EBS elements in ACC-treated WT plants (Fig. 6E and fig. S14, C and D). These findings establish that EIN3/EIL1 activates TOPPs expression by promoter binding, amplifying ethylene signaling in plants.

Fig. 6. — (A) Tissue-specific expression of *pTOPPs-GUS/ein3-1 eil1-1* etiolated seedlings treated with ±100 μM ACC (8 hours). Scale bar, 100 μm. (B) Dual luciferase assays showing EIN3-GFP binding to *TOPP4* and *TOPP5* promoters and enhancing their activity in tobacco leaves. (C) Y1H assays demonstrating the EIN3 interaction with full-length (full)/truncated (A, B, and C)/mutated (Am and Cm) *TOPP4* and *TOPP5* promoters. (D) Determination of the association of EIN3 with *TOPP4* and *TOPP5* promoters using the EMSA method. Recombinant EIN3-MBP and double-stranded oligonucleotide probes containing MBS sequences from the *TOPP4* and *TOPP5* promoter were subject to probe binding reactions. (E) ChIP-qPCR assay with EIN3 antibody was performed in WT and *ein3-1 eil1-1* etiolated seedlings treated with ±100 μM ACC (8 hours) to validate the EIN3-*TOPPs* promoter interaction. DNA amounts were normalized to untreated WT. Asterisks indicated statistical significance as determined by Student’s t test (***P < 0.001; NS, not significant).

DISCUSSION

TOPPs serve as central regulatory nodes in plants, orchestrating critical physiological responses through dynamic dephosphorylation of key substrates involved in hormone signaling (10–14), stress response (16–18), and immunity (15, 20, 21). Ethylene developmental roles are well characterized (24, 25). Crucially, as the master switch in ethylene signaling, EIN2 activity and subcellular trafficking are highly dependent on the phosphorylation status, yet corresponding phosphatases remain unidentified. This study demonstrates that TOPPs directly interact with EIN2 and dephosphorylate it at the S655 site, promoting its stability and EIN2 CEND nuclear accumulation. This facilitates stable EIN3-mediated transcriptional activation of ERF1 and TOPPs, establishing a self-reinforcing “TOPPs→EIN2→EIN3→TOPPs” positive feedback loop that augments ethylene responses (Fig. 7, A and B). Our study uncovers reciprocal interactions between phosphatase TOPPs and ethylene signaling, demonstrating that TOPPs mediate EIN2 dephosphorylation. These findings expand the canonical “receptor–phosphorylation cascade–transcriptional output” framework in ethylene signaling, elucidating precise PTM regulatory mechanisms in this pathway. Therefore, TOPPs are the key phosphatases found in ethylene signaling.

Fig. 7. — (A) In the absence of ethylene, receptor-activated CTR1 phosphorylates EIN2, enabling its interaction with F-box proteins ETP1/2. This facilitates EIN2 degradation via the 26S proteasome through ETP1/2-mediated ubiquitination (Ub; orange), thus suppressing ethylene responses. (B) When ethylene is present, CTR1 activity is inhibited. Subsequently, TOPP family phosphatases (TOPPs; blue) counteract ubiquitination-mediated degradation by binding to and dephosphorylating EIN2 at the S655 residue (green highlight), promoting EIN2 CEND nuclear accumulation, which enhances EIN3/EIL1 stability to activate *ERF1* and *TOPPs* expression. Ultimately, a self-reinforcing “TOPPs→EIN2→EIN3→*TOPPs*” signal feedback amplification circuit is formed to enhance the ethylene response. As a possibility, TOPPs may additionally regulate EIN3 stability through unidentified mechanisms. In addition, redundant protein phosphatases (PPs) may complement TOPPs-mediated regulation by targeting distinct EIN2 phosphorylation sites, creating synergistic control mechanisms in ethylene signaling. Pi, phosphorylation; PO₄³⁻, phosphate group; S/T/Y (serine/threonine/tyrosine), serine/threonine/tyrosine diversity sites; −Pi, dephosphorylation at the S/T/Y site; Mn²⁺, manganese ions. Solid arrows indicate canonical signaling progression, and dashed arrows denote putative modulatory interactions.

Consensus holds that each functional TOPPs enzyme consists of a highly conserved catalytic subunit and a regulatory subunit targeting catalytic subunit to specific subcellular compartment, modulate substrate specificity, and trigger specific biological responses (39). Although our study has revealed the critical role of the TOPPs-EIN2 complex in ethylene signaling, the specific regulatory subunits and mechanism controlling TOPPs-mediated dephosphorylation of EIN2 remain to be elucidated. Compared to the ein2-5 mutant, the topp1/4/5 mutant exhibited much weaker insensitivity, suggesting that other factors/mechanisms may contribute to the regulation of ethylene signal transduction by TOPPs. First, Arabidopsis has around 1000 kinases and 150 phosphatases that form a reversible phosphorylation network (40). EIN2 phosphorylation regulation in ethylene signaling demonstrates multilayered complexity. CTR1 phosphorylates EIN2 at S645/S924 (34), target of rapamycin (TOR) phosphorylates EIN2 at Thr⁶⁵⁷ (41), and Medicago truncatula compact root architecture 2 (MtCRA2) receptor-like kinase phosphorylates MtEIN2 at S643/S924 in plants (42). Accordingly, other paralogous protein phosphatases may also redundantly regulate EIN2 dephosphorylation. We found that TOPPs controlled EIN2 dephosphorylation at the EIN2 S655 site. The in vitro dephosphorylation assays revealed TOPP4 dephosphorylates CTR1-mediated EIN2 CEND-MBP phosphorylation, suggesting that CTR1 might regulate the S655 site. Moreover, AlphaFold model predictions and mass spectrometry analysis indicated that TOPPs selectively dephosphorylated EIN2 at the S655 site rather than S645, S648, or S659. Therefore, the functional interplay between CTR1 and TOPPs in regulating EIN2 phosphorylation is highly intricate, and their potential role as molecular switches modulating EIN2 functions in ethylene signaling remains to be further determined. Inevitably, other phosphatases may also specifically engage the EIN2 phosphorylation level by dephosphorylating multiple known or unknown serine, threonine, or tyrosine sites of EIN2, establishing hierarchical control over ethylene signal amplification. Furthermore, signaling pathways use multilayered regulatory checkpoints to maintain signal transduction and prevent overactivation. Our study demonstrates that EIN3 transcriptionally activates TOPPs expression, forming a self-reinforcing loop with the TOPPs-EIN2 module to potentiate ethylene signaling. In addition, our analyses revealed the EIN3 protein was notably elevated in the TOPP4 overexpression line but reduced in the topp1/4/5 mutant, contrasting with this mutant’s partial ethylene insensitivity, suggesting that TOPPs not only directly regulate EIN2 dephosphorylation but may also modulate negative regulatory factors affecting EIN2 activity or localization or target other key components (e.g., EIN3 and EBF1/2) in ethylene signaling.

The dynamic stability, subcellular localization, and cleavage of EIN2 into multiple EIN2 CEND fragments of varying sizes are beneficial to the complexity of its role in ethylene signaling (32, 43, 44). PTMs are essential for signaling cascades by precisely regulating central proteins (1). In Arabidopsis, CTR1-controlled phosphorylation of EIN2 triggers its ubiquitination-dependent degradation (30–34), with glucose-activated TOR kinase promoting EIN2 phosphorylation to block its nuclear localization (41), whereas Solanum lycopersicum O-glycosylation enhances the stability and nuclear accumulation of SlEIN2 in tomato (45). Our data show that the S655 site phosphorylation status did not alter EIN2-ETP1/2 interaction; it may be related to the previously discovered fact that ethylene can suppresses ETP1/2 protein levels (30). However, TOPPs can still inhibit EIN2 ubiquitination by dephosphorylating it at the S655 site, thereby mediating EIN2 stability and promoting EIN2 CEND nuclear accumulation. The above findings suggest that these PTMs of EIN2 should be intrinsically interrelated. Expressing phospho-deficient EIN2^S655A-YFP in ein2-5 exhibits enhanced salt tolerance, positioning TOPPs and ethylene as coordinated stress regulators. These findings establish that PTMs serve as molecular switches in precise stress-response networks, highlighting their significant potential for stress-resistant crop breeding.

As the proteolytic product of underphosphorylated EIN2, EIN2 CEND functions in guaranteeing EIN3/EIL1 stability in the ethylene signaling, although it is located both in the cytoplasm and nucleus (46). We found that TOPPs directly dephosphorylate EIN2 at the S655 site, facilitating nuclear translocation of most EIN2-YFP, leaving only a small amount of EIN2 in the cytoplasm in EIN2^S655A-YFP/ein2-5 lines. How the numerous EIN2 CEND fragments are sorted into the cytoplasm and nucleus is worthy of further study. In addition, EIN2 phosphorylation sites cluster within the IDR of EIN2 CEND, a structural feature known to regulate protein structure and function through PTMs (47). Therefore, exploring the potential relationship among EIN2 dephosphorylation, IDR conformational dynamics, and cytoplasmic p-body is of great significance for fully clarifying the spatial regulation of ethylene signaling.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia was used as the WT control in this study, and all mutants and transgenic plants have been described in table S2.

Plant growth conditions and hypocotyl measurement

Arabidopsis and N. benthamiana were grown in controlled chambers (22°C, 16-hour/8-hour light/dark) under white fluorescent light. For seedling assay, seeds were surface sterilized and grown vertically on 1/2 MS (Murashige and Skoog) agar plates containing 0 or 10 μM ACC (Sigma-Aldrich). Hypocotyl lengths were quantified from digital images using ImageJ software (NIH).

RT-qPCR analysis of TOPPs, EIN2, and ERF1 mRNA abundance

Four-day-old etiolated seedlings were treated with 10 μM ACC for 8 hours. Total RNA was extracted using a Plant RNA Purification Reagent (Omega), followed by genomic DNA removal using DNase I (Promega). Reverse transcription (RT) was performed using the PrimeScript RT reagent kit (TaKaRa), and qPCR analysis was conducted with the SYBR Green Premix Ex Taq II (TaKaRa) on a StepOnePlus Real-Time PCR System. UBQ10 (Ubiquitin10) served as the endogenous control. The primers used are listed in table S3.

GUS staining

Four-day-old etiolated seedlings were treated with or without 10 μM ACC for 8 hours and then subjected to GUS staining [50 mM Na-phosphate (pH 7.0), 1 mM EDTA, chloramphenicol (100 mg/ml), 2 mM ferricyanide, 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (1 mg/ml), and 0.1% Triton X-100] at 37°C. Following staining, seedlings were destained in 70% ethanol until sufficiently cleared and imaged with a Leica stereomicroscope.

Protein isolation and immunoblot analysis

For protein extraction, Arabidopsis samples were cryogenically ground in liquid nitrogen and homogenized in protein extraction buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5% β-mercaptoethanol, 1 mM EDTA, 10% glycerol, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× Cocktail] after 30-min ice incubation, and the lysates were centrifuged at 12,000g (4°C, 20 min). The clarified supernatant was immediately mixed with SDS loading buffer at room temperature. Then proteins were subjected to 8% SDS–polyacrylamide gel electrophoresis (PAGE) and analyzed by immunoblotting using anti-EIN2 (Agrisera, AS12 1865; dilutions: 1:1000; rabbit), anti-EIN3 (Agrisera, AS19 4273; dilutions: 1:1000; rabbit), anti-actin (D110007-0200, dilutions: 1:2000; rabbit), and anti-H2B (Abmart, T55848; dilutions: 1:5000; rabbit).

Mass spectrometry assay

To prepare samples for mass spectrometry analysis, at least 2 g of transgenic material seedlings was collected. The target protein complex was immunoprecipitated with anti-GFP beads (D153-11, MBL), and the immunoprecipitates were separated by SDS-PAGE. Subsequently, the sample requires a proteolytic step with trypsin before injecting to a mass spectrometer. Samples were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) connected to an EASY-nano-LC system. Raw files were analyzed together using Maxquant (1.6.2.10).

The phosphopeptide containing pS645 (sequence: APTSNFTVGSDGPPsPR), pS648/pS655 (sequence: sLSGEGGsGTGSLSR), and pS659 (sequence: SLSGEGGSGTGsLSR) were synthesized (Sangon Biotech, Shanghai, China). The phosphopeptide (5 μg) was incubated with 1 mM GST and TOPP4-GST in 100 μl of phosphatase assay buffer at 30°C for 1 hour. After removing GST and TOPP4-GST in an ultrafiltration filter device (10-kDa cutoff; Amicon, Sigma-Aldrich), the treated phosphopeptides were subjected to mass spectrometry.

Mating-based split-ubiquitin system and Y2H assay

TOPPs coding sequences were cloned into the pX-NubWTgate vector (THY.AP5 strain), whereas the full-length EIN2 coding sequence was inserted into the pMetYCgate vector (THY.AP4 strain), with empty vectors as negative controls. Positive transformants were selected on SD/-Trp-Leu medium, and protein interactions were assayed on SD/-Trp-Leu-His-Ade medium. Yeast colony growth was examined following 4-day incubation at 30°C.

For Y2H assay, the C-terminal domain of EIN2 was fused to the AD vector, whereas cDNAs of TOPP superfamily genes were cloned into the BD vector. Both constructs were cotransformed into yeast strain Y2H Gold. Transformants were selected on SD/-Trp-Leu medium at 30°C for 48 hours. Protein-protein interactions were assessed by growth on SD/-Trp-Leu-His-Ade plates supplemented with X-α-Gal (20 mg/ml) under identical conditions.

In vitro protein expression and pull-down assay

The target proteins were expressed in Escherichia coli BL21 cells using an isopropyl-β-d-thiogalactopyranoside–inducible vector. Postinduction, cells were lysed via sonication in binding buffers [GST: 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄ (pH 7.3); MBP: 20 mM tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 7.4)] and clarified by centrifugation 12,000g (30 min, 4°C). Proteins were affinity purified using either Glutathione Sepharose 4B beads (GE Healthcare) for GST-tagged proteins or PurKine MBP-Tag Dextrin Resin 6FF (Abbkine) for MBP-tagged proteins. After washing with tag-specific buffers [GST: 50 mM tris-HCl and 10 mM glutathione (pH 8.0); MBP: 20 mM tris-HCl, 1 mM EDTA, and 10 mM maltose (pH 7.4)].

Purified bait protein (GST-tagged) was immobilized on glutathione-Sepharose beads by incubating at 4°C for 1 hour. After blocking with bovine serum albumin (BSA), prey protein (MBP-tagged) was added and incubated for 2 hours in binding buffer [20 mM Hepes (pH 7.4), 150 mM NaCl, and 0.1% Triton X-100]. Beads were washed three times to remove unbound proteins and then boiled in SDS loading buffer. Eluted complexes were analyzed by SDS-PAGE and Western blotting using anti-GST (Abmart, M20007; dilutions: 1:5000; mouse) and anti-MBP (Proteintech, 66003-1-1g; dilutions: 1:5000; mouse) to confirm the interaction. The antibodies used are listed in table S4.

BiFC and fluorescence microscopy

For BiFC-based interaction mapping, truncated or full-length EIN2 and TOPPs were fused with nYFP and cYFP, respectively. Constructs were transformed into Agrobacterium tumefaciens strain GV3101 and coexpressed in N. benthamiana. For confocal imaging, leaf sections (2 mm by 2 mm) were harvested 36 to 48 hours postinfiltration and examined using a Nikon A1+ confocal laser scanning microscope with 20× objective. For live-cell imaging, root meristematic cells from 4-day-old Arabidopsis seedlings were observed using a Nikon confocal microscope with a 40× water objectives. YFP was excited with a 488-nm laser and detected between 500 and 550 nm.

Co-IP assay

The Co-IP assay was performed as previously described (19). In brief, 4-day-old etiolated seedlings (WT or TOPPs-OE transgenic) were lysed in ice-cold IP buffer [10 mM Hepes (pH 7.5), 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, and 1× Cocktail]. Lysates were incubated with anti-GFP magnetic beads at 4°C for 3 hours, followed by three cold IP buffer washes. Proteins were separated by SDS-PAGE and analyzed by anti-GFP (Abmart, M20004; dilutions: 1:5000; mouse) and anti-EIN2.

In vitro dephosphorylation and cell-free phosphorylation/dephosphorylation assay

CTR1-MBP was incubated with recombinant EIN2 CEND-MBP in kinase assay buffer [25 mM tris-HCl (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% Triton X-100, 5 mM MgCl₂, 5 mM MnCl₂, 1 mM PMSF, and 1× Cocktail] at 30°C for 1 hour, followed by reaction termination with 10 mM EDTA. Purified TOPPs-GST or GST was then added to the mixture and incubated for 30 min. Reactions were halted with 1× SDS-PAGE loading buffer. Phosphorylated EIN2 CEND-MBP was detected via immunoblotting using a pSer antibody (anti-pSer) (Abcam, ab9332; dilution: 1:1000; mouse). Protein bands were visualized by coomassie brilliant blue (CBB) staining.

Cell-free phosphorylation assays were performed as previously described (48, 49). Briefly, 4-day-old etiolated seedlings of WT and topp1/4/5 mutants treated with or without ACC were flash-frozen in liquid nitrogen. Total proteins were extracted using kinase buffer. EIN2 CEND-MBP was incubated with seedling extracts in kinase buffer at 30°C for 45 min, followed by reaction termination with 10 mM EDTA. For dephosphorylation, recombinant TOPPs-GST or GST (control) proteins were added to phosphorylated EIN2 CEND-MBP or variants (CEND^{S645A/S648A/S655A/S659A}) in buffer [50 mM Hepes (pH 7.4), 0.1% Triton X-100, 1 mM NaCl, 0.1% DTT, 5 mM EDTA, 1 mM adenosine triphosphate (ATP), and 10 mM MnCl₂] and incubated at 30°C for 90 min. Reactions were halted by adding SDS loading buffer and boiling for 5 min. Proteins were resolved on 10% SDS-PAGE gels and immunoblotted using anti-pSer.

AlphaFold 3 analysis

Protein interaction between EIN2 and TOPPs and ETP1/2 were predicted by the AlphaFold 3 model (https://alphafoldserver.com/). Full protein sequences of EIN2 and TOPPs, ETP1/2, and Mn²⁺ or Zn²⁺ were entered as input for the prediction. Final models were imported and modified with PyMOL V3.1.0 (https://pymol.org/). The interface prediction template modeling score (iPTM) and predicted template modeling score (pTM) showed high interaction confidence (pTM + ipTM > 0.75).

Cell-free degradation assay

Total proteins were extracted from 4-day-old WT and topp1/4/5 etiolated seedlings treated with or without ACC were frozen in liquid nitrogen, and total proteins were extracted with 1× degradation buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 10 mM NaF, 2 mM Na₃VO₄, 25 mM β-glycerophosphate, 10% (w/v) glycerin, 2 mM DTT, 1 mM PMSF, and 1× Cocktail]. Each individual assay used recombinant EIN2 CEND-MBP protein and 1 mM ATP incubated in 100 μl of total proteins extracts and then incubated at 28°C and sampled at the indicated intervals for Western blotting.

Ubiquitination assays

For semi–in vivo ubiquitination assay, total proteins were extracted from 3-day-old WT etiolated seedlings treated with or without 100 μM ACC and 50 μM MG132 (proteasome inhibitor) for 12 hours and then incubated with EIN2 CEND-MBP variants (EIN2 CEND-MBP, EIN2 CEND^S655A-MBP, and EIN2 CEND^S655D-MBP) with 1 mM ATP in 50 μl of total proteins extracts at 28°C for 90 min, respectively. Then, samples were added with 5× SDS loading buffer and analyzed by immunoblotting using anti-ubiquitin (CST, 3936; dilutions: 1:1000; mouse), anti-MBP, and anti-actin (loading control).

As previously reported (48, 49), endogenous ubiquitination of EIN2 variants was analyzed in 6-day-old transgenic ein2-5 plants expressing EIN2-YFP variants (EIN2-YFP, EIN2^S655A-YFP, and EIN2^S655D-YFP) after 12 hours post–ACC treatment, and the seedlings were treated with 50 μM MG132 for 4 hours. Total proteins were extracted in lysis buffer containing 50 μM MG132 and subjected to immunoaffinity purification using anti-GFP beads at 4°C for 2 hours. Beads were washed three to four times with lysis buffer, and bound proteins were eluted with 50 μl of 2× SDS loading buffer. Ubiquitination status was analyzed by immunoblotting with anti-ubiquitin, anti-GFP, and anti-actin antibodies.

Physiological measurements

Total chlorophyll was extracted from 50 mg of leaf sample with 80% acetone. The homogenate was centrifuged at 12,000g for 10 min at 4°C after 12 hours of dark incubation. Absorbance of the supernatant was measured at 649 and 665 nm using a spectrophotometer.

The MDA content was determined using commercial kits (BC005, Solarbio, China) according to the manufacturer’s instructions.

For the relative ion leakage, leaves were immersed in 10 ml of deionized water for 30 min. The initial electrical conductivity (EC₁) was measured using a conductivity meter. Samples were boiled for 20 min and cooled to 25°C, and the final conductivity (EC₂) was recorded. Relative ion leakage (%) was calculated as (EC₁/EC₂) × 100.

Nitro blue tetrazolium staining for superoxide (O₂⁻) detection

Leaf samples from unstressed and salt-stressed plants were vacuum infiltrated with 0.1% (w/v) nitro blue tetrazolium (NBT) in 0.05 M phosphate-buffered saline (PBS) (pH 7.8) for 10 min, followed by 3-hour dark incubation at 37°C. Chlorophyll was removed by boiling in destaining solution (3:1:1 ethanol:lactic acid:glycerol) for 15 min. Blue formazan precipitates indicating superoxide accumulation were photographed under a Leica stereomicroscope.

Dual luciferase assay

Dual luciferase assays were performed to analyze protein-DNA interactions. Full-length EIN3 was fused to enhanced GFP, whereas the TOPPs promoter sequence were cloned upstream of the Luc (Luciferase) reporter gene. These constructs were cotransformed into N. benthamiana leaves for transient expression. Leaves were infiltrated with a luciferase substrate solution (Promega, USA) and kept in the dark for 5 min. Images were collected by a high-resolution, low-illumination digital cold camera (Tanon, China). At least three biological replicates were performed for each sample.

Y1H assay

For the Y1H assay, AD-fusion constructs and LacZ reporters were cotransformed into the EGY48 yeast strain. Transformants were selected and cultured on SD/-Trp-Ura medium. Yeast transformation and liquid assays were performed as described in the Yeast Protocols Handbook (BD Clontech).

EMSA with CY5-labeled probes

YFP-MBP and EIN3-MBP recombinant fusion proteins were expressed in E. coli BL21 (DE3) cells and purified using the PurKine MBP-Tag Dextrin Resin 6FF (Abbkine). A 36–base pair (bp) promoter region containing the motif (mutant motif) was synthesized, with the 5′-end labeled with CY5 fluorescence modification (Sangon Biotech). Anneal strands in annealing buffer [10 mM tris-HCl (pH 8.0), 50 mM NaCl, and 1 mM EDTA] by heating to 95°C for 5 min followed by gradual cooling. EMSAs were conducted incubate 20 nM CY5-labeled probe with 1 to 5 μg of recombinant EIN3-MBP protein in 10 μl of binding buffer [20 mM tris-HCl (pH 8.0), 10 mM NaCl, 2 mM DTT, 2 mM EDTA, and BSA (0.1 mg/ml)] for 30 min at 25°C. For competition assays, a 50- or 100-fold molar excess of unlabeled probe DNA was added. The reaction products were added to 5× loading buffer [40% glycerol, 0.5 mM tris-HCl (pH 6.8), and bromophenol blue (2 mg/ml)] and separated on 6% PAGE gel with 0.5× Tris-borate-EDTA buffer (TBE) buffer. Electrophoresis was carried out under constant pressure (90 V) in the dark at 4°C for 60 min. The resulting blots were visualized using the multifunctional laser imaging system (PharosFX, BIO-LAB). The primers used are detailed in table S3.

Chromatin immunoprecipitation–quantitative polymerase chain reaction

Chromatin isolation was performed using 4-day-old WT and ein3-1 eil1-1 etiolated seedlings treated with or without ACC for 8 hours. After resuspension, chromatin was sonicated at 4°C to produce 200- to 600-bp fragments. Immunoprecipitation was performed using monoclonal anti-EIN3 antibody, followed by washing, reverse cross-linking, and DNA amplification. About 10% of sonicated but nonimmunoprecipitated chromatin was reverse cross-linked and used as an input DNA control. Both immunoprecipitated DNA and input DNA were analyzed by RT-qPCR (Applied Biosystems). The primers used are detailed in table S3.

Acknowledgments

We thank X. Gou (Lanzhou University) providing mbSUS-related vectors and Q. Chen (China Agricultural University) for pCBC-DT1T2 and pHEE401 vectors. We are grateful to L. Yan, L. Peng, L. Guan, and Y. Gao (Core Facility at Life Science Research, Lanzhou University) for technical assistance.

Funding:

This work was supported by the National Natural Science Foundation of China Grants (32170340 and U25A20634), the National Key Research & Development Program of China Grant (2022YFD1201801), the Major Science and Technology Project of Gansu Province (22ZD6NA049), the Foundations of Science and Technology of Gansu Province (25JRRA708 and 25JRRA683), the Top leading talents project of Gansu Province and the Chang Jiang Scholars Program of China (2023) for S.H., and the Funding for high-end talents of Lanzhou city (127000-563225107).

Author contributions:

Conceptualization: M.S., Q.Q., and J.Z. Methodology: M.S. and Q.Q. Software: M.S. Validation: M.S. and A.Y. Formal analysis: M.S. Investigation: M.S. and Y.L. Resources: M.S. and Q.Q. Data curation: M.S. and Q.Q. Writing—original draft: M.S. and S.H. Writing—review and editing: M.S., S.H., and Q.Q. Visualization: M.S.. Supervision: S.H., S.W., M.S., and Q.Q. Project administration: S.H., M.S., and Q.Q. Funding acquisition: S.H., M.S., and Q.Q.

Competing interests:

The authors declare that they have no competing interests.

Data and materials availability:

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. Sequence data for the genes used in this study can be found in The Arabidopsis Information Resource (www.arabidopsis.org) under the following accession numbers: TOPP1 (AT2G29400), TOPP2 (AT5G59160), TOPP3 (AT1G64040), TOPP4 (AT2G39840), TOPP5 (AT3G46820), TOPP6 (AT4G11240), TOPP7 (AT5G43380), TOPP8 (AT5G27840), TOPP9 (AT3G05580), EIN2 (AT5G03280), EIN3 (AT3G20770), EIL1 (AT2G27050), ERF1(AT3G23240), CTR1 (AT5G03730), ETP1(AT3G18980), and ETP2 (AT3G18910). This study did not generate new materials.

Supplementary Materials

The PDF file includes:

Figs. S1 to S14

Tables S1 to S5

Legends for data S1 to S3

sciadv.aec5937_sm.pdf^{(2.2MB, pdf)}

Other Supplementary Material for this manuscript includes the following:

Data S1 to S3

sciadv.aec5937_data_s1_to_s3.zip^{(1.7MB, zip)}

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21.Yan J., Liu Y., Huang X., Li L., Hu Z., Zhang J., Qin Q. Q., Yan L. F., He K., Wang Y., Hou S. W., An unreported NB-LRR protein SUT1 is required for the autoimmune response mediated by type one protein phosphatase 4 mutation (topp4-1) in Arabidopsis. Plant J. 100, 357–373 (2019). [DOI] [PubMed] [Google Scholar]
22.He Y., Tang X., Fu H., Tang Y., Lin H., Deng X., Arabidopsis KNL1 recruits type one protein phosphatase to kinetochores to silence the spindle assembly checkpoint. Sci. Adv. 11, eadq4033 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Cho H.-Y., Chou M.-Y., Ho H.-Y., Chen W.-C., Shih M.-C., Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2. Sci. Adv. 8, eabm7863 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ecker J. R., The ethylene signal transduction pathway in plants. Science 268, 667–675 (1995). [DOI] [PubMed] [Google Scholar]
25.Zhao H., Yin C. C., Ma B., Chen S. Y., Zhang J. S., Ethylene signaling in rice and Arabidopsis: New regulators and mechanisms. J. Integr. Plant Biol. 63, 102–125 (2021). [DOI] [PubMed] [Google Scholar]
26.Huang W., Hu N., Xiao Z., Qiu Y., Yang Y., Yang J., Mao X., Wang Y., Li Z., Guo H., A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato. Plant Cell 34, 3280–3300 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Poór P., Nawaz K., Gupta R., Ashfaque F., Khan M. I. R., Ethylene involvement in the regulation of heat stress tolerance in plants. Plant Cell 41, 675–698 (2022). [DOI] [PubMed] [Google Scholar]
28.Guo H. W., Ecker J. R., The ethylene signaling pathway: New insights. Curr. Opin. Plant Biol. 7, 40–49 (2004). [DOI] [PubMed] [Google Scholar]
29.Merchante C., Alonso J. M., Stepanova A. N., Ethylene signaling: Simple ligand, complex regulation. Curr. Opin. Plant Biol. 16, 554–560 (2013). [DOI] [PubMed] [Google Scholar]
30.Qiao H., Chang K. N., Yazaki J., Ecker J. R., Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. 23, 512–521 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Li W., Ma M., Feng Y., Li H., Wang Y., Ma Y., Li M., An F., Guo H., EIN2-directed translational regulation of ethylene signaling in Arabidopsis. Cell 163, 670–683 (2015). [DOI] [PubMed] [Google Scholar]
32.Wen X., Zhang C., Ji Y., Zhao Q., He W., An F., Jiang L., Guo H., Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus. Cell Res. 22, 1613–1616 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Qiao H., Shen Z., Huang S. S., Schmitz R. J., Urich M. A., Briggs S. P., Ecker J. R., Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science 338, 390–393 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Ju C., Yoon G. M., Shemansky J. M., Lin D. Y., Ying Z. I., Chang J., Garrett W. M., Kessenbrock M., Groth G., Tucker M. L., Cooper B., Kieber J. J., Chang C., CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 109, 19486–19491 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Riyazuddin R., Verma R., Singh K., Nisha N., Keisham M., Bhati K. K., Kim S. T., Gupta R., Ethylene: A master regulator of salinity stress tolerance in plants. Biomolecules 10, 959–981 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Zheng X., Fang A., Qiu S., Zhao G., Wang J., Wang S., Wei J., Gao H., Yang J., Mou B., Cui F., Zhang J., Liu J., Sun W., Ustilaginoidea virens secretes a family of phosphatases that stabilize the negative immune regulator OsMPK6 and suppress plant immunity. Plant Cell 34, 3088–3109 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Chang K. N., Zhong S., Weiraach M. T., Hon G., Pelizzola M., Li H., Huang S. S., Schmitz R. J., Urich M. A., Kuo D., Nery J. R., Qiao H., Yang A., Jamali A., Chen H., Ideker T., Ren B., Bar-Joseph Z., Hughes T. R., Ecker J. R., Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis. Elife 2, e00675 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Wang L. K., Ko E. E., Tran J., Qiao H., TREE1-EIN3-mediated transcriptional repression inhibits shoot growth in response to ethylene. Proc. Natl. Acad. Sci. U.S.A. 117, 29178–29189 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Shi Y. G., Serine/threonine phosphatases: Mechanism through structure. Cell 139, 468–484 (2009). [DOI] [PubMed] [Google Scholar]
40.Smoly I., Shemesh N., Ziv-Ukelson M., Ben-Zvi A., Yeger-Lotem E., An asymmetrically balanced organization of kinases versus phosphatases across eukaryotes determines their distinct impacts. PLOS Comput. Biol. 13, e1005221 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Fu L., Liu Y., Qin G., Wu P., Zi H., Xu Z., Zhao X., Wang Y., Li Y., Yang S., Peng C., Wong C. C. L., Yoo S. D., Zuo Z., Liu R., Cho Y. H., Xiong Y., The TOR-EIN2 axis mediates nuclear signalling to modulate plant growth. Nature 591, 288–292 (2021). [DOI] [PubMed] [Google Scholar]
42.Zhu F., Deng J., Chen H., Liu P., Zheng L., Ye Q., Li R., Brault M., Wen J., Frugier F., Dong J., Wang T., A CEP peptide receptor-like kinase regulates auxin biosynthesis and ethylene signaling to coordinate root growth and symbiotic nodulation in Medicago truncatula. Plant Cell 32, 2855–2877 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Su M. F., Hou S. W., Ethylene insensitive 2 (EIN2) destiny shaper: The post translational modification. J. Plant Physiol. 295, 154190–154198 (2024). [DOI] [PubMed] [Google Scholar]
44.Zhang J., Chen Y., Lu J., Zhang Y., Wen C.-K., Uncertainty of EIN2^{Ser645/Ser924} inactivation by CTR1-mediated phosphorylation reveals the complexity of ethylene signaling. Plant Commun. 1, 100046–100064 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Xu J., Liu S., Cai L., Wang L., Dong Y., Qi Z., Yu J., Zhou Y., SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato. Plant Biotechnol. J. 21, 219–231 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Usher E. T., Fossat M. J., Holehouse A. S., Phosphorylation of disordered proteins tunes local and global intramolecular interactions. Biophys. J. 123, 4082–4096 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Wang Z., Yang Q., Zhang D., Lu Y., Wang Y., Pan Y., Qiu Y., Men Y., Yan W., Xiao Z., Sun R., Li W., Huang H., Guo H., A cytoplasmic osmosensing mechanism mediated by molecular crowding-sensitive DCP5. Science 386, eadk9067 (2024). [DOI] [PubMed] [Google Scholar]
48.Liu C., Lin J. Z., Wang Y., Tian Y., Zheng H. P., Zhou Z. K., Zhou Y. B., Tang X. D., Zhao X. H., Wu T., Xu S. L., Tang D. Y., Zuo Z. C., He H., Bai L. Y., Yang Y. Z., Liu X. M., The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H₂O₂ homeostasis and salt tolerance in rice. Plant Cell 35, 3604–3625 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Ni L., Fu X. P., Zhang H., Li X., Cai X., Zhang P. P., Liu L., Wang Q., Sun M., Wang Q. W., Zhang A., Zhang Z. G., Jiang M. Y., Abscisic acid inhibits rice protein phosphatase PP45 via H₂O₂ and relieves repression of the Ca²⁺/CaM dependent protein kinase DMI3. Plant Cell 31, 128–152 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

Figs. S1 to S14

Tables S1 to S5

Legends for data S1 to S3

sciadv.aec5937_sm.pdf^{(2.2MB, pdf)}

Data S1 to S3

sciadv.aec5937_data_s1_to_s3.zip^{(1.7MB, zip)}

Data Availability Statement

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[R20] 20.Liu Y. Q., Yan J., Qin Q. Q., Zhang J., Chen Y., Zhao L., He K., Hou S. W., Type one protein phosphatases (TOPPs) contribute to the plant defense response in Arabidopsis. J. Integr. Plant Biol. 62, 360–377 (2019). [DOI] [PubMed] [Google Scholar]

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[R25] 25.Zhao H., Yin C. C., Ma B., Chen S. Y., Zhang J. S., Ethylene signaling in rice and Arabidopsis: New regulators and mechanisms. J. Integr. Plant Biol. 63, 102–125 (2021). [DOI] [PubMed] [Google Scholar]

[R26] 26.Huang W., Hu N., Xiao Z., Qiu Y., Yang Y., Yang J., Mao X., Wang Y., Li Z., Guo H., A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato. Plant Cell 34, 3280–3300 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] 27.Poór P., Nawaz K., Gupta R., Ashfaque F., Khan M. I. R., Ethylene involvement in the regulation of heat stress tolerance in plants. Plant Cell 41, 675–698 (2022). [DOI] [PubMed] [Google Scholar]

[R28] 28.Guo H. W., Ecker J. R., The ethylene signaling pathway: New insights. Curr. Opin. Plant Biol. 7, 40–49 (2004). [DOI] [PubMed] [Google Scholar]

[R29] 29.Merchante C., Alonso J. M., Stepanova A. N., Ethylene signaling: Simple ligand, complex regulation. Curr. Opin. Plant Biol. 16, 554–560 (2013). [DOI] [PubMed] [Google Scholar]

[R30] 30.Qiao H., Chang K. N., Yazaki J., Ecker J. R., Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev. 23, 512–521 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Li W., Ma M., Feng Y., Li H., Wang Y., Ma Y., Li M., An F., Guo H., EIN2-directed translational regulation of ethylene signaling in Arabidopsis. Cell 163, 670–683 (2015). [DOI] [PubMed] [Google Scholar]

[R32] 32.Wen X., Zhang C., Ji Y., Zhao Q., He W., An F., Jiang L., Guo H., Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus. Cell Res. 22, 1613–1616 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R33] 33.Qiao H., Shen Z., Huang S. S., Schmitz R. J., Urich M. A., Briggs S. P., Ecker J. R., Processing and subcellular trafficking of ER-tethered EIN2 control response to ethylene gas. Science 338, 390–393 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Ju C., Yoon G. M., Shemansky J. M., Lin D. Y., Ying Z. I., Chang J., Garrett W. M., Kessenbrock M., Groth G., Tucker M. L., Cooper B., Kieber J. J., Chang C., CTR1 phosphorylates the central regulator EIN2 to control ethylene hormone signaling from the ER membrane to the nucleus in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 109, 19486–19491 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R35] 35.Riyazuddin R., Verma R., Singh K., Nisha N., Keisham M., Bhati K. K., Kim S. T., Gupta R., Ethylene: A master regulator of salinity stress tolerance in plants. Biomolecules 10, 959–981 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Zheng X., Fang A., Qiu S., Zhao G., Wang J., Wang S., Wei J., Gao H., Yang J., Mou B., Cui F., Zhang J., Liu J., Sun W., Ustilaginoidea virens secretes a family of phosphatases that stabilize the negative immune regulator OsMPK6 and suppress plant immunity. Plant Cell 34, 3088–3109 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R37] 37.Chang K. N., Zhong S., Weiraach M. T., Hon G., Pelizzola M., Li H., Huang S. S., Schmitz R. J., Urich M. A., Kuo D., Nery J. R., Qiao H., Yang A., Jamali A., Chen H., Ideker T., Ren B., Bar-Joseph Z., Hughes T. R., Ecker J. R., Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis. Elife 2, e00675 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Wang L. K., Ko E. E., Tran J., Qiao H., TREE1-EIN3-mediated transcriptional repression inhibits shoot growth in response to ethylene. Proc. Natl. Acad. Sci. U.S.A. 117, 29178–29189 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Shi Y. G., Serine/threonine phosphatases: Mechanism through structure. Cell 139, 468–484 (2009). [DOI] [PubMed] [Google Scholar]

[R40] 40.Smoly I., Shemesh N., Ziv-Ukelson M., Ben-Zvi A., Yeger-Lotem E., An asymmetrically balanced organization of kinases versus phosphatases across eukaryotes determines their distinct impacts. PLOS Comput. Biol. 13, e1005221 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R41] 41.Fu L., Liu Y., Qin G., Wu P., Zi H., Xu Z., Zhao X., Wang Y., Li Y., Yang S., Peng C., Wong C. C. L., Yoo S. D., Zuo Z., Liu R., Cho Y. H., Xiong Y., The TOR-EIN2 axis mediates nuclear signalling to modulate plant growth. Nature 591, 288–292 (2021). [DOI] [PubMed] [Google Scholar]

[R42] 42.Zhu F., Deng J., Chen H., Liu P., Zheng L., Ye Q., Li R., Brault M., Wen J., Frugier F., Dong J., Wang T., A CEP peptide receptor-like kinase regulates auxin biosynthesis and ethylene signaling to coordinate root growth and symbiotic nodulation in Medicago truncatula. Plant Cell 32, 2855–2877 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R43] 43.Su M. F., Hou S. W., Ethylene insensitive 2 (EIN2) destiny shaper: The post translational modification. J. Plant Physiol. 295, 154190–154198 (2024). [DOI] [PubMed] [Google Scholar]

[R44] 44.Zhang J., Chen Y., Lu J., Zhang Y., Wen C.-K., Uncertainty of EIN2^{Ser645/Ser924} inactivation by CTR1-mediated phosphorylation reveals the complexity of ethylene signaling. Plant Commun. 1, 100046–100064 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R45] 45.Xu J., Liu S., Cai L., Wang L., Dong Y., Qi Z., Yu J., Zhou Y., SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato. Plant Biotechnol. J. 21, 219–231 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] 46.Usher E. T., Fossat M. J., Holehouse A. S., Phosphorylation of disordered proteins tunes local and global intramolecular interactions. Biophys. J. 123, 4082–4096 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R47] 47.Wang Z., Yang Q., Zhang D., Lu Y., Wang Y., Pan Y., Qiu Y., Men Y., Yan W., Xiao Z., Sun R., Li W., Huang H., Guo H., A cytoplasmic osmosensing mechanism mediated by molecular crowding-sensitive DCP5. Science 386, eadk9067 (2024). [DOI] [PubMed] [Google Scholar]

[R48] 48.Liu C., Lin J. Z., Wang Y., Tian Y., Zheng H. P., Zhou Z. K., Zhou Y. B., Tang X. D., Zhao X. H., Wu T., Xu S. L., Tang D. Y., Zuo Z. C., He H., Bai L. Y., Yang Y. Z., Liu X. M., The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H₂O₂ homeostasis and salt tolerance in rice. Plant Cell 35, 3604–3625 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R49] 49.Ni L., Fu X. P., Zhang H., Li X., Cai X., Zhang P. P., Liu L., Wang Q., Sun M., Wang Q. W., Zhang A., Zhang Z. G., Jiang M. Y., Abscisic acid inhibits rice protein phosphatase PP45 via H₂O₂ and relieves repression of the Ca²⁺/CaM dependent protein kinase DMI3. Plant Cell 31, 128–152 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Type one protein phosphatases (TOPPs) catalyze EIN2 dephosphorylation to regulate ethylene signaling in Arabidopsis

Meifei Su

Qianqian Qin

Jing Zhang

Yingdong Li

Ailin Ye

Suomin Wang

Suiwen Hou

Roles

Abstract

INTRODUCTION

RESULTS

TOPPs are involved in ethylene signaling

Fig. 1. TOPPs are involved in ethylene signaling.

Genetic association and physical interactions between TOPPs and EIN2

Fig. 2. TOPPs are genetically upstream of EIN2 and physically interact with EIN2.

TOPPs dephosphorylate EIN2 at the S655 residue

Fig. 3. TOPPs specifically dephosphorylate EIN2 at the S655 site.

Dephosphorylation at the S655 site is important for EIN2-mediated ethylene signaling

Fig. 4. S655 dephosphorylation is important for EIN2-mediated ethylene signaling.

EIN2 dephosphorylation is conducive to salt tolerance in Arabidopsis

Fig. 5. EIN2 dephosphorylation at the S655 site is conducive to salt tolerance in Arabidopsis.

Ethylene-induced TOPPs expression depends on EIN3/EIL1-mediated transcriptional activation

Fig. 6. EIN3/EIL1 binds to the EBS element in the TOPPs promoter and activates its expression in response to ethylene.

DISCUSSION

Fig. 7. A proposed working model illustrates how TOPPs regulate ethylene signaling by dephosphorylating EIN2.

MATERIALS AND METHODS

Plant materials and growth conditions

Plant growth conditions and hypocotyl measurement

RT-qPCR analysis of TOPPs, EIN2, and ERF1 mRNA abundance

GUS staining

Protein isolation and immunoblot analysis

Mass spectrometry assay

Mating-based split-ubiquitin system and Y2H assay

In vitro protein expression and pull-down assay

BiFC and fluorescence microscopy

Co-IP assay

In vitro dephosphorylation and cell-free phosphorylation/dephosphorylation assay

AlphaFold 3 analysis

Cell-free degradation assay

Ubiquitination assays

Physiological measurements

Nitro blue tetrazolium staining for superoxide (O2−) detection

Dual luciferase assay

Y1H assay

EMSA with CY5-labeled probes

Chromatin immunoprecipitation–quantitative polymerase chain reaction

Acknowledgments

Funding:

Author contributions:

Competing interests:

Data and materials availability:

Supplementary Materials

The PDF file includes:

Other Supplementary Material for this manuscript includes the following:

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Nitro blue tetrazolium staining for superoxide (O₂⁻) detection