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Enterics Part 1

Initiation and amplification of SnRK2 activation in abscisic acid signaling

Abstract

The phytohormone abscisic acid (ABA) is crucial for plant responses to environmental challenges. The SNF1-regulated protein kinase 2s (SnRK2s) are key components in ABA-receptor coupled core signaling, and are rapidly phosphorylated and activated by ABA. Recent studies have suggested that Raf-like protein kinases (RAFs) participate in ABA-triggered SnRK2 activation. In vitro kinase assays also suggest the existence of autophosphorylation of SnRK2s. Thus, how SnRK2 kinases are quickly activated during ABA signaling still needs to be clarified. Here, we show that both B2 and B3 RAFs directly phosphorylate SnRK2.6 in the kinase activation loop. This transphosphorylation by RAFs is essential for SnRK2 activation. The activated SnRK2s then intermolecularly trans-phosphorylate other SnRK2s that are not yet activated to amplify the response. High-order Arabidopsis mutants lacking multiple B2 and B3 RAFs show ABA hyposensitivity. Our findings reveal a unique initiation and amplification mechanism of SnRK2 activation in ABA signaling in higher plants.

Introduction

Environmental challenges like drought, cold, and high salinity induce the accumulation of abscisic acid (ABA), a major stress phytohormone that triggers multiple stress responses in plants1,2,3,4,5,6. ABA controls stomatal closure, seed dormancy and germination, senescence, growth, and development1,2,3,7. The ABA receptor-coupled core signaling pathway has been uncovered8,9,10 and consists of three key components: the ABA receptors, the PYRABACTIN RESISTANCE (PYR)/PYR-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family proteins; the negative regulator clade A type 2 C protein phosphatases (PP2Cs); and the positive regulator SNF1-related protein kinase 2 s (SnRK2s).

Phosphorylation and dephosphorylation determines the activation of SnRK2s and therefore ABA signaling. In the model plant Arabidopsis thaliana, there are ten members of the SnRK2 protein kinase family. Three of them, SnRK2.2, SnRK2.3, and SnRK2.6, are quickly activated within minutes after application of exogenous ABA, while all SnRK2s except SnRK2.9 are activated by osmotic stresses11,12,13,14. SnRK2.6, also known as OPEN STOMATA 1 (OST1), is mainly expressed in guard cells, while SnRK2.2 and SnRK2.3 are universally expressed15,16. The ost1/snrk2.6 mutant shows constitutive stomatal opening and is thus hypersensitive to water deficit15. The snrk2.2/2.3/2.6 triple (snrk2-triple) mutant is resistant to ABA and germinates and grows normally at very high concentrations of ABA13,14. At least two phosphosites, Ser171 and Ser175, which are located in the activation loop of SnRK2.6, are required for SnRK2.6 activation upon ABA treatment17,18. Other phosphosites like the N-terminal Ser7 and Ser29 may also contribute to the activation of SnRK2.619,20. The clade A PP2C phosphatases are the negative regulators of SnRK2s8,9,10,21,22,23. Under normal growth conditions, PP2Cs inhibit SnRK2.6 by directly binding to and dephosphorylating the Ser175 in the activation loop of SnRK2.6 and block the ABA signaling20,21,22.

ABA binds to PYR/PYL/RCARs, and then the ABA-receptor complex inhibits the activity of PP2C phosphatases, resulting in the release of SnRK2s from PP2C-mediated inhibition9,10,20,24,25. Using recombinant SnRK2.6 purified from E. coli, Ng et al. (2011) reported that the phosphorylation and activation of SnRK2.6 mainly depends on its autophosphorylation activity26. However, whether SnRK2s are auto-activatable in vivo is still debatable since recombinant SnRK2.6 purified from E. coli is already highly phosphorylated and active. Most recently, several studies suggested that dephosphorylated SnRK2.6 and SnRK2.4 have no self-activation activity, and that transphosphorylation of SnRK2s by Raf-like protein kinases (RAFs) is required for SnRK2 activation27,28,29,30. B2/B3 and B4 RAFs are also called Osmotic stress-activated protein Kinase-100 kDa (OK100) and Osmotic stress-activated protein Kinase-130 kDa (OK130), respectively, because of their rapid activation by osmotic stress and their molecular weights observed from in-gel kinase assays27. The B4 subgroup RAFs (OK130) interact with and phosphorylate ABA-independent SnRK2s27. In a null mutant of B4 subgroup RAFs, OK130-null(raf16/raf40/raf24/raf18/ raf20/raf35/raf42), or raf18/raf20/raf24, the osmotic stress-induced activation of ABA-independent SnRK2s is completely abolished27,31. Interestingly, the B3 and B2 RAFs (OK100) phosphorylate SnRK2.2, SnRK2.3, and SnRK2.6 in vitro27,28,30,32. The high-order mutant OK-quatdec, containing mutations in four B2, three B3, and seven B4 RAFs, shows weak ABA insensitivity in seed germination and root growth27. A triple mutant of B3 RAF kinases, m3kδ1/δ6/δ7 (raf5/raf4/raf3), is slightly insensitive to ABA and is impaired in ABA-mediated SnRK2 activation28. However, compared to the complete abolishment of ABA responses in snrk2-triple, pyr1pyl1pyl2pyl4pyl5pyl8 (pyl112458), or pyl-duodecuple (pyr1pyl1pyl2pyl3pyl4pyl5pyl7pyl8py9pyl10pyl11pyl12, pyl-duodec) mutants13,14,33,34, the ABA-insensitivity is much weaker in m3kδ1/δ6/δ7 or OK-quatdec mutants27,28. Thus, the role of RAFs in ABA signaling still needs further investigation.

Here, we show that the B2 and B3 subgroup RAFs phosphorylate Ser171 and Ser175 in SnRK2.6 with different specificity and that transphosphorylation is essential for initiating SnRK2.6 phosphorylation and activation. After phosphorylation by RAFs, the activated SnRK2.6 can quickly autophosphorylate (intermolecularly) and activate more SnRK2 proteins. We also generate a series of high-order mutants carrying null mutations in the B2, B3, or both B2 and B3 subgroup RAFs. From phenotypic assays of these high-order mutants, we find that both B2 and B3 subgroup RAFs are essential for ABA signaling. ABA-induced activation of SnRK2.2/2.3/2.6 and ABA-induced gene expression are strongly impaired in OK100-oct and OK100-nonu mutants lacking 8 and 9 members, respectively, of the B2 and B3 subgroups. OK100-oct and OK100-nonu also exhibit ABA hyposensitivity and can germinate and grow under extremely high ABA concentrations. We find that ABA does not activate B2 and B3 RAFs; instead, the basal level of RAF kinase activity is essential for SnRK2 activation and initiation of ABA signaling. Our results reveal a crucial RAF-SnRK2 cascade in ABA receptor-coupled core signaling and unique activation machinery for initiating and amplifying stress signaling in higher plants.

Results

SnRK2.6 activation requires transphosphorylation by B2 and B3 RAFs in vitro

Recent studies suggested that several B3 RAFs (RAF3-6) and one B2 RAF (RAF10) can phosphorylate and activate dephosphorylated SnRK2.6 in vitro27,28,30,32. In vitro kinase assays and subsequent mass spectrometry revealed that Ser171 and Ser175 of SnRK2.6 might be the major sites for phosphorylation by B2 and B3 subgroup RAFs27. To further dissect the role of B2 and B3 RAFs in SnRK2 activation, we first tested the ability and specificity of the recombinant kinase domains (KDs) of B2 and B3 subgroup RAFs in SnRK2.6 phosphorylation. Out of 12 tested B2/B3 RAFs, KDs of 10 RAFs, RAF1-7, and RAF10-12, strongly phosphorylated SnRK2.6KR, a kinase-dead form of SnRK2.6 (Fig. 1a). The recombinant KDs of RAF8 and RAF9 had no detectable kinase activity (Supplementary Fig. 1a). The KDs of B2 and B3 RAFs showed distinct specificity for Ser171 and Ser175 of SnRK2.6 in the in vitro assay: the B2 RAFs, RAF7 and RAF10-12, targeted Ser171, while the B3 RAFs, RAF1-6, preferred Ser175 (Fig. 1a).

a Recombinant RAF kinase domains (KDs) were used to phosphorylate SnRK2.6KR (SnRK2.6K50R, a kinase-dead form of SnRK2.6), SnRK2.6KR with Ser171Ala mutation (SnRK2.6KR-S171A), SnRK2.6KR with Ser175Ala mutation (SnRK2.6KR-S175A), or SnRK2.6KR proteins with Ser171AlaSer175Ala mutations (SnRK2.6KR-AA), in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-RAF-KD and HIS-SnRK2.6KR. b GST-SnRK2.6, HIS-SUMO-RAF3-KD, and HIS-SUMO-RAF10-KD phosphorylate HIS-SnRK2.6KR in vitro. Recombinant undephosphorylated GST-SnRK2.6 was used to phosphorylate HIS-SnRK2.6KR in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6, HIS-SUMO-RAF3-KD, HIS-SUMO-RAF10-KD, and HIS-SnRK2.6KR. c SnRK2.6M94G but not wild type SnRK2.6 can use N6-Benzyl-ATPγS to thiophosphorylate substrate. d RAF3-KD and RAF10-KD trigger the autophosphorylation of pre-dephosphorylated GST-SnRK2.6M94G (de-SnRK2.6M94G). e RAF3-KD activates pre-dephosphorylated GST-SnRK2.6M94G (de-SnRK2.6M94G) and the reactivated SnRK2.6M94G phosphorylates itself and ABF2. f RAF3-KD and RAF10-KD activate HIS-SUMO-SnRK2.2M96G and HIS-SUMO-SnRK2.3M95G. For d to f, Anti-γ-S immunoblot (upper) and Coomassie staining (lower) show thiophosphorylation and loading, respectively, of recombinant GST-RAF3-KD, GST-RAF10-KD, GST-SnRK2.6M94G, HIS-SUMO-SnRK2.2M96G, HIS-SUMO-SnRK2.3M95G, and GST-ABF2. Asterisks indicate preincubation of SnRK2.6M94G with N6-Benzyl-ATPγS for 30 min before further reaction. Arrows indicate degraded fragments co-purified with GST-SnRK2.6M94G. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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Ser175 was previously suggested to be a SnRK2.6 autophosphorylation site26. Supporting this notion, recombinant SnRK2.6 intermolecularly trans-phosphorylated SnRK2.6KR (Fig. 1b), though the phosphorylation was much weaker than the transphosphorylation by RAF3 and RAF10. Thus, SnRK2.6 can be either transphosphorylated by RAFs or transphosphorylated intermolecularly by other SnRK2.6 molecules.

To further evaluate the role of RAFs in SnRK2.6 activation, we designed an adenosine triphosphate (ATP) analog-based in vitro kinase assay system that distinguishes trans- and autophosphorylation of SnRK2.6 (Fig. 1c). A Met94Gly (M94G) mutation in SnRK2.6 enlarges its ATP binding pocket (Fig. 1c). SnRK2.6M94G can use the ATP analog N6-Benzyl-ATPγS to thiophosphorylate itself or its substrate (Supplementary Fig. 1b). Neither RAF3-KD nor wild-type SnRK2.6 can use the N6-Benzyl-ATPγS as a thiophosphate donor (Fig. 1d, Supplementary Fig. 1b). By this method, thiophosphorylation by activated SnRK2.6M94G can be detected with an anti-thiophosphate ester antibody that only recognizes the thiophosphorylation (Supplementary Fig. 1b). Pre-dephosphorylated SnRK2.6M94G (de-SnRK2.6M94G) had no auto-thiophosphorylation activity (Fig. 1d, lanes 2–6, Fig. 1e, lanes 4–5). Application of recombinant RAF3-KD or RAF10-KD quickly induced the auto-thiophosphorylation of SnRK2.6M94G in a time-dependent manner (Fig. 1d, lanes 7–14), suggesting that transphosphorylation by RAF3 and RAF10 is essential for SnRK2.6M94G auto-thiophosphorylation activity.

We then examined SnRK2.6M94G activity by detecting the thiophosphorylation of ABA‐RESPONSIVE ELEMENT‐BINDING FACTOR 2 (ABF2), a well-studied SnRK2 substrate. Adding RAF3-KD initiated the thiophosphorylation of both SnRK2.6M94G and ABF2 (Fig. 1e, lanes 10–12). Preincubation with RAF3 significantly enhanced the kinase activity of recombinant SnRK2.6M94G (Fig. 1e, lanes 10–12 compared to lanes 2–3). We also measured the activation effect of RAF3 and RAF10 on SnRK2.2M96G and SnRK2.3M95G, the mutated forms that can use the N6-Benzyl-ATPγS. Similar to previous study26, the recombinant SnRK2.2M96G and SnRK2.3M95G only had weak kinase activity that was rarely detectable in the thiophosphorylation assay (Fig. 1f, lanes 1, 2, 7, and 8). However, adding either RAF10-KD or RAF3-KD strongly enhanced the kinase activities of SnRK2.2M96G and SnRK2.3M95G, in the context of ABF2 and SnRK2 thiophosphorylation (Fig. 1f, lanes 4, 6 compared to lanes 2, and lanes 10, 12 compared to lane 8). Taking these results together, transphosphorylation by RAFs is required for the reactivation of SnRK2.2/2.3/2.6. The enhanced thiophosphorylation of SnRK2.6M94G, SnRK2.2M96G, and SnRK2.3M95G on themselves suggested that activated SnRK2s can quickly intermolecularly transphosphorylate and activate other SnRK2 molecules in vitro.

Intermolecular transphosphorylation amplifies SnRK2 activation

To further validate this amplification process, we measured the phosphorylation of SnRK2.6 on SnRK2.6KR by in vitro kinase assay. GST-SnRK2.6 showed weak phosphorylation of HIS-SnRK2.6KR (Fig. 2a). After preincubating with RAF10-KD and ATP for 30 min, and removing RAF10-KD after preincubation, the activated GST-SnRK2.6 (SnRK2.6*) then showed an enhanced ability to phosphorylate HIS-SnRK2.6KR (Fig. 2a, lane 6). The Ser171Ala or the Ser175Ala mutation significantly impaired, and Ser171AlaSer175Ala double mutation completely abolished, the HIS-SnRK2.6KR phosphorylation by pre-activated GST-SnRK2.6 (Fig. 2b). This result suggests that SnRK2.6 mainly transphosphorylates SnRK2.6 at Ser171 and Ser175 residues, the same major phosphosites of B2 and B3 RAFs.

a Recombinant SnRK2.6 and pre-activated SnRK2.6 (SnRK2.6*) phosphorylate HIS-SnRK2.6KR. Aliquot of SnRK2.6 was pre-incubated with HIS-SUMO-RAF10-KD coated on Ni-NTA beads for 30 min. After removal of HIS-SUMO-RAF10-KD by centrifugation, pre-activated SnRK2.6 (SnRK2.6*) was used to phosphorylate HIS-SnRK2.6KR, in the presence of [γ-32p]ATP. Same amount of SnRK2.6 without pre-activation was used as control (lanes 1–3). Lane 4 indicates no remaining RAF10-KD after removal. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6 and HIS-SnRK2.6KR. b Pre-activated SnRK2.6 (SnRK2.6*) phosphorylates SnRK2.6KR, SnRK2.6KR-S171A, SnRK2.6KR-S175A, or SnRK2.6KR-AA, in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6 and HIS-SnRK2.6KR proteins. c Transphosphorylation activity of pre-activated SnRK2.6M94G, SnRK2.6M94G-S171A, SnRK2.6M94G-S175A, and SnRK2.6M94G-AA on SnRK2.6KR. d Effect of recombinant SnRK2.6 and pre-activated SnRK2.6 (SnRK2.6*) on SnRK2.6M94G activity. For c to d, Anti-γ-S immunoblot (left) and Coomassie staining (right) show thiophosphorylation and loading, respectively, of recombinant GST-SnRK2.6M94G, HIS -SnRK2.6KR, and GST-ABF2. Arrow indicates partial degraded band of GST-SnRK2.6M94G. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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We further confirmed the trans- and autophosphorylation of Helium streamer by mass spectrometry analysis after in vitro kinase reaction. In such a reaction, γ-[18O]-ATP was used as the phosphate donor of RAF3 to trans-phosphorylate SnRK2.6M94G, and Benzyl-ATPγS was used as the thiophosphate donor for SnRK2.6M94G autophosphorylation. After 30 min of incubation, the reaction was subjected to mass spectrometry analysis. The result confirmed that Ser171 and Ser175 are both intermolecular auto-phosphosites (thio-phosphosites) and trans-phosphosites (18O-phosphosites in Supplementary Data 1). Beside Ser171 and Ser175, six other residues, Ser29, Ser43, Ser71, Thr176, Thr179, Try182, in SnRK2.6M94G were both intermolecular auto-phosphosites and trans-phosphosites (Supplementary Data 1). We then generated non-phosphorylatable mutations (Ser/Thr/Try to Ala) on these phosphosites in SnRK2.6 to elucidate their contributions to SnRK2 intermolecularly trans-phosphorylation activity. Mutating either Ser175 or Ser171 completely abolished the ability of SnRK2.6M94G to thiophosphorylate SnRK2.6KR (Fig. 2c), while Thr179 and Try182 may also contribute to the intermolecularly trans-phosphorylation activity of SnRK2.6 (Supplementary Fig. 2a).

We then further verified the ability of pre-activated SnRK2.6 to activate pre-dephosphorylated SnRK2.6M94G. We activated GST-SnRK2.6 by incubating it with RAF3-KD and RAF10-KD. After removing RAF3/10-KD, we used the pre-activated SnRK2.6 to phosphorylate SnRK2.6M94G. The kinase activity of SnRK2.6M94G was determined by thiophosphorylation of SnRK2.6M94G and ABF2. Incubating with pre-activated SnRK2.6 (SnRK2.6*), but not the inactive SnRK2.6, enhanced the kinase activity of SnRK2.6M94G (Fig. 2d). Pre-activated SnRK2.6 was also able to transphosphorylate SnRK2.2 and SnRK2.3 in the in vitro kinase assay (Supplementary Fig. 2b). Thus, after activation by RAF kinases, SnRK2.6 can intermolecularly autophosphorylate and activate other SnRK2s to amplify the response.

B2 and B3 RAFs function redundantly in ABA response in vivo

To validate the RAF-SnRK2 cascade in ABA signaling in planta, we used gene-editing technology to introduce mutations in B2 or B3 subgroup RAF genes in Arabidopsis Col-0 wild type (Fig. 3a, b). Genotyping thousands of transgenic seeds identified two high-order mutants. OK100-B3 [raf2/enhanced disease resistance 1(edr1);raf3;raf4; raf5/sugar insensitive 8 (sis8);raf6] contained null mutations in five of six B3 subgroup RAFs. raf1/constitutive triple response 1 (ctr1) was not included in the mutant due to a severe morphological phenotype35. OK100-B2 (raf7;raf8;raf9;raf10;raf11;raf12) contained null mutations in all six B2 subgroup RAF genes (Fig. 3a, b, Supplementary Fig. 3a, b). OK100-B3 had a low germination rate on half Murashige and Skoog (MS) medium without sucrose and had strong arrested growth even under normal conditions (Fig. 3c, Supplementary Fig. 4a, b). No ABA insensitivity in germination and seedling development in OK100-B3 was observed on half MS medium without sugar (Fig. 3c, upper panel, d, Supplementary Fig. 4a, b). Interestingly, addition of 1% sucrose strongly improved the germination and seedling development of the OK100-B3 mutant, which showed strong ABA insensitivity and geminated on medium containing up to 5 µM ABA (Fig. 3c, e, Supplementary Fig. 4c, d). OK100-B2 was hyposensitive to ABA and germinated on medium containing up to 10 µM ABA, with or without sucrose (Fig. 3c–e, Supplementary Fig. 4a–d). The OK130-null mutant [raf16;raf40/hydraulic conductivity of root 1(hcr1);raf24;raf18;raf35;raf42] did not show insensitivity to ABA (Fig. 3c–e, Supplementary Fig. 4a–d). OK100-B3 and OK100-B2 also showed impaired seed dormancy and fresh harvested OK100-B3 and OK100-B2 seeds had higher germination rates than fresh harvested wild-type seeds (Supplementary Fig. 4e). To measure the phosphorylation and activation of SnRK2s, we used the phosphorylation-specific antibodies recognizing the phosphoserines corresponding to Ser175 and Ser171 in SnRK2.6 (Supplementary Fig. 5 and Supplementary Data 2). ABA-induced phosphorylation of conserved serine residues corresponding to Ser171 and Ser175 in SnRK2.6 was markedly reduced in the OK100-B2, and relatively not affected in the OK100-B3 and OK130-null mutants (Fig. 3f). Thus, B2 and B3 RAFs may have partially redundant roles in ABA responses in plants.

a Mutations of RAF genes in the high-order mutants used in this study. b Photographs of seedlings after 4 weeks of growth in the soil. Bar = 1 cm. c Photographs of seedlings after 10 days of germination and growth on 1/2 MS medium containing indicated concentrations of ABA, without (upper panel) or with (bottom panel) 1% sucrose. The position of mutants in the image is shown in the box at the upper-right corner. d Photographs of seedlings after 10 days of germination and growth on 1/2 MS medium containing indicated concentrations of ABA without sucrose. e Photographs of seedlings after 10 days of germination and growth on 1/2 MS medium containing indicated concentrations of ABA with 1% sucrose. f The ABA-induced phosphorylation of Ser171 and Ser175 of SnRK2.6 in wild-type and the OK100-B2 and OK100-B3 mutants. The anti-pS171 and anti-pS175 immunoblots were used to show the phosphorylation of the conserved serine residues. Arrows indicate the non-specific bands recognized by anti-pS171 and anti-pS175 antibodies. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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B2 and B3 RAFs cooperate in ABA-induced SnRK2 activation

We also generated OK100-oct (raf3;raf4;raf5/sis8;raf7;raf8;raf9;raf10;raf11) and OK100-nonu (raf3;raf4;raf5/sis8;raf6;raf7;raf8;raf9;raf10;raf11) mutants containing null mutations in eight and nine RAF genes, respectively, of the B2 and B3 subgroups (Fig. 3a, Supplementary Fig. 6). Growth analysis showed that the OK100-oct and OK100-nonu mutants resembled the previously characterized snrk2-triple mutant and the high-order mutants of PYR/PYL/RCARs exhibiting a strong defect in growth and development (Fig. 4a, Supplementary Fig. 7a–c). By contrast, the OK100-quin mutant, which contains mutations in only two B2 and three B3 RAF genes, displayed a wild-type-like growth phenotype (Fig. 4a). The OK100-nonu mutant produced very few seeds and had a lower seed germination rate (Supplementary Fig. 7b, c). The OK100-oct and OK100-nonu mutants showed higher water loss than the wild type (Fig. 4b). Furthermore, ABA-induced stomatal closure was strongly impaired in the OK100-oct and OK100-nonu mutants, which resembles the snrk2-triple and pyl112458 mutants (Fig. 4c). These results suggested an essential role of the B2 and B3 subgroup RAFs in ABA signaling.

a Photographs of wild-type and mutant seedlings after 4 weeks of growth in the soil. Bar = 1 cm. b Water loss of 4-week-old wild-type and high-order mutants. Error bars, SEM (n = 5 biological replicates, each replicate has 5 or 6 individual seedlings). c Stomatal closure of 4-week-old wild type and mutants in response to ABA. Error bars, SEM (n = 59 or 60 individual stomates). d Photographs of seedlings after 10 days germination and growth on 1/2 MS medium containing different concentrations of ABA. The position of mutants in the image is shown in the gray box at the bottom-right corner. e Photographs of seedlings growing 10 days after transfer to 1/2 MS medium with or without 50 µM ABA. Bar = 1 cm. f In-gel kinase assay showing the activation of SnRK2s and RAFs in wild-type and different mutants with or without 15 min of ABA or mannitol treatment. Arrows indicate the OK100 band observed in Col-0, snrk2-triple, and pyl112458 mutants. g The ABA-induced phosphorylation of Ser171 and Ser175 acid production - Crack Key For U SnRK2.6 in wild-type and the OK100 high-order mutants. Arrows indicate the non-specific bands recognized by anti-pS171 and anti-pS175 antibodies. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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We further evaluated the role of B2 and B3 subgroup RAFs in ABA signaling by assaying seed germination in response to ABA. All tested mutants carrying high-order mutations in B2 and B3 subgroup RAFs, including OK100-oct and OK100-nonu, showed insensitivity to ABA in seed germination and post-germination seedling growth (Fig. 4d, e, Supplementary Fig. 7d–h). The order of insensitivity to ABA in seed germination was OK100-quin < OK100-oct = OK-quatdec < OK100-nonu < snrk2-triple, pyl112458, and pyl-duodec (Fig. 4d, Supplementary Fig. 7d). Higher-order RAF mutants were clearly more insensitive than lower-order RAF mutants to ABA in seed germination, which further suggests that the RAF members in the B2 and B3 subgroups have essential and partially redundant roles in ABA signaling. OK100-oct and OK100-nonu also exhibited hyposensitivity to ABA in seedling growth as indicated by root growth, fresh weight measurements, and leaf yellowing (Supplementary Fig. 7g–j).

We then measured ABA-induced SnRK2.2/2.3/2.6 activation in these mutants by in-gel kinase assay. The ABA-induced SnRK2.2/2.3/2.6 activation was almost completely abolished in the OK100-oct and OK100-nonu mutants, which resembles that in the snrk2-triple and pyl112458 mutants (Fig. 4f). Interestingly, we observed a weak OK100 band (indicated by arrows) in wild type, snrk2-triple and pyl112458 even without ABA or mannitol treatment, and this band was not induced by ABA, when compared to the strong induction by mannitol treatment (Fig. 4f, rightmost lane). Consistently, the immunoblot result showed that the ABA-induced phosphorylation of conserved serine residues corresponding to Ser171 and Ser175 in SnRK2.6 was markedly reduced in the OK100-oct and OK100-nonu mutants (Fig. 4g). Interestingly, perhaps due to the abolishment of ABA signaling, the ABA-induced rapid SnRK2 degradation was also abolished in the OK100-oct and OK100-nonu mutants (Fig. 4g). These results strongly indicate that the B2 and B3 subgroup RAFs are essential for ABA-induced SnRK2.2/2.3/2.6 activation.

To evaluate which members of the B2 and B3 subgroups have predominant roles in ABA-regulated seed germination and seedling establishment, we backcrossed OK100-nonu with Col-0 wild type and screened F2 populations on 1/2 MS medium containing 10 µM ABA and sucrose. By genotyping 103 individual F2 seedlings with strong ABA insensitivity, we found that each RAF might contribute to ABA hyposensitivity (Chi-square test, p < 0.05). RAF3, RAF4, RAF5, and RAF7-9 (closed linked together) might have predominant roles (p < 0.0001) in ABA-regulated germination and seedling establishment (Supplementary Table 1).

B2 and B3 RAFs are required for ABA-induced gene expression

To investigate the impact of RAF null mutations on ABA-induced gene expression, we performed transcriptomics analysis in WT and OK100-oct seedlings. We identified 1368 ABA-induced (>= 3-fold, p < 0.05) and 1256 ABA-repressed (>= 3-fold, p < 0.05) genes in the wild type. Among these differentially expressed genes (DEGs), only 674 genes were significantly induced (>= 3-fold, p < 0.05) and 399 genes were significantly repressed (>= 3-fold, p < 0.05) by ABA in the OK100-oct seedlings (Fig. 5a, Supplementary Data 3). The heatmap indicated that most ABA-induced and -repressed genes in wild type were less responsive in the OK100-oct mutant (Fig. 5b). Quantitative RT-PCR analysis of several ABA-inducible genes, including RESPONSIVE TO ABA 18 (RAB18), COLD-REGULATED 15A (COR15A), KINASE 1 (KIN1), and RESPONSIVE TO DESICCATION 29B (RD29B), also showed that the induction of these genes by ABA was dramatically impaired in both the OK100-oct and OK100-nonu mutants (Fig. 5c).

a Venn diagrams showing the overlaps of ABA-induced and ABA-repressed genes in the wild type and OK100-oct seedlings. b Heat map showing the expression levels of ABA-responsive genes in wild type and OK100-oct seedlings. c Expression of the ABA-inducible marker genes in acid production - Crack Key For U type, OK100-oct, and OK100-nonu seedlings after 6 h of ABA treatment. Error bars, SEM (n = 3 biological replicates). Two-tailed paired t-tests, *p < 0.05, **p < 0.01, ***p < 0.001. d The activation assay of the ABA-responsive RD29B-LUC reporter gene in wild type and mutants. The protoplasts were transformed with the reporter plasmid and incubated with or without 5 µM ABA for 5 h under light. Error bars, SEM (n = 3 individual transfections). e Add-back assay testing RAF1 to RAF12 in activating the reporter gene in the protoplasts of OK100-oct. Error bars, SEM (n = 4 individual transfections). f Activation of the reporter gene by the combinations of RAFs with SnRK2.2, SnRK2.3, or SnRK2.6 in the protoplasts of OK100-oct. The ratio of RD29B-LUC expression in the protoplasts with 5 µM ABA relative to that without ABA treatment was used to indicate the activation activity of RAF-SnRK2 pairs. Error bars, SEM (n = 4 individual transfections). g Activation of the reporter gene by RAF3 or RAF7 in the protoplasts of wild type, pyl112458, or snrk2-triple. Error bars, SEM (n = 3 individual transfections). Source data are provided as Source Data files.

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Among the DEGs between wild type and the mutant, 67 genes showed significantly higher expression (>= 3-fold, p < 0.05) in the OK100-oct mutant under control conditions. Gene Ontology (GO) analysis indicated that these genes are enriched in plant response to chitin, fungus, bacterium, and oxidative stress, suggesting a potential role of the B2 and B3 RAFs in these biotic stress responses (Supplementary Data 4).

To further evaluate the role of B2 and B3 RAFs in ABA-induced gene expression, we used transient activation assays. We used the LUCIFERASE (LUC) reporter gene driven by the ABA-responsive RD29B promoter as an indicator of ABA response8. In the wild type, ABA clearly induced the expression of RD29B-LUC in the mesophyll cell protoplasts, while ABA-induced RD29B-LUC expression was completely abolished in the protoplasts of OK100-oct, snrk2-triple, pyl112458, and abi1-1 mutants (Fig. 5d). Co-expression of RAF3, RAF5, or RAF11 fully rescued the ABA-induced expression of RD29B-LUC, and co-expression of RAF4, RAF6, RAF7, RAF9, or RAF10 partially rescued the ABA-induced expression of RD29B-LUC in the protoplasts of OK100-oct mutant (Fig. 5e). No rescue was seen with co-expression of RAF1, RAF2, RAF8, or RAF12 (Fig. 5e).

We then co-transfected SnRK2.2, SnRK2.3, or SnRK2.6 in the transient activation assays with each of RAF1 to RAF12 to evaluate the specificity of different RAFs for different SnRK2 activation (Fig. 5f). Protoplasts co-expressing RAF4, RAF5, RAF6, or RAF9 with SnRK2.2 had higher ABA-induced RD29B-LUC expression, while the combination of RAF3, RAF7, RAF8, or RAF10 with SnRK2.6 showed higher ABA-induced RD29B-LUC expression in the OK100-oct protoplasts. These results suggest that different RAFs exhibit activation specificity for SnRK2.2, SnRK2.3, or SnRK2.6. In addition, RAF3 and RAF7 could not rescue ABA-induced RD29B-LUC expression in the pyl112458 protoplasts (Fig. 5g), which suggests that activation of SnRK2s by RAFs requires ABA-induced release of SnRK2s from PP2C inhibition.

ABA does not activate B2 and B3 RAFs in plants

Unlike the strong activation of RAFs by hyperosmolarity, the kinase activity of RAFs was not enhanced by ABA treatment (Fig. 4f). Consistently, the phosphorylation of pSTAGTPEWMAPEVLR, a conserved peptide located in the activation loop of RAF2/EDR1 and RAF3, was not affected by ABA treatment but highly induced by osmotic stress caused by mannitol treatment (Fig. 6a, b, Supplementary Fig. 8a). Multiple phosphosites in this region also could be detected without ABA treatment27,36 (Fig. 6b, Supplementary Fig. 8a, b, highlighted in Supplementary Data 5). SnRK2.6 showed clearly induced phosphorylation in the peptide containing the phosphorylation site Ser175 by both ABA and mannitol treatments (Supplementary Fig. 8c). We further tested whether RAF3 phosphorylation is required for its activity on SnRK2.6 by generating a non-phosphorylatable mutation of RAF3. Co-transfection of RAF3S763AS766AT770A, with Ser to Ala substitutions in the activation loop, did not rescue the ABA-induced RD29B-LUC expression in the protoplasts of OK100-oct (Fig. 6c). RAF3S763AS766AT770A completely lost its ability to phosphorylate SnRK2.6 in vitro (Fig. 6d, left panel). However, mutating these conserved residues of RAF10, producing RAF10T706AT709AT713A, hardly affected RAF10 activity in in vitro kinase or transient expression assays (Fig. 6d, right panel, Supplementary Fig. 8d). Therefore, the activation mechanism of RAF10 in the B2 RAF subgroup may differ from that of RAF3 in the B3 RAF subgroup. Taken together, these results indicate that B2 and B3 RAFs have basal levels of phosphorylation and activity under normal conditions and that application of ABA does not increase their phosphorylation.

a The phosphorylation of the conserved phosphosite in RAF2 and RAF3 showing enhanced phosphorylation by mannitol but not ABA treatment. The relative intensity of the phosphopeptide was obtained from previous phosphoproteomics results (n = 3 biological replicates). b Sequence alignment showing the conserved phosphosites (indicated by arrows) in the activation loop of Arabidopsis B2, B3, B4 RAFs, and PpARK/PpCTR1 from Physcomitrella patens. The conserved serine residues corresponding to Ser1029 in PpARK/PpCTR1 are highlighted by the red arrow. c Activation of the reporter gene by wild type and the non-phosphorylatable mutants of Ser763, Ser766, and Thr770 in RAF3 in transient reporter gene expression in protoplasts of OK100-oct. RAF3K636R (RAF3KR), a kinase-dead form of RAF3, is used as a control. Error bars, SEM (n = 3 individual transfections). d Phosphorylation of SnRK2.6KR by recombinant kinase domain of RAF3 and RAF10. Wild type and mutated recombinant GST-RAF3-KD (left panel) and GST-RAF10-KD (right panel) was used to determine the phosphorylation of SnRK2.6KR expressed and purified from E. coli in the presence of [γ-32P]ATP. GST-RAF3K636R (RAF3KR) and GST-RAF10K515R (RAF10KR) were used as negative controls. Autoradiograph (upper) and Coomassie staining (lower) show phosphorylation and loading, respectively, of purified GST-RAF3-KD, GST-RAF10-KD, and HIS-SnRK2.6KR. Images shown are representative of at least two independent experiments. Source data are provided in Source Acid production - Crack Key For U size image

Discussion

Subgroup B RAFs belong to the MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE (MAPKKK) family due to their similarity with animal B-Raf protein kinases37,38,39. In a canonical MAPK cascade, MAPKKKs are activated by extracellular signals and phosphorylate and activate MAPK KINASEs (MAPKKs), which then phosphorylate and activate MAPKs to regulate various cellular processes. Instead of phosphorylating MAPKKs, after rapid activation by osmotic stresses, B2, B3, and B4 RAFs phosphorylate and activate SnRK2s27,28,29,30,31. Plants use this noncanonical RAF-SnRK2 cascade to relay early osmotic stress signaling. Here we show a unique initiation-amplification mechanism of RAF-SnRK2 in ABA signaling to ensure rapid activation of SnRK2s with a basal level of Raf kinase activity (Fig. 7). Unlike the B4 subgroup RAFs, which are quickly activated by hyperosmolality27, the application of exogenous ABA does not enhance the phosphorylation or activity of B2 and B3 subgroup RAFs, as indicated by in-gel kinase assays and phosphoproteomics (Figs. 4 and 6). The phosphoproteomics and the in-gel kinase assay result also revealed the existence of basal-level phosphorylation and activity of B2 and B3 RAFs even without ABA (Figs. 4f and 6a, Supplementary Data 5). This basal level activation of RAFs might be necessary and sufficient for maintaining the SnRK2 activity and ABA signaling required for normal growth and development6. The upstream kinases or other mechanisms for this basal activity of RAFs need to be determined in the future. In the presence of ABA, the ABA and PYR1/PYL/RCAR complex releases SnRK2s from PP2C-mediated inhibition, resulting in the accumulation of uninhibited forms of SnRK2s. The RAFs quickly trans-phosphorylate uninhibited SnRK2s to initiate SnRK2 activation. The activated SnRK2s then intermolecularly transphosphorylate and activate other SnRK2 molecules not yet activated by RAFs, to amplify the ABA signaling (Fig. 7). By this activation-amplification mechanism, the basal level activity of RAF kinases is sufficient to quickly activate SnRK2s to transduce the ABA signal. It would be interesting to apply the ATP analog-based method to evaluate whether this activation-amplification mechanism also exists in other kinase cascades in plants or animal cells, as autophosphorylation is a general feature of many protein kinases.

Under unstressed conditions, PP2C binds to and inhibits SnRK2 protein to prevent the transphosphorylation by activated RAFs (left panel). In the presence of ABA, ABA receptor PYR/PYL/RCARs (PYL) complex binds to and inhibits PP2C and SnRK2 is released from PP2C-mediated inhibition. SnRK2 can then be quickly activated by RAFs. In the meantime, stress also activates RAFs by an unknown mechanism (middle panel). The activated SnRK2 can quickly trans-phosphorylate more SnRK2 proteins to amplify the activation and phosphorylate downstream substrates to mediate stress responses (right panel).

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Several B3 subgroup Raf-like kinases, M3Kδ6/SIS8/RAF5, M3Kδ7/RAF4, M3Kδ1/RAF3, and RAF6, phosphorylate Acid production - Crack Key For U, and are essential for ABA-induced SnRK2 activation27,28. In this study, we show that the B2 subgroup RAFs, together with B3 subgroup, have an essential role in the ABA core signaling pathway. OK100-nonu and OK100-oct show strong ABA-insensitivity in germination, leaf yellowing, and stomatal closure. The OK100-nonu seeds even germinate at an ABA concentration of up to 25 µM (Fig. 4). To our knowledge, OK100-oct and OK100-nonu are among the few mutants, including snrk2-triple, pyl112458 and pyl-duodec, that can germinate on such an extremely high concentration of ABA, further supporting the critical role of the B2 and B3 RAFs in ABA responses. However, ABA-insensitivity of OK100-nonu is still not identical with that of snrk2-triple and pyl high-order mutants. This suggests that, besides B2 and B3 RAFs, additional protein kinases also participate in ABA-induced SnRK2 activation. At least two protein kinases, BRASSINOSTEROID-INSENSITIVE2 (BIN2) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), can phosphorylate SnRK2s and are involved in ABA signaling40,41. BIN2 likely phosphorylates only SnRK2.2 and SnRK2.3, but not SnRK2.641, at the conserved threonine corresponding to Thr179 in SnRK2.6 (Supplementary Fig. 2). Thus, whether BIN2, or other members of the GSK family, cooperate with RAFs in amplifying ABA-triggered SnRK2 activation, needs to be further studied.

Although both B2 and B3 RAFs are essential for ABA-induced SnRK2 activation, they might have distinct roles Freemake Video Downloader 4.1.12.53 Crack+ Activation Key Free 2021 some ABA-regulated biological processes. OK100-B3, but not OK100-B2, has arrested growth in soil, suggesting a unique role of B3 subgroup RAFs in growth regulation (Fig. 3). OK100-B2 shows similar ABA-insensitivity on medium with or without sucrose, whereas OK100-B3 only shows strong ABA-insensitivity with exogenous sucrose (Fig. 3). Supporting this notion, the miRNA-M3K was screened from the medium containing sucrose28,42. RAF3-5 and RAF7-9 might have more dominant roles in germination and seedling establishment, while RAF3, RAF5, RAF7, and RAF11 rescue the ABA-induced RD29B-LUC expression more robustly. Several key regulators in ABA signaling and synthesis are also involved in sugar responses43,44. Mutants raf1/ctr1/sis1, and raf5/sis8, are resistant to high concentrations of sugar45,46. Together with these findings, our results suggest a crucial role of B3 subgroup RAFs in sucrose signaling and/or in seed development (e.g., in the accumulation of energy reserve in the seeds). It is notable that the role of RAFs in rescuing ABA-induced RD29B-LUC transcription is not identical to their contribution in gemination and seedling establishment, further indicating that different RAFs have various contributions to different ABA-mediated biological processes. Such functional diversity is also observed in the 14 PYR1/PYL/RCAR ABA receptors. Only pyl112458, but not 3791112 (pyl3/7/9/11/12) shows arrested growth under normal condition33. By contrast, pyl112458 has more predominant roles in ABA-mediated regulation of germination, stomatal movement, etc.33. In guard cell, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential47. PYL8 directly binds to the transcription factor MYB77 to regulate auxin responsive gene expression48. PYR1 especially participates in cross-talk between salicylic acid and ethylene, thereby redirecting defense disease resistance towards fungal Plectosphaerella cucumerina49. In Arabidopsis, 14 PYLs, at least 8 PP2Cs, three SnRK2s, and 12 members of the B2 and B3 RAF subgroups comprise a complex network in ABA sensing and signaling, which may ensure that plants precisely respond to ever-changing environments. The engineering of ABA receptors is an efficient way to improve stress resistance in both Arabidopsis and crops50,51,52. Our findings regarding B2 and B3 RAFs in stress signaling provide additional targets (e.g., ectopic expression of stress-inducible or constitutively activated forms of RAFs in guard cells) for engineering crops resistant to harsh environmental conditions.

Besides involvement in sugar and ABA signaling, CTR1/RAF1 is a crucial component in ethylene signaling. We excluded RAF1/CTR1 from the OK100 high-order mutants because the ctr1 mutant displays severe growth inhibition under normal conditions35. However, although the KD of RAF1/CTR1 strongly phosphorylates SnRK2.6 in vitro, neither the full-length RAF1/CTR1 nor RAF1/CTR1-KD rescued the ABA-induced expression of RD29B-LUC in the protoplasts of OK100-oct (Supplementary Fig. 8e). Thus, additional mechanisms may determine RAF1 specificity in vivo. Similarly, RAF2, RAF8, and RAF12 only show weak activities on the induction of RD29B-LUC expression in the protoplasts. The roles of these RAFs in ABA signaling therefore need to be further investigated.

The phosphorylation of Ser1029 of the ABA AND ABIOTIC STRESS-RESPONSIVE RAF-LIKE KINASE (PpARK)/PpCTR1 in Physcomitrella patens is induced by exogenous ABA in P. patens53,54,55, which is inconsistent with our observations on RAF3 and RAF10 (Fig. 6). Therefore, P. patens and higher plants may adopt different machinery to relay ABA signaling. In addition, non-phosphorylatable mutations at Ser1029 in PpARK/PpCTR1, or Ser763Ser766AThr770 in RAF3, abolished their kinase activities, suggesting phosphorylation-dependent activation of PpARK/PpCTR1 and RAF3. By contrast, the activation of RAF10 might be independent of phosphorylation. In animal cells, RAF kinases can be activated through phosphorylation, dimerization, or by binding of small GTPases, scaffold protein, 14-3-3 proteins, etc.56,57,58. Future work will investigate the phosphorylation or other activation mechanisms of RAF-SnRK2 cascades in different plant species and acid production - Crack Key For U roles in plant adaptive plasticity.

Methods

Seed germination and plant growth assay

Seeds were surface-sterilized in 70% ethanol for 10 min, followed by four times washing with sterile-deionized water. For the germination assay, seeds were sown on 1/2 Murashige and Skoog (MS) medium (0.75% agar, pH 5.7) with or without the indicated concentrations of ABA and 1% sucrose. Plates were kept at 4 °C for 3 days in darkness for stratification and then shifted to a plant growth chamber set at 23 °C and a 16 h light/8 h dark photoperiod. After 72 h of transfer, radical emergence was examined, and photographs of seedlings were taken at the times indicated. For growth assays, seeds were placed on 1/2 MS medium (0.75% agar, pH 5.7) and plates were placed vertically in a plant growth chamber after 3 days of stratification. After 3–4 days, the seedlings were transferred to medium with or without the indicated concentrations of ABA. Root length and fresh weight were measured at the indicated days. For seed dormancy assays, fresh seeds were harvested and sown on 1/2 MS medium (0.75% agar, pH 5.7) and plates were placed in a plant growth chamber. Radical emergence was measured 48 h after transferring.

Generation of OK100 high-order mutants

The clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9) and guide RNA fragment from pCAMBIA-2300-11RAFs27 was cloned into pCAMBIA-1300. The resulting vectors containing sgRNAs targeting B2, B3, or B2/B3 RAFs were used to transform wild type to generate OK100-B2, OK100-B3, OK100-oct, and OK100-nonu. The transgenic plants were screened for hygromycin resistance. The T1 transformants were identified by sequencing the fragments with the RAF target regions, which were amplified by PCR using primer pairs listed in Supplementary Data 6.

In-gel kinase assay

For in-gel kinase assays, 20 µg extract of total proteins were electrophoresed on 10% Acid production - Crack Key For U embedded with histone in the separating gel as a substrate for kinase. The gel was then washed three times at room temperature for 30 min each with washing buffer (25 mM Tris-Cl, pH 7.5, 0.5 mM Dithiothreitol (DTT), 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg/mL BSA, and 0.1% Triton X-100). The kinase was allowed to renature in renaturing buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF) and incubated at 4 °C overnight with three changes of renaturing buffer. The gel was further incubated at room temperature in 30 mL reaction buffer (25 mM Tris-Cl, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, and 0.1 mM Na3VO4) with 200 nM ATP plus 50 µCi of [γ-32P]ATP for 90 min. The reaction was stopped by transferring the gel into 5% (w/v) trichloroacetic acid and 1% (w/v) sodium pyrophosphate. The gel was then washed to remove unincorporated [γ-32P]ATP in the same solution for at least 5 h with five changes. Radioactivity was detected with a Personal Molecular Imager (Bio-Rad).

RNA sequencing and data analysis

Total RNA was isolated from two-week-old seedlings of Col-0 and OK100-oct mutant, with and without ABA treatment, using RNeasy Plant Mini Kit (Qiagen). Total RNA (1 µg) was used for library preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7765) following the manufacturer’s instructions. Prepared libraries were assessed for fragment size using NGS High-Sensitivity kit on a Fragment Analyzer (AATI), and for quantity using Qubit 2.0 fluorometer (Thermo Fisher Scientific) and KAPA Library Quantification Kit (Kapa, KK4824). All libraries were sequenced in paired-end 150 bases protocol (PE150) on an Illumina Nova sequencer.

The paired-end reads were cleaned by Trimmomatic59 (version 0.39). After trimming the adapter sequence, removing low quality bases, and filtering short reads, clear read pairs were retained for further analysis. The Arabidopsis thaliana reference genome sequence was downloaded from TAIR10. Clean reads were mapped to the genome sequence by HISAT (2.1.0)60 with default parameters. Number of reads that were mapped to each gene was calculated with the htseq-count script in HTSeq (0.11.2)61. EdgeR62 was used to identify genes that were differentially expressed. Genes with at least three-fold change in expression and with an FDR < 0.05 were considered differentially expressed genes (DEGs).

Analysis of gene expression by qRT-PCR

Total RNA was extracted from two-week-old wild-type, OK100-oct, andOK100-nonu seedlings with or without 50 µM ABA treatment for 6 h. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Genomic DNA was removed using RNase-free DNase and subsequently, 1 µg of total RNA was reverse transcribed using the iScriptTM gDNA Clear cDNA Synthesis Kit (Bio-Rad) following the manufacturer’s instructions. The actin gene was used as an internal control. Quantification was performed using three independent biological replicates.

Water loss measurement

The water loss was estimated on detached rosette leaves of 4-week-old plants by weighing using a weighing dish. Leaves were then kept on the laboratory bench for at least 30 min. Fresh weight was monitored before and after the procedure and at the times indicated. Water loss was expressed as a percentage of initial fresh weight.

Stomatal bioassay

For stomatal aperture assay, rosette leaves of 4-week-old Arabidopsis seedlings were taken. Epidermal strips were peeled out and incubated in buffer containing 50 mM KCl, 10 mM MES, pH 6.15, in a plant growth chamber for 3 h before ABA treatment. Stomatal apertures were measured 2 h after the addition of 5 μM ABA. The apertures of about 60 stomata per sample were measured by quantifying the pore width of stomata using Image J software (1.51 K). All the experiments were repeated at least three times.

Protein purification and in vitro kinase assay

For in vitro kinase assays, full-length coding sequence of SnRK2.6 and kinase domains of RAFs were cloned into either pGEX-4T-1, pET28a or pET-SUMO vectors and transformed into BL21 or ArecticExpression cells. The recombinant proteins were expressed and purified using standard protocols. For the phosphorylation assay, recombinant kinase domains of RAFs (aa541-821 for RAF1-KD, aa650-933 for RAF2-KD, aa600-880 for RAF3-KD, aa710-992 for RAF4-KD, aa733-1030 for RAF5-KD, aa645-956 for RAF6-KD, aa470-773 for RAF7-KD, aa424-671 for RAF8-KD, aa436-730 for RAF9-KD, aa466-767 for RAF10-KD, aa472-765 for RAF11-KD, aa457-735 for RAF12-KD) were incubated with “kinase-dead” forms of SnRK2.6 with or without Ser to Ala mutations at Ser171 and Ser175 in reaction buffer (25 mM Tris HCl, pH 7.4, 12 mM MgCl2, 2 mM DTT), with 1 μM ATP plus 1 µCi of [γ-32P] ATP for 30 min at 30 °C. Reactions were stopped by boiling in SDS sample buffer and proteins were separated by 10% SDS-PAGE.

For the dephosphorylation assay, SnRK2.6M94G coated on Glutathione Sepharose (Cytiva) were dephosphorylated with Lambda Protein Phosphatase (λPP, NEB, P0753S) for 30 min and the λPP was removed by washing three times with protein acid production - Crack Key For U (25 mM Tris HCl, pH 7.4, 150 mM NaCl). To detect the effects of RAF3-KD and RAF10-KD on SnRK2.6M94G, SnRK2.2M96G, and SnRK2.3M95G thiophosphorylation and activity, recombinant GST-RAF3/10-KD was incubated with pre-dephosphorylated SnRK2.6M94G, SnRK2.2M96G, or SnRK2.3M95G for 30 min in reaction buffer (25 mM Tris HCl, pH 7.4, 12 mM MgCl2, 2 mM MnCl2, 0.5 mM DTT, 50 μM ATP, 50 μM N6-Benzyl-ATPγS). Then ABF2 was added to the reaction and incubated for an additional 30 min. This phosphorylation reaction was stopped by adding EDTA to a final concentration of 25 μM. A final concentration of 2.5 mM p-nitrobenzyl mesylate (Abcam, ab138910) was added to proceed the alkylating reaction for 1 h at room temperature. Samples with SDS sample buffer were boiled and separated by SDS-PAGE, transferred to Polyvinylidene fluoride (PVDF) membrane, and immunoblotted with antibodies against thiophosphate ester (Abcam, ab92570). To pre-activate SnRK2.6, HIS-SUMO-RAF-KD proteins were coated on the Ni-NTA beads and incubated with SnRK2.6 (in solution) in the presence of ATP. HIS-SUMO-RAF-KD were removed by centrifuging after the reaction.

Protoplast isolation and transactivation assay

Protoplasts were isolated from leaves of 4-week-old plants grown under a short photoperiod (10 h light at 23 °C/14 h dark at 20 °C). Leaf strips were excised from the middle parts of young rosette leaves, dipped in enzyme solution containing cellulase R10 (Yakult Pharmaceutical Industry) and macerozyme R10 (Yakult Pharmaceutical Industry) and incubated at room temperature in the dark. The protoplast solution was diluted with an equal volume of W5 solution (2 mM MES, pH 5.7, 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl) and filtered through a nylon mesh. The flow-through was centrifuged at 100 g for 2 min to pellet the protoplasts. Protoplasts were resuspended in W5 solution and incubated for 30 min. 100 μL of protoplasts suspended in MMG solution (4 mM MES, pH 5.7, 0.4 M mannitol, and 15 mM MgCl2) were mixed with the plasmid mix and added to 110 μL PEG solution (40% w/v PEG-4000, 0.2 M mannitol, and 100 mM CaCl2). The transfection mixture was mixed completely by gently tapping the tube followed by incubation at room temperature for 5 min. The protoplasts were washed twice with 1 mL W5 solution. After transfection, protoplasts were left for incubation for a further 5 h under light in washing and incubation solution (0.5 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7) with or without 5 μM ABA. The RD29B-LUC (7 μg of plasmid per transfection) and ZmUBQ-GUS (1 μg per transfection) were used as an ABA-responsive reporter gene and as an internal control, respectively. For wild-type and mutated RAF, SnRK2 plasmids, 3 μg per transfection were used. After transfection, protoplasts were incubated for 5 h under light in washing and incubation solution (0.5 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7) with or without 5 μM ABA. The mutations were introduced into wild-type RAFs using the primers listed in Supplementary Data 6.

Generation of anti-pS171-SnRK2.6 antibody

The anti-pS171-SnRK2.6 antibody was generated by ABcloneal. Phosphopeptide C-KSSVLHpSQPK was synthesized and used as an antigen to immunize rabbit and generate the polyclonal anti-phosphorylation antibody. Phosphorylation-non-specific antibody was removed using peptide C-KSSVLHSQPK.

Immunoblotting

30 mg seedling samples of wild-type, OK100-oct, and OK100-nonu were ground into fine powder in liquid nitrogen. Total proteins were extracted in 100 μL protein extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF, 5 μg/mL leupeptin, 5 μg/mL antipain, 5 μg/mL aprotinin, and 5% glycerol). Cell debris was removed by centrifugation at 12,000 g at 4 °C for 40 min and supernatant was collected. 20 µg protein were separated by 10% SDS/PAGE, and the proteins were transferred to PVDF membrane. Blots were probed with primary antibodies against SnRK2.2/2.3/2.6 (Agrisera) at a dilution of 1:5000, p-S175-SnRK2.6 at a dilution of 1:500033, and p-S171-SnRK2.6 (ABclonal) at a dilution of 1:5000. Anti-actin antibody (ABclonal) was used as the loading control at a dilution of 1:10,000. Secondary anti-rabbit antibodies at a dilution of 1:20,000 were used to detect antibodies in conjugation with secondary horseradish peroxidase and enhanced chemoluminescence reagent (ShengEr).

Reporting summary

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

Data availability

The RNA sequencing data were deposited to the GEO database with the dataset identifier GSE152691. Source data are provided with this paper.

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Источник: https://www.nature.com/articles/s41467-021-22812-x

Phosphoric acid

Chemical compound

This article is about orthophosphoric acid. For other acids commonly called "phosphoric acid", see Phosphoric acids and phosphates.

Not to be confused with Phosphorous acid.

Structural formula of phosphoric acid, showing dimensions
Ball-and-stick model
Space-filling model
Names
IUPAC name

Phosphoric acid

Other names

Orthophosphoric acid

Identifiers

CAS Number

3D model (JSmol)

ChEBI
ChEMBL
ChemSpider
ECHA InfoCard100.028.758Edit this at Wikidata
EC Number
E numberE338 (antioxidants. .)
KEGG

PubChemCID

RTECS number
UNII
UN number1805

CompTox Dashboard(EPA)

InChI

  • InChI=1S/H3O4P/c1-5(2,3)4/h(H3,1,2,3,4) checkY
    Key: NBIIXXVUZAFLBC-UHFFFAOYSA-N checkY
  • InChI=1/H3O4P/c1-5(2,3)4/h(H3,1,2,3,4)

    Key: NBIIXXVUZAFLBC-UHFFFAOYAI

Properties

Chemical formula

H
3PO
4
Molar mass97.994 g·mol−1
Appearance white solid
OdorOdorless
Density1.6845  g⋅cm−3 (25 °C, 85%),[1] 1.834  g⋅cm−3 (solid)[2]
Melting point 40–42.4 °C (104.0–108.3 °F; 313.1–315.5 K)[6]
Boiling point 

Solubility in water

  • 392.2 g/100 g (−16.3 °C)
  • 369.4 g/100 mL (0.5 °C)
  • 446 g/100 mL (15 °C)[5]
  • 548 g/100 mL (20 °C)[6]
SolubilitySoluble in ethanol
log P−2.15[7]
Vapor pressure0.03 mmHg (20 °C)[8]
Conjugate baseDihydrogen phosphate

Magnetic susceptibility (χ)

−43.8·10−6 cm3/mol[10]

Refractive index (nD)

  • 1.3420 (8.8% w/w aq. soln.)[11]
  • 1.4320 (85% aq. soln) 25 °C
Viscosity2.4–9.4 cP (85% aq. soln.)
147 cP (100%)
Structure

Crystal structure

Monoclinic

Molecular shape

Tetrahedral
Thermochemistry[12]

Heat capacity(C)

145.0 J/mol⋅K

Std molar
entropy(S298)

150.8 J/mol⋅K

Std enthalpy of
formation(ΔfH298)

−1271.7 kJ/mol

Gibbs free energy(ΔfG˚)

-1123.6 kJ/mol
Hazards
Safety data sheetICSC 1008
GHS pictogramsGHS05: Corrosive[13]
GHS Signal wordDanger

GHS hazard statements

H290, H314[13]

GHS precautionary statements

P280, P305+351+338, P310[13]
NFPA 704 (fire diamond)
Flash pointNon-flammable
Lethal dose or concentration (LD, LC):

LD50 (median dose)

1530 mg/kg (rat, oral)[14]
NIOSH (US health exposure limits):

PEL (Permissible)

TWA 1 mg/m3[8]

REL (Recommended)

TWA 1 mg/m3 ST 3 mg/m3[8]

IDLH (Immediate danger)

1000 mg/m3[8]
Related compounds

Related phosphorusoxoacids

Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

☒N verify (what is checkY☒N ?)
Infobox references

Chemical compound

Phosphoric acid, also known as orthophosphoric acid or phosphoric(V) acid, is a weak acid with the chemical formulaH
3PO
4. The pure compound is a colorless solid.

All three hydrogens are acidic to varying degrees and can be lost from the molecule as H+ ions (protons). When all three H+ ions are removed, the result is an orthophosphate ion PO43−, commonly called "phosphate". Removal of one or two protons gives dihydrogen phosphate ion H
2PO
4, and the hydrogen phosphate ion HPO2−
4, respectively. Orthophosphoric acid also forms esters, called organophosphates.[15]

Phosphoric acid is commonly encountered in chemical laboratories as an 85% aqueous solution, which is a colourless, odourless, and non-volatile syrupy liquid. Although phosphoric acid does not meet the strict definition of a strong acid, the 85% solution can still severely irritate the skin and damage the eyes.

The name "orthophosphoric acid" can be used to distinguish this specific acid from other "phosphoric acids", such as pyrophosphoric acid. Nevertheless, the term "phosphoric acid" acid production - Crack Key For U means this specific compound; and that is the current IUPAC nomenclature.

Manufacture[edit]

Phosphoric acid is produced industrially by two general routes.[16] In the wet process a phosphate-containing mineral such as calcium hydroxyapatite is treated with sulfuric acid.[17]

{\displaystyle {\ce {Ca5(PO4)3OH + 5H2SO4 -> 3H3PO4 + 5CaSO4v + H2O}}}

Fluoroapatite is an alternative feedstock, in which case fluoride is removed as the insoluble compound Na2SiF6. The phosphoric acid solution usually contains 23–33% P2O5 (32–46% H3PO4). It may be concentrated to produce commercial- or merchant-grade phosphoric acid, which contains about 54–62% P2O5 (75–85% H3PO4). Further removal of water yields superphosphoric acid with a P2O5 concentration above 70% (corresponding to nearly 100% H3PO4). Calcium sulfate (gypsum) is produced as a by-product and is removed as phosphogypsum.

To produce food-grade phosphoric acid, phosphate ore is first reduced with coke in an electric arc furnace, to make elemental phosphorus. Silica is also added, resulting in the production of calcium silicate slag. Elemental phosphorus is distilled out of the furnace and burned with air to produce high-purity phosphorus pentoxide, which is dissolved in water to make phosphoric acid.

The phosphoric acid from both processes may be further purified by removing compounds of arsenic and other potentially toxic impurities.

Acidic properties[edit]

All three hydrogens are acidic, with dissociation constants pKa1 = 2.14, pKa2 = 7.20, and pKa3 = 12.37. It follows that, in water solutions, phosphoric acid is mostly dissociated into some combination of its three anions, except at very low pH. The equilibrium equations are:

H3PO4   + H2O ⇌ H3O+ + H2PO4      Ka1= 7.25×10−3 [pKa1 = 2.14]
H2PO4+ H2O ⇌ H3O+ + HPO42−       Ka2= 6.31×10−8 [pKa2 = 7.20]
HPO42−+ H2O ⇌ H3O+ +  PO43−        Ka3= 3.98×10−13 [pKa3 = 12.37]

Uses[edit]

The dominant use of phosphoric acid is for fertilizers, consuming approximately 90% of production.[18]

Application Demand (2006) in thousands of tons Main phosphate derivatives
Soaps and detergents1836STPP
Food industry309STPP (Na5P3O10), SHMP, TSP, SAPP, SAlP, MCP, DSP (Na2HPO4), H3PO4
Water treatment164SHMP, STPP, TSPP, MSP (NaH2PO4), DSP
Toothpastes68DCP (CaHPO4), IMP, SMFP
Other applications287STPP (Na3P3O9), TCP, APP, DAP, zinc phosphate (Zn3(PO4)2), aluminium phosphate (AlPO4, H3PO4)

Food-grade phosphoric acid (additive E338[19]) is used to acidify foods and beverages such as various colas and jams, providing a tangy or sour taste. The phosphoric acid also serves as a preservative.[20] Soft drinks containing phosphoric acid, which would include Coca-Cola, are sometimes called phosphate sodas or phosphates. Phosphoric acid in soft drinks has the potential to cause dental erosion.[21] Phosphoric acid also has the potential to contribute to the formation of kidney stones, especially in those who have had kidney stones previously.[22]

Specific applications of phosphoric acid include:

Safety[edit]

A link has been shown between long-term regular cola intake and osteoporosis in later middle age in women (but not men).[28] This was thought to be due to the presence of phosphoric acid, and the risk for women was found to be greater for sugared and caffeinated colas than diet and decaffeinated variants, with a higher intake of cola correlating with lower bone density.

At moderate concentrations phosphoric acid solutions are irritating to the skin. Contact with concentrated solutions can cause severe skin burns and permanent eye damage.[29]

See also[edit]

References[edit]

  1. ^Christensen, J. H. & Reed, R. B. (1955). "Design and Analysis Data—Density of Aqueous Solutions of Phosphoric Acid Measurements at 25 °C". Ind. Eng. Chem. 47 (6): 1277–1280. doi:10.1021/ie50546a061.
  2. ^"CAMEO Chemicals Datasheet – Phosphoric Acid". Archived from the original on 15 August 2019. Retrieved 15 August 2019.
  3. ^"Phosphoric acid". www.chemspider.com. Archived from the original on 12 March 2020. Retrieved 3 March 2020.
  4. ^Brown, Earl H.; Whitt, Carlton D. (1952). "Vapor Pressure of Phosphoric Acids". Industrial & Engineering Chemistry. 44 (3): 615–618. doi:10.1021/ie50507a050.
  5. ^Seidell, Atherton; Linke, William F. (1952). Solubilities of Inorganic and Organic Compounds. Van Nostrand. Archived from the original on 11 March 2020. Retrieved 2 June 2014.
  6. ^ abHaynes, p. 4.80
  7. ^"phosphoric acid_msds". Archived from the original on 4 July 2017. Retrieved 2 May 2018.
  8. ^ abcdNIOSH Pocket Guide to Chemical Hazards. "#0506". National Institute for Occupational Safety and Health (NIOSH).
  9. ^Haynes, p. 5.92
  10. ^Haynes, p. 4.134
  11. ^Edwards, O. W.; Dunn, R. L. & Hatfield, J. D. (1964). "Refractive Index of Phosphoric Acid Solutions at 25 C.". J. Chem. Eng. Data. 9 (4): 508–509. doi:10.1021/je60023a010.
  12. ^Haynes, p. 5.13
  13. ^ abcSigma-Aldrich Co., Phosphoric acid.
  14. ^"Phosphoric acid". Immediately Dangerous to Life or Health Concentrations (IDLH). National Institute for Occupational Safety and Health (NIOSH).
  15. ^Westheimer, F.H. (6 June 1987). "Why nature chose phosphates". Science. 235 (4793): 1173–1178 (see pp. 1175–1176). Bibcode:1987Sci.235.1173W. CiteSeerX 10.1.1.462.3441. doi:10.1126/science.2434996. PMID 2434996.
  16. ^Becker, Pierre (1988). Phosphates and phosphoric acid. New York: Marcel Dekker. ISBN .
  17. ^Greenwood, Norman N.; Earnshaw, Alan (1997). Chemistry of the Elements (2nd ed.). Butterworth-Heinemann. pp. 520–522. ISBN .
  18. ^Schrödter, Klaus; Bettermann, Gerhard; Staffel, Thomas; Wahl, Friedrich; Klein, Thomas; Hofmann, Thomas (2008). "Phosphoric Acid and Phosphates". Ullmann's Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a19_465.pub3.
  19. ^"Current EU approved additives and their E Numbers". Foods Standards Agency. 14 March 2012. Archived from the original on 19 July 2013. Retrieved 22 July 2012.
  20. ^"Why is phosphoric acid used in some Coca‑Cola drinks?
     

    Through this website we are seeking historical materials relating to fuel cells. We have constructed the site to gather information from people already familiar with the technology–people such as inventors, researchers, manufacturers, electricians, and marketers. This Basics section presents a general overview of fuel cells for casual visitors.

    What is a fuel cell?

    A fuel cell is a device that generates electricity by a chemical reaction. Every fuel cell has two electrodes called, respectively, the anode and cathode. The reactions that produce electricity take place at the electrodes.

    Every fuel cell also has an electrolyte, which carries electrically charged particles from one electrode to the other, and a catalyst, which speeds the reactions at the electrodes.

    Hydrogen is the basic fuel, but fuel cells also require oxygen. One great appeal of fuel cells is that they generate electricity with very little pollution–much of the hydrogen and oxygen used in generating electricity ultimately combine to form a harmless byproduct, namely water.

    One detail of terminology: a single fuel cell generates a tiny amount of direct current (DC) electricity. In practice, many fuel cells are usually assembled into a stack. Cell or stack, the principles are the same.

    Top

    How do fuel cells work?

    The purpose of a fuel cell is to produce an electrical current that can be directed outside the cell to do work, such as powering an electric motor or illuminating a light bulb or a city. Because of the way electricity behaves, this current returns to the fuel cell, completing an electrical circuit. (To learn more about electricity and electric power, visit "Throw The Switch" on the Smithsonian website Powering a Generation of Change.) The chemical reactions that produce this current are the key to how a fuel cell works.

    There are several kinds of fuel cells, and each operates a bit differently. But in general terms, hydrogen atoms enter a fuel cell at the anode where a chemical reaction strips them of their electrons. The hydrogen atoms are now "ionized," and carry a positive electrical charge. The negatively charged electrons provide the current through wires to do work. If alternating current (AC) is needed, the DC output of the fuel cell must be routed through a conversion device called an inverter.

    animated image showing the function of a PEM fuel cell
    Graphic by Marc Marshall, Schatz Energy Research Center

    Oxygen enters the fuel cell at the cathode and, in some cell types (like the one illustrated above), it there combines with electrons returning from the electrical circuit and hydrogen ions that have traveled through the electrolyte from the anode. In other cell types the oxygen picks up electrons and then travels through the electrolyte to the anode, where it combines with hydrogen ions.

    The electrolyte plays a key role. It must permit only the appropriate ions to pass between the anode and cathode. If free electrons or other substances could travel through the electrolyte, they would disrupt the chemical reaction.

    Whether they combine at anode or cathode, together hydrogen and oxygen form water, which drains from the cell. As long as a fuel cell is supplied with hydrogen and oxygen, it will generate electricity.

    Even better, since fuel cells create electricity chemically, rather than by combustion, they are not subject to the thermodynamic laws that limit a conventional power plant (see "Carnot Limit" in the glossary). Therefore, fuel cells are more efficient in extracting energy from a fuel. Waste heat from some cells can also be harnessed, boosting system efficiency still further.

    Top

    So why can't I go out and buy a fuel cell?

    The basic workings of a fuel cell may not be difficult to illustrate. But building inexpensive, efficient, reliable fuel cells is a far more complicated business.

    Scientists and inventors have designed many different types and sizes of fuel cells in the search for greater efficiency, and the technical details of each kind vary. Many of the choices facing fuel cell developers are constrained by the choice of electrolyte. The design of electrodes, for example, and the materials used to make them depend on the electrolyte. Today, the main electrolyte types are alkali, molten carbonate, phosphoric acid, proton exchange membrane (PEM) and solid oxide. The first three are liquid electrolytes; the last two are solids.

    The type of fuel also depends on the electrolyte. Some cells need pure hydrogen, and therefore demand extra equipment such as a "reformer" to purify the fuel. Other cells can tolerate some impurities, but might need higher temperatures to run efficiently. Liquid electrolytes circulate in some cells, which requires pumps. The type of electrolyte also dictates a cell's operating temperature–"molten" carbonate cells run hot, just as the name implies.

    Each type of fuel cell has advantages and drawbacks compared to the others, and none is yet cheap and efficient enough to widely replace traditional ways of generating power, such coal-fired, hydroelectric, or even nuclear power plants.

    The following list describes the five main types of fuel cells. More detailed information can be found in those specific areas of this site.

    Top

    Different types of fuel cells.

    drawing of an Alkali fuel cell
    Drawing of an alkali cell.
    Alkali fuel cells operate on compressed hydrogen and oxygen. They generally use a solution of potassium hydroxide (chemically, KOH) in water as their electrolyte. Efficiency is about 70 percent, and operating temperature is 150 to 200 degrees C, (about 300 to 400 degrees F). Cell output ranges from 300 watts (W) to 5 kilowatts (kW). Alkali cells were used in Apollo spacecraft to provide both electricity and drinking water. They require pure hydrogen fuel, however, and their platinum electrode catalysts are expensive. And like any container filled with liquid, they can leak.
    drawing of molten carbonate fuel cell
    Drawing of a molten carbonate cell
    Molten Carbonate fuel cells (MCFC) use high-temperature compounds of salt (like sodium or magnesium) carbonates (chemically, CO3) as the electrolyte. Efficiency ranges from 60 to 80 percent, and operating temperature is about 650 degrees C (1,200 degrees F). Units with output up to 2 megawatts (MW) have been constructed, and designs exist for units up to 100 MW. The high temperature limits damage from carbon monoxide "poisoning" of the cell and waste heat can be recycled to make additional electricity. Their nickel electrode-catalysts are inexpensive compared to the platinum used in other cells. But the high temperature also limits the materials and safe uses of MCFCs–they would probably be too hot for home use. Also, carbonate ions from the electrolyte are used up in the reactions, making it necessary to inject carbon dioxide to compensate.

    Phosphoric Acid fuel cells (PAFC) use phosphoric acid as the electrolyte. Efficiency ranges from 40 to 80 percent, and operating temperature is between 150 to 200 degrees C (about 300 to 400 degrees F). Existing phosphoric acid cells have outputs up to 200 kW, and 11 MW units have been tested. PAFCs tolerate a carbon monoxide concentration of about 1.5 percent, which broadens the choice of fuels they can use. If gasoline is used, the sulfur must be removed. Platinum electrode-catalysts are needed, and internal parts must be able to withstand the corrosive acid.

    drawing of how both phosphoric acid and PEM fuel cells operate
    Drawing of how both phosphoric acid and PEM fuel cells operate.

    Proton Exchange Membrane (PEM) fuel cells work with a polymer electrolyte in the form of a thin, permeable sheet. Efficiency is about 40 to 50 percent, and operating temperature is about 80 degrees C (about 175 degrees F). Cell outputs generally range from 50 to 250 kW. The solid, flexible electrolyte will not leak or crack, and these cells operate at a low enough temperature to make them suitable for homes and cars. But their fuels must be purified, and a platinum catalyst is used on both sides of the membrane, raising costs.

    drawing of solid oxide fuel cell
    Drawing of a solid oxide cell
    Solid Oxide fuel cells (SOFC) use a hard, ceramic compound of metal (like calcium or zirconium) oxides (chemically, O2) as electrolyte. Efficiency is about 60 percent, and operating temperatures are about 1,000 degrees C (about 1,800 degrees F). Cells output is up to 100 kW. At such high temperatures a reformer is not required to extract hydrogen from the fuel, and waste heat can be recycled to make additional electricity. However, the high temperature limits applications of SOFC units and they tend to be rather large. While solid electrolytes cannot leak, they can crack.

    More detailed information about each fuel cell type, including histories and current applications, can be found on their specific parts of this site. We have also provided a glossary of technical terms–a link is provided at the top of each technology page.

    Top

    ©2017 Smithsonian Institution
    (Copyright Statement)
     

     
    Источник: https://americanhistory.si.edu/fuelcells/basics.htm
    protein synthesis definition and steps

    Protein synthesis
    n., plural: protein syntheses
    Definition: the creation of protein.

    Protein synthesis is the process of creating protein molecules. In biological systems, it involves amino acid synthesis, transcription, translation, and post-translational events. In amino acidsynthesis, there is a set of biochemical processes that produce amino acids from carbon sources like glucose. Not all amino acids are produced by the body; other amino acids are obtained from diet. Within the cells, proteins are generated involving transcription and translation processes. In brief, transcription is the process by which the mRNA template is transcribed from DNA. The template is used for the succeeding step, translation. In translation, the amino acids are linked together in a particular order based on the genetic code. After translation, the newly formed protein undergoes further processing, such as proteolysis, post-translational modification, and protein folding.

     


    Proteins are made up of amino acids that are arrainged in orderly fashion. Discover how the cell organizes protein synthesis with acid production - Crack Key For U help of the RNAs. You’re more than welcome to join us in our Forum discussion: What does mRNA do in protein synthesis?


    Protein synthesis definition

    protein synthesis - schematic diagram

    Protein synthesis is the creation of proteins. In biological systems, it is carried out inside the cell. In prokaryotes, it occurs in the cytoplasm. In eukaryotes, it initially occurs in the nucleus to create a transcript (mRNA) of the coding region of the DNA. The transcript leaves the nucleus and reaches the ribosomes for translation into a protein molecule with a specific sequence of amino acids.

    Protein synthesis is the creation of proteins by cells that uses DNA, RNA, and various enzymes. It generally includes transcription, translation, and post-translational events, such as protein folding, modifications, and proteolysis.

    Etymology

    The term protein came from Late Greek prōteios, prōtos, meaning “first”. The word synthesis came from Greek sunthesis, from suntithenai, meaning “to put together”. Variant: protein biosynthesis.

    Forum Question: Where does protein synthesis take place?    Best Answer!

     

    Prokaryotic vs. eukaryotic protein synthesis

    Proteins are a major type of biomolecule that all living things require to thrive. Both prokaryotes and eukaryotes produce various proteins for multifarious processes and functions. Some proteins are used for structural purposes while others act as catalysts for biochemical reactions. Prokaryotic and eukaryotic protein syntheses have distinct differences. For instance, protein synthesis in prokaryotes occurs in the cytoplasm. In eukaryotes, the first step (transcription) occurs in the nucleus. When the transcript (mRNA) is formed, it proceeds to the cytoplasm where ribosomes are located. Here, the mRNA is translated into an amino acid chain. In the table below, differences between prokaryotic and eukaryotic protein syntheses are shown.

    Prokaryotic protein synthesisEukaryotic protein synthesis
    Translation occurs even before transcription of mRNA endsTranscription occurs followed by translation
    Except in archaebacterial, bacterial mRNA formation does not include the addition of a cap and a poly A tailmRNA formation includes the addition of 5′ cap and a poly A tail at the 3′ end of mRNA transcript
    Translation begins at AUG codonTranslation begins via the 5′ cap, binding the mRNA to the ribosomal unit at the first AUG codon
    Initiating factors: PIF-1, PIF-2, PIF-3Initiating factors: eIF1-6, eIF4B, eIF4C, eIF4D, eIF4F

    Genetic code

    RNA codon amino acid chart

    In biology, a codon refers to the trinucleotides that specify for a particular amino acid. For example, Guanine-Cytosine-Cytosine (GCC) codes for the amino acid alanine. The Guanine-Uracil-Uracil (GUU) codes for valine. Uracil-Adenine-Adenine (UAA) is a stop codon. The codon of the mRNA complements the trinucleotide (called anticodon) in the tRNA.


    What is the Genetic Code? “The genetic code is the system that combines different components of protein synthesis, like DNA, mRNA, tRNA…” More FAQ answered by our biology expert in the Forum: What does mRNA do in protein synthesis? Come join us now!


     

    mRNA, tRNA, and rRNA

    mRNA, tRNA, and rRNA are the three major types of RNA involved in protein synthesis. The mRNA (or messenger RNA) carries the code for making a protein. In eukaryotes, it is formed inside the nucleus and consists of a 5′ cap, 5’UTR region, coding region, 3’UTR region, and poly(A) tail. The copy of a DNA segment for gene expression is located in its coding region. It begins with a start codon at 5’end and a stop codon at the 3′ end.
    tRNA (or transfer RNA), as the name implies, transfers the specific amino acid to the ribosome to be added to the growing chain of amino acid. It consists of two major sites: (1) anticodon arm and (2) acceptor stem. The anticodon arm contains the anticodon that complementary base pairs with the codon of the mRNA. The acceptor stem is the site where a specific amino acid is attached (in this case, the tRNA with amino acid is called aminoacyl-tRNA). A peptidyl-tRNA is the tRNA that holds the growing polypeptide chain.
    Unlike the first two, rRNA (or ribosomal RNA) does not carry genetic information. Rather, it serves as one of the components of the ribosome. The ribosome is a cytoplasmic structure in cells of prokaryotes and eukaryotes that are known for serving as a site of protein synthesis. The ribosomes can be used to determine a prokaryote from a eukaryote. Prokaryotes have 70S ribosomes whereas eukaryotes have 80S ribosomes. Both types, though, are each made up of two subunits of differing sizes. The larger subunit serves as the ribozyme that catalyzes the peptide bond formation between amino acids. rRNA has three binding sites: A, P, and E sites. The A (aminoacyl) site is where aminoacyl-tRNA docks. The P (peptidyl) site is where peptidyl-tRNA binds. The E (exit) site is where the tRNA leaves the ribosome.

    Protein biosynthesis steps

    Transcription

    Transcription is the process by which mRNAtemplate, encoding the sequence of the protein in the form of a trinucleotide code, is transcribed from DNA to provide a template for translation through the help of the enzyme, RNA polymerase. Thus, transcription is regarded as the first step of gene expression. Similar to DNA replication, the transcription proceeds in the 5′ → 3′ direction. But unlike DNA replication, transcription needs no primer to initiate the process and, instead of thymine, uracil pairs with adenine.
    The steps of transcription are as follows: (1) Initiation, (2) Promoter escape, (3) Elongation, and (4) Termination. The first step, initiation, is when the RNA polymerase, with the assistance of certain transcription factors, binds to the promoter of DNA. This leads to the opening (unwinding) of DNA at the promoter region, forming a transcription bubble. A transcription start site in the transcription bubble binds to the RNA polymerase, particularly to the latter’s initiating NTP and an extending NTP. A phase of abortive cycles of synthesis occurs resulting in the release of short mRNA transcripts (about 2 to 15 nucleotides). The next step is for the RNA polymerase to escape the promoter so that it can enter into the elongation step. During elongation, RNA polymerase traverses the template strand of the DNA and base pairs with the nucleotides on the template (noncoding) strand. This results in mRNA transcript containing a copy of the coding strand of DNA, except for thymines that are replaced by uracils. The sugar-phosphate backbone forms through RNA polymerase. The last step is termination. During this phase, the hydrogen bonds of the RNA-DNA helix break. In eukaryotes, the mRNA transcript goes through further processing. It goes through polyadenylation, capping, and splicing.

    Translation

    Translation is the process in which amino acids are linked together in a specific order according to the rules specified by the genetic code. It occurs in the cytoplasm where the ribosomes are located. It consists of four phases: (1) activation (the amino acid is covalently bonded to the tRNA), (2) initiation (the small subunit of the ribosome binds to 5′ end of mRNA with the help of initiation factors), (3) elongation (the next aminoacyl-tRNA in line binds to the ribosome along with GTP and an elongation factor), and (4) termination (the A site of the ribosome faces a stop codon).

    Post-translation

    Following protein synthesis are events, e.g. proteolysis and protein folding. Proteolysis refers to the cleavage of proteins by proteases. Through it, N-terminal, C-terminal, or the internal amino-acid residues are removed from the polypeptide. Post-translational modification refers to the enzymatic processing of a polypeptide chain following translation and peptide bond formation. The ends and the side chains of the polypeptide may be modified in order to ensure proper cellular localization and function. Protein folding is the folding of the polypeptide chains to assume secondary and tertiary structures.


    Has this info helped you understand the topic? Got any question? How about hearing answers directly from our community? Join us in our Forum: What does mRNA do in protein synthesis? Let’s keep it fun and simple!


     

    See also

    References

    1. Protein Synthesis. (2019). Retrieved from Elmhurst.edu website: http://chemistry.elmhurst.edu/vchembook/584proteinsyn.html
    2. Protein Synthesis. (2019). Retrieved from Estrellamountain.edu website: https://www2.estrellamountain.edu/faculty/farabee/biobk/BioBookPROTSYn.html
    3. Protein Synthesis. (2019). Retrieved from Nau.edu website: http://www2.nau.edu/lrm22/lessons/protein-synthesis/protein-synthesis.htm

    © Biology Online. Content provided and moderated by Biology Online Editors


    Last updated on November 5th, 2021

    Источник: https://www.biologyonline.com/dictionary/protein-synthesis

    Meth Awareness In The News

    Methamphetamine abuse has become a tremendous challenge for the entire Nation. Education, prevention, and community involvement are key parts of our National Strategy to reduce the demand for meth. People who know about the destructive effects of meth on the user and the community, are far less likely to use meth.

    Please share what you learn about meth with everyone you know and together we will end the scourge of methamphetamine.

    What is methamphetamine?
    How is meth made?
    How does meth affect a user?
    How does meth affect everyone else?

    WHAT IS METHAMPHETAMINE?

    Methamphetamine is a powerful, highly addictive stimulant drug that dramatically affects the central nervous system.  It is usually illegally produced and distributed.

    Meth comes in several forms, including powder, crystal, rocks, and tablets.   When it comes in the crystal form it is called “crystal meth.” 

    Ice Methamphetamine Ice Methamphetamine
    Meth needle

    Meth can be taken by swallowing, snorting, smoking, or injecting it with a hypodermic needle. 

    HOW IS METH MADE?

    Unlike drugs such as marijuana, cocaine, and heroin, which are derived from plants, meth can be manufactured using a variety of store bought chemicals.

    The most common ingredient in meth is pseudoephedrine or ephedrine, commonly found in cold medicine.  Through a cooking process the pseudoephedrine or ephedrine is chemically changed into meth.  The ingredients that are used in the process of making meth can include: ether, paint thinner, Freon®, acetone, anhydrous ammonia, iodine crystals, red phosphorus, drain cleaner, battery acid, and lithium (taken from inside batteries). 

    Photo of a home meth lab Photo of a home meth lab
    Photo of a home meth lab

    Meth is often manufactured or “cooked” in very crude laboratories.  Many of these labs are not sophisticated operations and do not require sophisticated chemistry equipment.  And the people who cook the meth usually do not have any chemistry training.  Cooking meth is relatively simple, but highly dangerous and toxic. 

    There are two basic categories of meth labs:

    Superlabs produce large quantities of meth and supply organized drug trafficking groups that sell the drug in communities across the U.S. Most of the larger labs are controlled by Mexican Drug Trafficking Organizations operating in the U.S. and Mexico.

    Small Toxic Labs produce smaller quantities of meth.  These labs can be set up in homes, motel rooms, inside automobiles, and in parks or rural areas -- really almost anywhere.

    Photo of a home meth lab

    HOW DOES METH AFFECT A USER?

    Using meth causes an increase in energy and alertness, a decrease in appetite, and an intense euphoric “rush.”  That’s in the short term. 

    With sustained use, a meth user can develop a tolerance to it.  The user may take increasingly higher doses of meth trying to catch that high she first experienced.  She may take it more frequently Windows 7 Crack + Serial Code Free Download 2021 or may go on binges.  She may change the way she takes meth.  For example a user may have started by taking a pill, but as she develops a tolerance she may begin injecting it.  Addiction is likely.

    In the long term, a person using meth may experience irritability, fatigue, headaches, anxiety, sleeplessness, confusion, aggressive feelings, violent rages, cravings for more meth, and depression.  They may become psychotic and experience paranoia, auditory hallucinations, mood disturbances, and delusions.  The paranoia may lead to homicidal or suicidal thoughts.

    A fairly common hallucination experienced by meth users is the so-called crank bug.  The user gets the sensation that there are insects creeping on top of, or underneath, her skin.  The user will pick at or scratch her skin trying to get rid of the imaginary bugs.  This scratching can create open sores that may become infected.

    Photo of a Face of Meth (Left) and 11 Months later (right) Photo of a Meth Hands

    Photos courtesy of Sheriff’s Acid production - Crack Key For U, Multnomah County, Oregon

     

    Meth reduces the amount of protective saliva around the teeth. Meth users also consume excess sugared, carbonated soft drinks, tend to neglect personal hygiene, grind their teeth and clench their jaws, leading to what is commonly called �meth mouth.� Teeth can eventually fall out of users� mouths�even as they do simple things like eating a sandwich.

    Photo of a Meth Mouth Photo of a Meth Mouth
    Photos courtesy of: Sharlee Shirley, RDH, MPH; Jim Cecil, DMD, MPH, University of Kentucky, School of Utorrent pro free - Crack Key For U doses of meth can elevate body temperature to dangerous, sometimes lethal, levels.  High doses can also cause convulsions. 

    People can die as a result of using meth.

    Because meth is so addictive, the distance between the short and long term effects may not be very long. 

    HOW DOES METH AFFECT EVERYONE ELSE?

    As you can imagine, all those toxic chemicals used in the meth manufacturing process take a toll on the environment.  Every pound of meth made can generate up to five pounds of toxic waste that may seep into the soil and groundwater. 

    The manufacturing process also generates toxic fumes.  These fumes can severely harm anyone exposed to them.  Meth labs also generate highly explosive gases.

    Meth also has a very serious impact on children.  Many children are rescued from homes with meth labs or meth using parents.  Meth, chemicals, and syringes are all within reach of these children.  Parents high on meth neglect their children.  And the mental, physical, and emotional consequences for these Drug Endangered Children are often severe.

    Millions of our tax dollars are spent each year to clean up meth labs, to care for Drug Endangered Children, and to pay for law enforcement to deal with the meth problem.

    Photo of a home meth lab Photo of a play area

    Photo provided by: Cheyenne Albro, Director of the Pennyrile Narcotics Task Force, (Marshall Co. lab); Kentucky Drug Endangered Children Training Network.

    Photo provided by: Det. Tim Ahumada & Det. Joyell Lucero, Phoenix Police Dept.

    Photo of a home meth lab

     

    Now that you know about meth, pass it on.

    SEND A LINK TO A FRIEND

    Источник: https://www.justice.gov/archive/olp/methawareness/

    Do you start group chats about the best scalp treatments? Google AHA vs. BHA exfoliants until the wee hours? You're our people. And we know you're going to love The Science of Beauty, a series on Allure.com that goes deep into the how and why behind your favorite products. For even more nerdiness, check outThe Science of Beauty podcast, produced by our editors.

    Have you ever found yourself staring into the mirror wondering how you could possibly be breaking out while having dry skin on your face? If so, dehydrated skin might be what's ailing your complexion — even if you might normally self-identify as someone with oily skin. 

    In fact, everyone's complexions are susceptible to dehydration (which is different from dry skin), regardless of skin type, New York City-based dermatologist Y. Claire Chang shared on the hydration episode of Allure's The Science of Beauty podcast. "Those with oily skin can still have dehydrated skin, which means it can have high sebum levels, but low water content," she explained. Plus, acne treatments can be particularly drying or even irritating, so skin hydration is even more important for those dealing with breakouts and oiliness, Chang added. 

    Identifying Dehydrated Skin Versus Dry Skin

    You can typically tell if your skin is dehydrated if you pinch one of your cheeks and it wrinkles with gentle pressure instead of holding its shape, Ross C. Radusky, a board-certified dermatologist at SoHo Skin & Laser Dermatology, tells Allure. Dehydrated skin will also feel tight and appear duller than usual, he adds. You may also notice more exaggerated wrinkles acid production - Crack Key For U ones in places you don't remember having them, along with deeper dark circles. 

    Dry skin, on the other hand, is characterized by lack of oil, Radusky says. Skin peeling and itchy, flaky skin typically happen as a result. Basically, general discomfort is a major sign of dryness. "The worst areas are typically near the eyebrows and around the corners of the nose and mouth," Radusky adds.

    Most of us understand the negative impacts of dehydration on our overall health, but not enough of us are aware of its potential to wreak such visible havoc on our complexions. In order to understand the difference between dryness and dehydration, we consulted experts on how to best address dehydration for happier, healthier, and more hydrated skin. 

    How to Treat Dehydration and Dryness

    Both dry and dehydrated skin have some treatments in common — however, you may be neglecting some of the more obvious ones if your complexion is on the oily side. If you're not positive whether you're dealing with dry or dehydrated skin, the good news is that the topical treatments for both are essentially the same. These six steps should help you see improvement in how your skin looks and feels either way. 

    Moisturize, then moisturize some more

    Dry and dehydrated skin can be soothed in a number of ways, but making sure you're finishing off your day and nighttime skin-care routines with a moisturizer rich in emollients, humectants, and ceramides is the most obvious. For those unfamiliar with the latter ingredient, ceramides are lipids (aka fat molecules) that "help the skin retain moisture and allow [for] proper function," New York City-based dermatologist Sejal Shah previously told Allure. When your skin lacks them, dryness and irritation occur. But a great moisturizer will replenish your skin's ceramide levels and hydrate it in the process.

    Источник: https://www.allure.com/story/dry-vs-dehydrated-skin-whats-the-difference

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    Phosphoric acid

    Chemical compound

    This article is about orthophosphoric acid. For other acids commonly called "phosphoric acid", see Phosphoric acids and phosphates.

    Not to be confused with Phosphorous acid.

    Structural formula of phosphoric acid, showing dimensions
    Ball-and-stick model
    Space-filling model
    Names
    IUPAC name

    Phosphoric acid

    Other names

    Orthophosphoric acid

    Identifiers

    CAS Number

    3D model (JSmol)

    ChEBI
    ChEMBL
    ChemSpider
    ECHA InfoCard100.028.758Edit this at Wikidata
    EC Number
    E numberE338 (antioxidants, ...)
    KEGG

    PubChemCID

    RTECS number
    UNII
    UN number1805

    CompTox Dashboard(EPA)

    InChI

    • InChI=1S/H3O4P/c1-5(2,3)4/h(H3,1,2,3,4) checkY
      Key: NBIIXXVUZAFLBC-UHFFFAOYSA-N checkY
    • InChI=1/H3O4P/c1-5(2,3)4/h(H3,1,2,3,4)

      Key: NBIIXXVUZAFLBC-UHFFFAOYAI

    Properties

    Chemical formula

    H
    3PO
    4
    Molar mass97.994 g·mol−1
    Appearance white solid
    OdorOdorless
    Density1.6845  g⋅cm−3 (25 °C, 85%),[1] 1.834  g⋅cm−3 (solid)[2]
    Melting point 40–42.4 °C (104.0–108.3 °F; 313.1–315.5 K)[6]
    Boiling point 

    Solubility in water

    • 392.2 g/100 g (−16.3 °C)
    • 369.4 g/100 mL (0.5 °C)
    • 446 g/100 mL (15 °C)[5]
    • 548 g/100 mL (20 °C)[6]
    SolubilitySoluble in ethanol
    log P−2.15[7]
    Vapor pressure0.03 mmHg (20 °C)[8]
    Conjugate baseDihydrogen phosphate

    Magnetic susceptibility (χ)

    −43.8·10−6 cm3/mol[10]

    Refractive index (nD)

    • 1.3420 (8.8% w/w aq. soln.)[11]
    • 1.4320 (85% aq. soln) 25 °C
    Viscosity2.4–9.4 cP (85% aq. soln.)
    147 cP (100%)
    Structure

    Crystal structure

    Monoclinic

    Molecular shape

    Tetrahedral
    Thermochemistry[12]

    Heat capacity(C)

    145.0 J/mol⋅K

    Std molar
    entropy(S298)

    150.8 J/mol⋅K

    Std enthalpy of
    formation(ΔfH298)

    −1271.7 kJ/mol

    Gibbs free energy(ΔfG˚)

    -1123.6 kJ/mol
    Hazards
    Safety data sheetICSC 1008
    GHS pictogramsGHS05: Corrosive[13]
    GHS Signal wordDanger

    GHS hazard statements

    H290, H314[13]

    GHS precautionary statements

    P280, P305+351+338, P310[13]
    NFPA 704 (fire diamond)
    Flash pointNon-flammable
    Lethal dose or concentration (LD, LC):

    LD50 (median dose)

    1530 mg/kg (rat, oral)[14]
    NIOSH (US health exposure limits):

    PEL (Permissible)

    TWA 1 mg/m3[8]

    REL (Recommended)

    TWA 1 mg/m3 ST 3 mg/m3[8]

    IDLH (Immediate danger)

    1000 mg/m3[8]
    Related compounds

    Related phosphorusoxoacids

    Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

    ☒N verify (what is checkY☒N ?)
    Infobox references

    Chemical compound

    Phosphoric acid, also known as orthophosphoric acid or phosphoric(V) acid, is a weak acid with the chemical formulaH
    3PO
    4. The pure compound is a colorless solid.

    All three hydrogens are acidic to varying degrees and can be lost from the molecule as H+ ions (protons). When all three H+ ions are removed, the result is an orthophosphate ion PO43−, commonly called "phosphate". Removal of one or two protons gives dihydrogen phosphate ion H
    2PO
    4, and the hydrogen phosphate ion HPO2−
    4, respectively. Orthophosphoric acid also forms esters, called organophosphates.[15]

    Phosphoric acid is commonly encountered in chemical laboratories as an 85% aqueous solution, which is a colourless, odourless, and non-volatile syrupy liquid. Although phosphoric acid does not meet the strict definition of a strong acid, the 85% solution can still severely irritate the skin and damage the eyes.

    The name "orthophosphoric acid" can be used to distinguish this specific acid from other "phosphoric acids", such as pyrophosphoric acid. Nevertheless, the term "phosphoric acid" often means this specific compound; and that is the current IUPAC nomenclature.

    Manufacture[edit]

    Phosphoric acid is produced industrially by two general routes.[16] In the wet process a phosphate-containing mineral such as calcium hydroxyapatite is treated with sulfuric acid.[17]

    {\displaystyle {\ce {Ca5(PO4)3OH + 5H2SO4 -> 3H3PO4 + 5CaSO4v + H2O}}}

    Fluoroapatite is an alternative feedstock, in which case fluoride is removed as the insoluble compound Na2SiF6. The phosphoric acid solution usually contains 23–33% P2O5 (32–46% H3PO4). It may be concentrated to produce commercial- or merchant-grade phosphoric acid, which contains about 54–62% P2O5 (75–85% H3PO4). Further removal of water yields superphosphoric acid with a P2O5 concentration above 70% (corresponding to nearly 100% H3PO4). Calcium sulfate (gypsum) is produced as a by-product and is removed as phosphogypsum.

    To produce food-grade phosphoric acid, phosphate ore is first reduced with coke in an electric arc furnace, to make elemental phosphorus. Silica is also added, resulting in the production of calcium silicate slag. Elemental phosphorus is distilled out of the furnace and burned with air to produce high-purity phosphorus pentoxide, which is dissolved in water to make phosphoric acid.

    The phosphoric acid from both processes may be further purified by removing compounds of arsenic and other potentially toxic impurities.

    Acidic properties[edit]

    All three hydrogens are acidic, with dissociation constants pKa1 = 2.14, pKa2 = 7.20, and pKa3 = 12.37. It follows that, in water solutions, phosphoric acid is mostly dissociated into some combination of its three anions, except at very low pH. The equilibrium equations are:

    H3PO4   + H2O ⇌ H3O+ + H2PO4      Ka1= 7.25×10−3 [pKa1 = 2.14]
    H2PO4+ H2O ⇌ H3O+ + HPO42−       Ka2= 6.31×10−8 [pKa2 = 7.20]
    HPO42−+ H2O ⇌ H3O+ +  PO43−        Ka3= 3.98×10−13 [pKa3 = 12.37]

    Uses[edit]

    The dominant use of phosphoric acid is for fertilizers, consuming approximately 90% of production.[18]

    Application Demand (2006) in thousands of tons Main phosphate derivatives
    Soaps and detergents1836STPP
    Food industry309STPP (Na5P3O10), SHMP, TSP, SAPP, SAlP, MCP, DSP (Na2HPO4), H3PO4
    Water treatment164SHMP, STPP, TSPP, MSP (NaH2PO4), DSP
    Toothpastes68DCP (CaHPO4), IMP, SMFP
    Other applications287STPP (Na3P3O9), TCP, APP, DAP, zinc phosphate (Zn3(PO4)2), aluminium phosphate (AlPO4, H3PO4)

    Food-grade phosphoric acid (additive E338[19]) is used to acidify foods and beverages such as various colas and jams, providing a tangy or sour taste. The phosphoric acid also serves as a preservative.[20] Soft drinks containing phosphoric acid, which would include Coca-Cola, are sometimes called phosphate sodas or phosphates. Phosphoric acid in soft drinks has the potential to cause dental erosion.[21] Phosphoric acid also has the potential to contribute to the formation of kidney stones, especially in those who have had kidney stones previously.[22]

    Specific applications of phosphoric acid include:

    Safety[edit]

    A link has been shown between long-term regular cola intake and osteoporosis in later middle age in women (but not men).[28] This was thought to be due to the presence of phosphoric acid, and the risk for women was found to be greater for sugared and caffeinated colas than diet and decaffeinated variants, with a higher intake of cola correlating with lower bone density.

    At moderate concentrations phosphoric acid solutions are irritating to the skin. Contact with concentrated solutions can cause severe skin burns and permanent eye damage.[29]

    See also[edit]

    References[edit]

    1. ^Christensen, J. H. & Reed, R. B. (1955). "Design and Analysis Data—Density of Aqueous Solutions of Phosphoric Acid Measurements at 25 °C". Ind. Eng. Chem. 47 (6): 1277–1280. doi:10.1021/ie50546a061.
    2. ^"CAMEO Chemicals Datasheet – Phosphoric Acid". Archived from the original on 15 August 2019. Retrieved 15 August 2019.
    3. ^"Phosphoric acid". www.chemspider.com. Archived from the original on 12 March 2020. Retrieved 3 March 2020.
    4. ^Brown, Earl H.; Whitt, Carlton D. (1952). "Vapor Pressure of Phosphoric Acids". Industrial & Engineering Chemistry. 44 (3): 615–618. doi:10.1021/ie50507a050.
    5. ^Seidell, Atherton; Linke, William F. (1952). Solubilities of Inorganic and Organic Compounds. Van Nostrand. Archived from the original on 11 March 2020. Retrieved 2 June 2014.
    6. ^ abHaynes, p. 4.80
    7. ^"phosphoric acid_msds". Archived from the original on 4 July 2017. Retrieved 2 May 2018.
    8. ^ abcdNIOSH Pocket Guide to Chemical Hazards. "#0506". National Institute for Occupational Safety and Health (NIOSH).
    9. ^Haynes, p. 5.92
    10. ^Haynes, p. 4.134
    11. ^Edwards, O. W.; Dunn, R. L. & Hatfield, J. D. (1964). "Refractive Index of Phosphoric Acid Solutions at 25 C.". J. Chem. Eng. Data. 9 (4): 508–509. doi:10.1021/je60023a010.
    12. ^Haynes, p. 5.13
    13. ^ abcSigma-Aldrich Co., Phosphoric acid.
    14. ^"Phosphoric acid". Immediately Dangerous to Life or Health Concentrations (IDLH). National Institute for Occupational Safety and Health (NIOSH).
    15. ^Westheimer, F.H. (6 June 1987). "Why nature chose phosphates". Science. 235 (4793): 1173–1178 (see pp. 1175–1176). Bibcode:1987Sci...235.1173W. CiteSeerX 10.1.1.462.3441. doi:10.1126/science.2434996. PMID 2434996.
    16. ^Becker, Pierre (1988). Phosphates and phosphoric acid. New York: Marcel Dekker. ISBN .
    17. ^Greenwood, Norman N.; Earnshaw, Alan (1997). Chemistry of the Elements (2nd ed.). Butterworth-Heinemann. pp. 520–522. ISBN .
    18. ^Schrödter, Klaus; Bettermann, Gerhard; Staffel, Thomas; Wahl, Friedrich; Klein, Thomas; Hofmann, Thomas (2008). "Phosphoric Acid and Phosphates". Ullmann's Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a19_465.pub3.
    19. ^"Current EU approved additives and their E Numbers". Foods Standards Agency. 14 March 2012. Archived from the original on 19 July 2013. Retrieved 22 July 2012.
    20. ^"Why is phosphoric acid used in some Coca‑Cola drinks? Frequently Asked Questions

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      Acid Rain

      Inorganic Reactions Experiment

      Authors: Rachel Casiday and Regina Frey
      Department of Chemistry, Washington University
      St. Louis, MO 63130


      Natural Acidity of Rainwater

      Pure water has a pH of 7.0 (neutral); however, natural, unpolluted rainwater actually has a pH of about 5.6 (acidic).[Recall from Experiment 1 that pH is a measure of the hydrogen ion (H+) concentration.] The acidity of rainwater comes from the natural presence of three substances (CO2, NO, and SO2) found in the troposphere (the lowest layer of the atmosphere). As is seen in Table I, carbon dioxide (CO2) is present in the greatest concentration and therefore contributes the most to the natural acidity of rainwater.

      Gas

      Natural Sources

      Concentration

      Carbon dioxide
      CO2
      Decomposition 355 ppm
      Nitric oxide
      NO
      Electric discharge 0.01 ppm
      Sulfur dioxide
      SO2
      Volcanic gases 0-0.01 ppm

      Table 1

      Carbon dioxide, produced in the decomposition of organic material, is the primary source of acidity in unpolluted rainwater.

      NOTE: Parts per million (ppm) is a common concentration measure used in environmental chemistry. The formula for ppm is given by:

      Carbon dioxide reacts with water to form carbonic acid (Equation 1). Carbonic acid then dissociates to give the hydrogen ion (H+) and the hydrogen carbonate ion (HCO3-) (Equation 2). The ability of H2CO3 to deliver H+ is what classifies this molecule as an acid, thus lowering the pH of a solution.


      (1)

       


      (2)

      Nitric oxide (NO), which also contributes to the natural acidity of rainwater, is formed during lightning storms by the reaction of nitrogen and oxygen, two common atmospheric gases (Equation 3). In air, NO is oxidized to nitrogen dioxide (NO2) (Equation 4), which in turn reacts with water to give nitric acid (HNO3) (Equation 5). This acid dissociates in water to yield hydrogen ions and nitrate ions (NO3-) in a reaction analagous to the dissociation of carbonic acid shown in Equation 2, again lowering the pH of the solution.


      (3)

       


      (4)

       


      (5)

      Acidity of Polluted Rainwater

      Unfortunately, human industrial activity produces additional acid-forming compounds in far greater quantities than the natural sources of acidity described above. In some areas of the United States, the pH of rainwater can be 3.0 or lower, approximately 1000 times more acidic than normal rainwater. In 1982, the pH of a fog on the West Coast of the United States was measured at 1.8! When rainwater is too acidic, it can cause problems ranging from killing freshwater fish and damaging crops, to eroding buildings and monuments.


      Questions on Acidity of Rainwater

      1. List two or more ways that you could test the acidity of a sample of rainwater.

      2. Write a balanced chemical equation for the dissociation of nitric acid in water. (HINT: Draw an analogy with Equation 2.)

      3. The gaseous oxides found in the atmosphere, including CO2 and NO are nonmetal oxides. What would happen to the pH of rainwater if the atmosphere contained metal oxides instead? (HINT: Think back to Experiment 1.) Briefly, explain your answer.


      Sources of Excess Acidity in Rainwater

      What causes such a dramatic increase in the acidity of rain relative to pure water? The answer lies within the concentrations of nitric oxide and sulfur dioxide in polluted air. As shown in Table II and Figure 1, the concentrations of these oxides are much higher than in clean air.

      Gas

      Non-Natural Sources

      Concentration

      Nitric oxide
      NO
      Internal Combustion0.2 ppm
      Sulfur dioxide
      SO2
      Fossil-fuel Combustion 0.1 - 2.0 ppm

      Table II

      Humans cause many combustion processes that dramatically increase the concentrations of acid-producing oxides in the atmosphere. Although CO2 is present in a much higher concentration than NO and SO2, CO2 does not form acid to the same extent as the other two gases. Thus, a large increase in the concentration of NO and SO2 significantly affects the pH of rainwater, even though both gases are present at much lower concentration than CO2.

      Figure 1

      Comparison of the concentrations of NO and SO2 in clean and polluted air.

      About one-fourth of the acidity of rain is accounted for by nitric acid (HNO3). In addition to the natural processes that form small amounts of nitric acid in rainwater, high-temperature air combustion, such as occurs in car engines and power plants, produces large amounts of NO gas. This gas then forms nitric acid via Equations 4 and 5. Thus, a process that occurs naturally at levels tolerable by the environment can harm the environment when human activity causes the process (e.g., formation of nitric acid) to occur to a much greater extent.

      What about the other 75% of the acidity of rain? Most is accounted for by the presence of sulfuric acid (H2SO4) in rainwater. Although sulfuric acid may be produced naturally in small quantities from biological decay and volcanic activity (Figure 1), it is produced almost entirely by human activity, especially the combustion of sulfur-containing fossil fuels in power plants. When these fossil fuels are burned, the sulfur contained in them reacts with oxygen from the air to form sulfur dioxide (SO2). Combustion of fossil fuels accounts for approximately 80% of the total atmospheric SO2 in the United States. The effects of burning fossil fuels can be dramatic: in contrast to the unpolluted atmospheric SO2 concentration of 0 to 0.01 ppm, polluted urban air can contain 0.1 to 2 ppm SO2, or up to 200 times more SO2! Sulfur dioxide, like the oxides of carbon and nitrogen, reacts with water to form sulfuric acid (Equation 6).


      (6)

      Sulfuric acid is a strong acid, so it readily dissociates in water, to give an H+ ion and an HSO4- ion (Equation 7). The HSO4- ion may further dissociate to give H+ and SO42- (Equation 8). Thus, the presence of H2SO4 causes the concentration of H+ ions to increase dramatically, and so the pH of the rainwater drops to harmful levels.


      (7)

       


      (8)


      Questions on Sources of Acidity in Rainwater

      4. At sea level and 25oC, one mole of air fills a volume of 24.5 liters, and the density of air is 1.22x10-6 g/ml. Compute the mole fraction (i.e.,moles of component /total moles) and molarity of SO2 when the atmospheric concentration of SO2 is 2.0 ppm (see note in Table I).

      5.One strategy for limiting the amount of acid pollution in the atmosphere is scrubbing. In particular, calcium oxide (CaO) is injected into the combustion chamber of a power plant, where it reacts with the sulfur dioxide produced, to yield solid calcium sulfite.

      a. Write a balanced chemical equation for this reaction. (HINT: Consult the table of common ions in the tutorial assignment for Experiment 1 to view the structure and formula for sulfite; also, use your knowledge of the periodic table to deduce the charge of the calcium ion. Using these facts, you can deduce the formula for calcium sulfite.)

      b. Approximately one ton, or 9.0x102 kg, of calcium sulfite is generated each year for every person served by a power plant. How much sulfur dioxide (in moles) is prevented from entering the atmosphere when this much calcium sulfite is generated? Show your calculation.

      c. The final stage in the scrubbing process is to treat the combustion gases with a slurry of solid CaO in water, in order to trap any remaining SO2 and convert it to calcium sulfite. A slurry is a thick suspension of an insoluble precipitate in water. Using the solubility guidelines provided in the lab manual for this experiment, predict whether this stage of the scrubbing process will produce a slurry (i.e., precipitate) or a solution (i.e., no precipitate) of calcium sulfite .

      d. If MgO, rather than CaO, were used for scrubbing, would the product of the final stage be a slurry or a solution of magnesium sulfite? (Assume that a very large quantity of magnesium sulfite, relative to the amount of water, is produced.)


      Environmental Effects of Acid Rain

      Acid rain triggers a number of inorganic and biochemical reactions with deleterious environmental effects, making this a growing environmental problem worldwide.

      • Many lakes have become so acidic that fish cannot live in them anymore.
      • Degradation of many soil minerals produces metal ions that are then washed away in the runoff, causing several effects:
        • The release of toxic ions, such as Al3+, into the water supply.
        • The loss of important minerals, such as Ca2+, from the soil, killing trees and damaging crops.
      • Atmospheric pollutants are easily moved by wind currents, so acid-rain effects are felt far from where pollutants are generated.

      Stone Buildings and Monuments in Acid Rain

      Marble and limestone have long been preferred materials for constructing durable buildings and monuments. The Saint Louis Art Museum, the Parthenon in Greece, the Chicago Field Museum, and the United States Capitol building are all made of these materials. Marble and limestone both consist of calcium carbonate (CaCO3), and differ only in their crystalline structure. Limestone consists of smaller crystals and is more porous than marble; it is used more extensively in buildings. Marble, with its larger crystals and smaller pores, can attain a high polish and is thus preferred for monuments and statues. Although these are recognized as highly durable materials, buildings and outdoor monuments made of marble and limestone are now being gradually eroded away by acid rain.

      How does this happen? A chemical reaction (Equation 9) between calcium carbonate and sulfuric acid (the primary acid component of acid rain) results in the dissolution of CaCO3 to give aqueous ions, which in turn are washed away in the water flow.


      (9)

      This process occurs at the surface of the buildings or monuments; thus acid rain can easily destroy the details on relief work (e.g., the faces on a statue), but generally does not affect the structural integrity of the building. The degree of damage is determined not only by the acidity of the rainwater, but also by the amount of water flow that a region of the surface receives. Regions exposed to direct downpour of acid rain are highly susceptible to erosion, but regions that are more sheltered from water flow (such as under eaves and overhangs of limestone buildings) are much better preserved. The marble columns of the emperors Marcus Aurelius and Trajan, in Rome, provide a striking example: large volumes of rainwater flow directly over certain parts of the columns, which have been badly eroded; other parts are protected by wind effects from this flow, and are in extremely good condition even after nearly 2000 years!

      Even those parts of marble and limestone structures that are not themselves eroded can be damaged by this process (Equation 9). When the water dries, it leaves behind the ions that were dissolved in it. When a solution containing calcium and sulfate ions dries, the ions crystallize as CaSO4l 2H2O, which is gypsum. Gypsum is soluble in water, so it is washed away from areas that receive a heavy flow of rain. However, gypsum accumulates in the same sheltered areas that are protected from erosion, and attracts dust, carbon particles, dry-ash, and other dark pollutants. This results in blackening of the surfaces where gypsum accumulates.

      An even more serious situation arises when water containing calcium and sulfate ions penetrates the stone's pores. When the water dries, the ions form salt crystals within the pore system. These crystals can disrupt the crystalline arrangement of the atoms in the stone, causing the fundamental structure of the stone to be disturbed. If the crystalline structure is disrupted sufficiently, the stone may actually crack. Thus, porosity is an important factor in determining a stone's durability.


      Questions on Effects of Acid Rain

      6. Based on the information described above about the calcium ion, and the formula of calcium carbonate (CaCO3), deduce the charge of the carbonate ion. Also, in the structure of the carbonate ion, are any of the oxygens bonded to one another, or all the oxygens bonded to the carbon atom? (HINT: Consult the structure of the common ions given in the tutorial for Experiment 1).

      7. In water, H2SO4 can dissociate to yield two H+ ions and one SO42- ion. Write the net ionic equation for the reaction of calcium carbonate and sulfuric acid. (See the introduction to Experiment 2 in the lab manual for a discussion of net ionic equations.)

      8. Which is a more durable building material, limestone or marble? Briefly, explain your reasoning.

      Additional Links:


      References:

      Brown, Lemay, and Buster. Chemistry: the Central Science, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 673-5.

      Charola, A. "Acid Rain Effects on Stone Monuments," J. Chem. Ed.64 (1987), p. 436-7.

      Petrucci and Harwood. General Chemistry: Principles and Modern Applications, 7th ed. Upper Saddle River, NJ: Prentice Hall, 1997. p. 614-5.

      Walk, M. F. and P.J. Godfrey. "Effects of Acid Deposition on Surface Waters," J. New England Water Works Assn. Dec. 1990, p. 248-251.

      Zumdahl, S.. Chem. Principles, 3rd ed. Boston: Houghton Mifflin, 1998. p. 174-6.

      Stryer, L. Biochemistry, 4th ed., W.H. Freeman and Co., New York, 1995, p. 332-339.


      Acknowledgements:

      The authors thank Dewey Holten (Washington University) for many helpful suggestions in the writing of this tutorial.

      The development of this tutorial was supported by a grant from the Howard Hughes Medical Institute, through the Undergraduate Biological Sciences Education program, Grant HHMI# 71192-502004 to Washington University.

      Copyright 1998, Washington University, All Rights Reserved.

      Источник: http://www.chemistry.wustl.edu/~edudev/LabTutorials/Water/FreshWater/acidrain.html

      All MAGIX products are designed to be as user-friendly as possible. However, if you have questions about your software, you can find initial support and various ways to get in touch with the MAGIX technical support here.

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      Do you start group chats about the best scalp treatments? Google AHA vs. BHA exfoliants until the wee hours? You're our people. And we know you're going to love The Science of Beauty, a series on Allure.com that goes deep into the how and why behind your favorite products. For even more nerdiness, check outThe Science of Beauty podcast, produced by our editors.

      Have you ever found yourself staring into the mirror wondering how you could possibly be breaking out while having dry skin on your face? If so, dehydrated skin might be what's ailing your complexion — even if you might normally self-identify as someone with oily skin. 

      In fact, everyone's complexions are susceptible to dehydration (which is different from dry skin), regardless of skin type, New York City-based dermatologist Y. Claire Chang shared on the hydration episode of Allure's The Science of Beauty podcast. "Those with oily skin can still have dehydrated skin, which means it can have high sebum levels, but low water content," she explained. Plus, acne treatments can be particularly drying or even irritating, so skin hydration is even more important for those dealing with breakouts and oiliness, Chang added. 

      Identifying Dehydrated Skin Versus Dry Skin

      You can typically tell if your skin is dehydrated if you pinch one of your cheeks and it wrinkles with gentle pressure instead of holding its shape, Ross C. Radusky, a board-certified dermatologist at SoHo Skin & Laser Dermatology, tells Allure. Dehydrated skin will also feel tight and appear duller than usual, he adds. You may also notice more exaggerated wrinkles or ones in places you don't remember having them, along with deeper dark circles. 

      Dry skin, on the other hand, is characterized by lack of oil, Radusky says. Skin peeling and itchy, flaky skin typically happen as a result. Basically, general discomfort is a major sign of dryness. "The worst areas are typically near the eyebrows and around the corners of the nose and mouth," Radusky adds.

      Most of us understand the negative impacts of dehydration on our overall health, but not enough of us are aware of its potential to wreak such visible havoc on our complexions. In order to understand the difference between dryness and dehydration, we consulted experts on how to best address dehydration for happier, healthier, and more hydrated skin. 

      How to Treat Dehydration and Dryness

      Both dry and dehydrated skin have some treatments in common — however, you may be neglecting some of the more obvious ones if your complexion is on the oily side. If you're not positive whether you're dealing with dry or dehydrated skin, the good news is that the topical treatments for both are essentially the same. These six steps should help you see improvement in how your skin looks and feels either way. 

      Moisturize, then moisturize some more

      Dry and dehydrated skin can be soothed in a number of ways, but making sure you're finishing off your day and nighttime skin-care routines with a moisturizer rich in emollients, humectants, and ceramides is the most obvious. For those unfamiliar with the latter ingredient, ceramides are lipids (aka fat molecules) that "help the skin retain moisture and allow [for] proper function," New York City-based dermatologist Sejal Shah previously told Allure. When your skin lacks them, dryness and irritation occur. But a great moisturizer will replenish your skin's ceramide levels and hydrate it in the process.

      Источник: https://www.allure.com/story/dry-vs-dehydrated-skin-whats-the-difference

      Initiation and amplification of SnRK2 activation in abscisic acid signaling

      Abstract

      The phytohormone abscisic acid (ABA) is crucial for plant responses to environmental challenges. The SNF1-regulated protein kinase 2s (SnRK2s) are key components in ABA-receptor coupled core signaling, and are rapidly phosphorylated and activated by ABA. Recent studies have suggested that Raf-like protein kinases (RAFs) participate in ABA-triggered SnRK2 activation. In vitro kinase assays also suggest the existence of autophosphorylation of SnRK2s. Thus, how SnRK2 kinases are quickly activated during ABA signaling still needs to be clarified. Here, we show that both B2 and B3 RAFs directly phosphorylate SnRK2.6 in the kinase activation loop. This transphosphorylation by RAFs is essential for SnRK2 activation. The activated SnRK2s then intermolecularly trans-phosphorylate other SnRK2s that are not yet activated to amplify the response. High-order Arabidopsis mutants lacking multiple B2 and B3 RAFs show ABA hyposensitivity. Our findings reveal a unique initiation and amplification mechanism of SnRK2 activation in ABA signaling in higher plants.

      Introduction

      Environmental challenges like drought, cold, and high salinity induce the accumulation of abscisic acid (ABA), a major stress phytohormone that triggers multiple stress responses in plants1,2,3,4,5,6. ABA controls stomatal closure, seed dormancy and germination, senescence, growth, and development1,2,3,7. The ABA receptor-coupled core signaling pathway has been uncovered8,9,10 and consists of three key components: the ABA receptors, the PYRABACTIN RESISTANCE (PYR)/PYR-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) family proteins; the negative regulator clade A type 2 C protein phosphatases (PP2Cs); and the positive regulator SNF1-related protein kinase 2 s (SnRK2s).

      Phosphorylation and dephosphorylation determines the activation of SnRK2s and therefore ABA signaling. In the model plant Arabidopsis thaliana, there are ten members of the SnRK2 protein kinase family. Three of them, SnRK2.2, SnRK2.3, and SnRK2.6, are quickly activated within minutes after application of exogenous ABA, while all SnRK2s except SnRK2.9 are activated by osmotic stresses11,12,13,14. SnRK2.6, also known as OPEN STOMATA 1 (OST1), is mainly expressed in guard cells, while SnRK2.2 and SnRK2.3 are universally expressed15,16. The ost1/snrk2.6 mutant shows constitutive stomatal opening and is thus hypersensitive to water deficit15. The snrk2.2/2.3/2.6 triple (snrk2-triple) mutant is resistant to ABA and germinates and grows normally at very high concentrations of ABA13,14. At least two phosphosites, Ser171 and Ser175, which are located in the activation loop of SnRK2.6, are required for SnRK2.6 activation upon ABA treatment17,18. Other phosphosites like the N-terminal Ser7 and Ser29 may also contribute to the activation of SnRK2.619,20. The clade A PP2C phosphatases are the negative regulators of SnRK2s8,9,10,21,22,23. Under normal growth conditions, PP2Cs inhibit SnRK2.6 by directly binding to and dephosphorylating the Ser175 in the activation loop of SnRK2.6 and block the ABA signaling20,21,22.

      ABA binds to PYR/PYL/RCARs, and then the ABA-receptor complex inhibits the activity of PP2C phosphatases, resulting in the release of SnRK2s from PP2C-mediated inhibition9,10,20,24,25. Using recombinant SnRK2.6 purified from E. coli, Ng et al. (2011) reported that the phosphorylation and activation of SnRK2.6 mainly depends on its autophosphorylation activity26. However, whether SnRK2s are auto-activatable in vivo is still debatable since recombinant SnRK2.6 purified from E. coli is already highly phosphorylated and active. Most recently, several studies suggested that dephosphorylated SnRK2.6 and SnRK2.4 have no self-activation activity, and that transphosphorylation of SnRK2s by Raf-like protein kinases (RAFs) is required for SnRK2 activation27,28,29,30. B2/B3 and B4 RAFs are also called Osmotic stress-activated protein Kinase-100 kDa (OK100) and Osmotic stress-activated protein Kinase-130 kDa (OK130), respectively, because of their rapid activation by osmotic stress and their molecular weights observed from in-gel kinase assays27. The B4 subgroup RAFs (OK130) interact with and phosphorylate ABA-independent SnRK2s27. In a null mutant of B4 subgroup RAFs, OK130-null(raf16/raf40/raf24/raf18/ raf20/raf35/raf42), or raf18/raf20/raf24, the osmotic stress-induced activation of ABA-independent SnRK2s is completely abolished27,31. Interestingly, the B3 and B2 RAFs (OK100) phosphorylate SnRK2.2, SnRK2.3, and SnRK2.6 in vitro27,28,30,32. The high-order mutant OK-quatdec, containing mutations in four B2, three B3, and seven B4 RAFs, shows weak ABA insensitivity in seed germination and root growth27. A triple mutant of B3 RAF kinases, m3kδ1/δ6/δ7 (raf5/raf4/raf3), is slightly insensitive to ABA and is impaired in ABA-mediated SnRK2 activation28. However, compared to the complete abolishment of ABA responses in snrk2-triple, pyr1pyl1pyl2pyl4pyl5pyl8 (pyl112458), or pyl-duodecuple (pyr1pyl1pyl2pyl3pyl4pyl5pyl7pyl8py9pyl10pyl11pyl12, pyl-duodec) mutants13,14,33,34, the ABA-insensitivity is much weaker in m3kδ1/δ6/δ7 or OK-quatdec mutants27,28. Thus, the role of RAFs in ABA signaling still needs further investigation.

      Here, we show that the B2 and B3 subgroup RAFs phosphorylate Ser171 and Ser175 in SnRK2.6 with different specificity and that transphosphorylation is essential for initiating SnRK2.6 phosphorylation and activation. After phosphorylation by RAFs, the activated SnRK2.6 can quickly autophosphorylate (intermolecularly) and activate more SnRK2 proteins. We also generate a series of high-order mutants carrying null mutations in the B2, B3, or both B2 and B3 subgroup RAFs. From phenotypic assays of these high-order mutants, we find that both B2 and B3 subgroup RAFs are essential for ABA signaling. ABA-induced activation of SnRK2.2/2.3/2.6 and ABA-induced gene expression are strongly impaired in OK100-oct and OK100-nonu mutants lacking 8 and 9 members, respectively, of the B2 and B3 subgroups. OK100-oct and OK100-nonu also exhibit ABA hyposensitivity and can germinate and grow under extremely high ABA concentrations. We find that ABA does not activate B2 and B3 RAFs; instead, the basal level of RAF kinase activity is essential for SnRK2 activation and initiation of ABA signaling. Our results reveal a crucial RAF-SnRK2 cascade in ABA receptor-coupled core signaling and unique activation machinery for initiating and amplifying stress signaling in higher plants.

      Results

      SnRK2.6 activation requires transphosphorylation by B2 and B3 RAFs in vitro

      Recent studies suggested that several B3 RAFs (RAF3-6) and one B2 RAF (RAF10) can phosphorylate and activate dephosphorylated SnRK2.6 in vitro27,28,30,32. In vitro kinase assays and subsequent mass spectrometry revealed that Ser171 and Ser175 of SnRK2.6 might be the major sites for phosphorylation by B2 and B3 subgroup RAFs27. To further dissect the role of B2 and B3 RAFs in SnRK2 activation, we first tested the ability and specificity of the recombinant kinase domains (KDs) of B2 and B3 subgroup RAFs in SnRK2.6 phosphorylation. Out of 12 tested B2/B3 RAFs, KDs of 10 RAFs, RAF1-7, and RAF10-12, strongly phosphorylated SnRK2.6KR, a kinase-dead form of SnRK2.6 (Fig. 1a). The recombinant KDs of RAF8 and RAF9 had no detectable kinase activity (Supplementary Fig. 1a). The KDs of B2 and B3 RAFs showed distinct specificity for Ser171 and Ser175 of SnRK2.6 in the in vitro assay: the B2 RAFs, RAF7 and RAF10-12, targeted Ser171, while the B3 RAFs, RAF1-6, preferred Ser175 (Fig. 1a).

      a Recombinant RAF kinase domains (KDs) were used to phosphorylate SnRK2.6KR (SnRK2.6K50R, a kinase-dead form of SnRK2.6), SnRK2.6KR with Ser171Ala mutation (SnRK2.6KR-S171A), SnRK2.6KR with Ser175Ala mutation (SnRK2.6KR-S175A), or SnRK2.6KR proteins with Ser171AlaSer175Ala mutations (SnRK2.6KR-AA), in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-RAF-KD and HIS-SnRK2.6KR. b GST-SnRK2.6, HIS-SUMO-RAF3-KD, and HIS-SUMO-RAF10-KD phosphorylate HIS-SnRK2.6KR in vitro. Recombinant undephosphorylated GST-SnRK2.6 was used to phosphorylate HIS-SnRK2.6KR in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6, HIS-SUMO-RAF3-KD, HIS-SUMO-RAF10-KD, and HIS-SnRK2.6KR. c SnRK2.6M94G but not wild type SnRK2.6 can use N6-Benzyl-ATPγS to thiophosphorylate substrate. d RAF3-KD and RAF10-KD trigger the autophosphorylation of pre-dephosphorylated GST-SnRK2.6M94G (de-SnRK2.6M94G). e RAF3-KD activates pre-dephosphorylated GST-SnRK2.6M94G (de-SnRK2.6M94G) and the reactivated SnRK2.6M94G phosphorylates itself and ABF2. f RAF3-KD and RAF10-KD activate HIS-SUMO-SnRK2.2M96G and HIS-SUMO-SnRK2.3M95G. For d to f, Anti-γ-S immunoblot (upper) and Coomassie staining (lower) show thiophosphorylation and loading, respectively, of recombinant GST-RAF3-KD, GST-RAF10-KD, GST-SnRK2.6M94G, HIS-SUMO-SnRK2.2M96G, HIS-SUMO-SnRK2.3M95G, and GST-ABF2. Asterisks indicate preincubation of SnRK2.6M94G with N6-Benzyl-ATPγS for 30 min before further reaction. Arrows indicate degraded fragments co-purified with GST-SnRK2.6M94G. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

      Full size image

      Ser175 was previously suggested to be a SnRK2.6 autophosphorylation site26. Supporting this notion, recombinant SnRK2.6 intermolecularly trans-phosphorylated SnRK2.6KR (Fig. 1b), though the phosphorylation was much weaker than the transphosphorylation by RAF3 and RAF10. Thus, SnRK2.6 can be either transphosphorylated by RAFs or transphosphorylated intermolecularly by other SnRK2.6 molecules.

      To further evaluate the role of RAFs in SnRK2.6 activation, we designed an adenosine triphosphate (ATP) analog-based in vitro kinase assay system that distinguishes trans- and autophosphorylation of SnRK2.6 (Fig. 1c). A Met94Gly (M94G) mutation in SnRK2.6 enlarges its ATP binding pocket (Fig. 1c). SnRK2.6M94G can use the ATP analog N6-Benzyl-ATPγS to thiophosphorylate itself or its substrate (Supplementary Fig. 1b). Neither RAF3-KD nor wild-type SnRK2.6 can use the N6-Benzyl-ATPγS as a thiophosphate donor (Fig. 1d, Supplementary Fig. 1b). By this method, thiophosphorylation by activated SnRK2.6M94G can be detected with an anti-thiophosphate ester antibody that only recognizes the thiophosphorylation (Supplementary Fig. 1b). Pre-dephosphorylated SnRK2.6M94G (de-SnRK2.6M94G) had no auto-thiophosphorylation activity (Fig. 1d, lanes 2–6, Fig. 1e, lanes 4–5). Application of recombinant RAF3-KD or RAF10-KD quickly induced the auto-thiophosphorylation of SnRK2.6M94G in a time-dependent manner (Fig. 1d, lanes 7–14), suggesting that transphosphorylation by RAF3 and RAF10 is essential for SnRK2.6M94G auto-thiophosphorylation activity.

      We then examined SnRK2.6M94G activity by detecting the thiophosphorylation of ABA‐RESPONSIVE ELEMENT‐BINDING FACTOR 2 (ABF2), a well-studied SnRK2 substrate. Adding RAF3-KD initiated the thiophosphorylation of both SnRK2.6M94G and ABF2 (Fig. 1e, lanes 10–12). Preincubation with RAF3 significantly enhanced the kinase activity of recombinant SnRK2.6M94G (Fig. 1e, lanes 10–12 compared to lanes 2–3). We also measured the activation effect of RAF3 and RAF10 on SnRK2.2M96G and SnRK2.3M95G, the mutated forms that can use the N6-Benzyl-ATPγS. Similar to previous study26, the recombinant SnRK2.2M96G and SnRK2.3M95G only had weak kinase activity that was rarely detectable in the thiophosphorylation assay (Fig. 1f, lanes 1, 2, 7, and 8). However, adding either RAF10-KD or RAF3-KD strongly enhanced the kinase activities of SnRK2.2M96G and SnRK2.3M95G, in the context of ABF2 and SnRK2 thiophosphorylation (Fig. 1f, lanes 4, 6 compared to lanes 2, and lanes 10, 12 compared to lane 8). Taking these results together, transphosphorylation by RAFs is required for the reactivation of SnRK2.2/2.3/2.6. The enhanced thiophosphorylation of SnRK2.6M94G, SnRK2.2M96G, and SnRK2.3M95G on themselves suggested that activated SnRK2s can quickly intermolecularly transphosphorylate and activate other SnRK2 molecules in vitro.

      Intermolecular transphosphorylation amplifies SnRK2 activation

      To further validate this amplification process, we measured the phosphorylation of SnRK2.6 on SnRK2.6KR by in vitro kinase assay. GST-SnRK2.6 showed weak phosphorylation of HIS-SnRK2.6KR (Fig. 2a). After preincubating with RAF10-KD and ATP for 30 min, and removing RAF10-KD after preincubation, the activated GST-SnRK2.6 (SnRK2.6*) then showed an enhanced ability to phosphorylate HIS-SnRK2.6KR (Fig. 2a, lane 6). The Ser171Ala or the Ser175Ala mutation significantly impaired, and Ser171AlaSer175Ala double mutation completely abolished, the HIS-SnRK2.6KR phosphorylation by pre-activated GST-SnRK2.6 (Fig. 2b). This result suggests that SnRK2.6 mainly transphosphorylates SnRK2.6 at Ser171 and Ser175 residues, the same major phosphosites of B2 and B3 RAFs.

      a Recombinant SnRK2.6 and pre-activated SnRK2.6 (SnRK2.6*) phosphorylate HIS-SnRK2.6KR. Aliquot of SnRK2.6 was pre-incubated with HIS-SUMO-RAF10-KD coated on Ni-NTA beads for 30 min. After removal of HIS-SUMO-RAF10-KD by centrifugation, pre-activated SnRK2.6 (SnRK2.6*) was used to phosphorylate HIS-SnRK2.6KR, in the presence of [γ-32p]ATP. Same amount of SnRK2.6 without pre-activation was used as control (lanes 1–3). Lane 4 indicates no remaining RAF10-KD after removal. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6 and HIS-SnRK2.6KR. b Pre-activated SnRK2.6 (SnRK2.6*) phosphorylates SnRK2.6KR, SnRK2.6KR-S171A, SnRK2.6KR-S175A, or SnRK2.6KR-AA, in the presence of [γ-32p]ATP. Autoradiograph (left) and Coomassie staining (right) show phosphorylation and loading, respectively, of purified GST-SnRK2.6 and HIS-SnRK2.6KR proteins. c Transphosphorylation activity of pre-activated SnRK2.6M94G, SnRK2.6M94G-S171A, SnRK2.6M94G-S175A, and SnRK2.6M94G-AA on SnRK2.6KR. d Effect of recombinant SnRK2.6 and pre-activated SnRK2.6 (SnRK2.6*) on SnRK2.6M94G activity. For c to d, Anti-γ-S immunoblot (left) and Coomassie staining (right) show thiophosphorylation and loading, respectively, of recombinant GST-SnRK2.6M94G, HIS -SnRK2.6KR, and GST-ABF2. Arrow indicates partial degraded band of GST-SnRK2.6M94G. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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      We further confirmed the trans- and autophosphorylation of SnRK2.6 by mass spectrometry analysis after in vitro kinase reaction. In such a reaction, γ-[18O]-ATP was used as the phosphate donor of RAF3 to trans-phosphorylate SnRK2.6M94G, and Benzyl-ATPγS was used as the thiophosphate donor for SnRK2.6M94G autophosphorylation. After 30 min of incubation, the reaction was subjected to mass spectrometry analysis. The result confirmed that Ser171 and Ser175 are both intermolecular auto-phosphosites (thio-phosphosites) and trans-phosphosites (18O-phosphosites in Supplementary Data 1). Beside Ser171 and Ser175, six other residues, Ser29, Ser43, Ser71, Thr176, Thr179, Try182, in SnRK2.6M94G were both intermolecular auto-phosphosites and trans-phosphosites (Supplementary Data 1). We then generated non-phosphorylatable mutations (Ser/Thr/Try to Ala) on these phosphosites in SnRK2.6 to elucidate their contributions to SnRK2 intermolecularly trans-phosphorylation activity. Mutating either Ser175 or Ser171 completely abolished the ability of SnRK2.6M94G to thiophosphorylate SnRK2.6KR (Fig. 2c), while Thr179 and Try182 may also contribute to the intermolecularly trans-phosphorylation activity of SnRK2.6 (Supplementary Fig. 2a).

      We then further verified the ability of pre-activated SnRK2.6 to activate pre-dephosphorylated SnRK2.6M94G. We activated GST-SnRK2.6 by incubating it with RAF3-KD and RAF10-KD. After removing RAF3/10-KD, we used the pre-activated SnRK2.6 to phosphorylate SnRK2.6M94G. The kinase activity of SnRK2.6M94G was determined by thiophosphorylation of SnRK2.6M94G and ABF2. Incubating with pre-activated SnRK2.6 (SnRK2.6*), but not the inactive SnRK2.6, enhanced the kinase activity of SnRK2.6M94G (Fig. 2d). Pre-activated SnRK2.6 was also able to transphosphorylate SnRK2.2 and SnRK2.3 in the in vitro kinase assay (Supplementary Fig. 2b). Thus, after activation by RAF kinases, SnRK2.6 can intermolecularly autophosphorylate and activate other SnRK2s to amplify the response.

      B2 and B3 RAFs function redundantly in ABA response in vivo

      To validate the RAF-SnRK2 cascade in ABA signaling in planta, we used gene-editing technology to introduce mutations in B2 or B3 subgroup RAF genes in Arabidopsis Col-0 wild type (Fig. 3a, b). Genotyping thousands of transgenic seeds identified two high-order mutants. OK100-B3 [raf2/enhanced disease resistance 1(edr1);raf3;raf4; raf5/sugar insensitive 8 (sis8);raf6] contained null mutations in five of six B3 subgroup RAFs. raf1/constitutive triple response 1 (ctr1) was not included in the mutant due to a severe morphological phenotype35. OK100-B2 (raf7;raf8;raf9;raf10;raf11;raf12) contained null mutations in all six B2 subgroup RAF genes (Fig. 3a, b, Supplementary Fig. 3a, b). OK100-B3 had a low germination rate on half Murashige and Skoog (MS) medium without sucrose and had strong arrested growth even under normal conditions (Fig. 3c, Supplementary Fig. 4a, b). No ABA insensitivity in germination and seedling development in OK100-B3 was observed on half MS medium without sugar (Fig. 3c, upper panel, d, Supplementary Fig. 4a, b). Interestingly, addition of 1% sucrose strongly improved the germination and seedling development of the OK100-B3 mutant, which showed strong ABA insensitivity and geminated on medium containing up to 5 µM ABA (Fig. 3c, e, Supplementary Fig. 4c, d). OK100-B2 was hyposensitive to ABA and germinated on medium containing up to 10 µM ABA, with or without sucrose (Fig. 3c–e, Supplementary Fig. 4a–d). The OK130-null mutant [raf16;raf40/hydraulic conductivity of root 1(hcr1);raf24;raf18;raf35;raf42] did not show insensitivity to ABA (Fig. 3c–e, Supplementary Fig. 4a–d). OK100-B3 and OK100-B2 also showed impaired seed dormancy and fresh harvested OK100-B3 and OK100-B2 seeds had higher germination rates than fresh harvested wild-type seeds (Supplementary Fig. 4e). To measure the phosphorylation and activation of SnRK2s, we used the phosphorylation-specific antibodies recognizing the phosphoserines corresponding to Ser175 and Ser171 in SnRK2.6 (Supplementary Fig. 5 and Supplementary Data 2). ABA-induced phosphorylation of conserved serine residues corresponding to Ser171 and Ser175 in SnRK2.6 was markedly reduced in the OK100-B2, and relatively not affected in the OK100-B3 and OK130-null mutants (Fig. 3f). Thus, B2 and B3 RAFs may have partially redundant roles in ABA responses in plants.

      a Mutations of RAF genes in the high-order mutants used in this study. b Photographs of seedlings after 4 weeks of growth in the soil. Bar = 1 cm. c Photographs of seedlings after 10 days of germination and growth on 1/2 MS medium containing indicated concentrations of ABA, without (upper panel) or with (bottom panel) 1% sucrose. The position of mutants in the image is shown in the box at the upper-right corner. d Photographs of seedlings after 10 days of germination and growth on 1/2 MS medium containing indicated concentrations of ABA without sucrose. e Photographs of seedlings after 10 days of germination and growth on 1/2 MS medium containing indicated concentrations of ABA with 1% sucrose. f The ABA-induced phosphorylation of Ser171 and Ser175 of SnRK2.6 in wild-type and the OK100-B2 and OK100-B3 mutants. The anti-pS171 and anti-pS175 immunoblots were used to show the phosphorylation of the conserved serine residues. Arrows indicate the non-specific bands recognized by anti-pS171 and anti-pS175 antibodies. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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      B2 and B3 RAFs cooperate in ABA-induced SnRK2 activation

      We also generated OK100-oct (raf3;raf4;raf5/sis8;raf7;raf8;raf9;raf10;raf11) and OK100-nonu (raf3;raf4;raf5/sis8;raf6;raf7;raf8;raf9;raf10;raf11) mutants containing null mutations in eight and nine RAF genes, respectively, of the B2 and B3 subgroups (Fig. 3a, Supplementary Fig. 6). Growth analysis showed that the OK100-oct and OK100-nonu mutants resembled the previously characterized snrk2-triple mutant and the high-order mutants of PYR/PYL/RCARs exhibiting a strong defect in growth and development (Fig. 4a, Supplementary Fig. 7a–c). By contrast, the OK100-quin mutant, which contains mutations in only two B2 and three B3 RAF genes, displayed a wild-type-like growth phenotype (Fig. 4a). The OK100-nonu mutant produced very few seeds and had a lower seed germination rate (Supplementary Fig. 7b, c). The OK100-oct and OK100-nonu mutants showed higher water loss than the wild type (Fig. 4b). Furthermore, ABA-induced stomatal closure was strongly impaired in the OK100-oct and OK100-nonu mutants, which resembles the snrk2-triple and pyl112458 mutants (Fig. 4c). These results suggested an essential role of the B2 and B3 subgroup RAFs in ABA signaling.

      a Photographs of wild-type and mutant seedlings after 4 weeks of growth in the soil. Bar = 1 cm. b Water loss of 4-week-old wild-type and high-order mutants. Error bars, SEM (n = 5 biological replicates, each replicate has 5 or 6 individual seedlings). c Stomatal closure of 4-week-old wild type and mutants in response to ABA. Error bars, SEM (n = 59 or 60 individual stomates). d Photographs of seedlings after 10 days germination and growth on 1/2 MS medium containing different concentrations of ABA. The position of mutants in the image is shown in the gray box at the bottom-right corner. e Photographs of seedlings growing 10 days after transfer to 1/2 MS medium with or without 50 µM ABA. Bar = 1 cm. f In-gel kinase assay showing the activation of SnRK2s and RAFs in wild-type and different mutants with or without 15 min of ABA or mannitol treatment. Arrows indicate the OK100 band observed in Col-0, snrk2-triple, and pyl112458 mutants. g The ABA-induced phosphorylation of Ser171 and Ser175 of SnRK2.6 in wild-type and the OK100 high-order mutants. Arrows indicate the non-specific bands recognized by anti-pS171 and anti-pS175 antibodies. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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      We further evaluated the role of B2 and B3 subgroup RAFs in ABA signaling by assaying seed germination in response to ABA. All tested mutants carrying high-order mutations in B2 and B3 subgroup RAFs, including OK100-oct and OK100-nonu, showed insensitivity to ABA in seed germination and post-germination seedling growth (Fig. 4d, e, Supplementary Fig. 7d–h). The order of insensitivity to ABA in seed germination was OK100-quin < OK100-oct = OK-quatdec < OK100-nonu < snrk2-triple, pyl112458, and pyl-duodec (Fig. 4d, Supplementary Fig. 7d). Higher-order RAF mutants were clearly more insensitive than lower-order RAF mutants to ABA in seed germination, which further suggests that the RAF members in the B2 and B3 subgroups have essential and partially redundant roles in ABA signaling. OK100-oct and OK100-nonu also exhibited hyposensitivity to ABA in seedling growth as indicated by root growth, fresh weight measurements, and leaf yellowing (Supplementary Fig. 7g–j).

      We then measured ABA-induced SnRK2.2/2.3/2.6 activation in these mutants by in-gel kinase assay. The ABA-induced SnRK2.2/2.3/2.6 activation was almost completely abolished in the OK100-oct and OK100-nonu mutants, which resembles that in the snrk2-triple and pyl112458 mutants (Fig. 4f). Interestingly, we observed a weak OK100 band (indicated by arrows) in wild type, snrk2-triple and pyl112458 even without ABA or mannitol treatment, and this band was not induced by ABA, when compared to the strong induction by mannitol treatment (Fig. 4f, rightmost lane). Consistently, the immunoblot result showed that the ABA-induced phosphorylation of conserved serine residues corresponding to Ser171 and Ser175 in SnRK2.6 was markedly reduced in the OK100-oct and OK100-nonu mutants (Fig. 4g). Interestingly, perhaps due to the abolishment of ABA signaling, the ABA-induced rapid SnRK2 degradation was also abolished in the OK100-oct and OK100-nonu mutants (Fig. 4g). These results strongly indicate that the B2 and B3 subgroup RAFs are essential for ABA-induced SnRK2.2/2.3/2.6 activation.

      To evaluate which members of the B2 and B3 subgroups have predominant roles in ABA-regulated seed germination and seedling establishment, we backcrossed OK100-nonu with Col-0 wild type and screened F2 populations on 1/2 MS medium containing 10 µM ABA and sucrose. By genotyping 103 individual F2 seedlings with strong ABA insensitivity, we found that each RAF might contribute to ABA hyposensitivity (Chi-square test, p < 0.05). RAF3, RAF4, RAF5, and RAF7-9 (closed linked together) might have predominant roles (p < 0.0001) in ABA-regulated germination and seedling establishment (Supplementary Table 1).

      B2 and B3 RAFs are required for ABA-induced gene expression

      To investigate the impact of RAF null mutations on ABA-induced gene expression, we performed transcriptomics analysis in WT and OK100-oct seedlings. We identified 1368 ABA-induced (>= 3-fold, p < 0.05) and 1256 ABA-repressed (>= 3-fold, p < 0.05) genes in the wild type. Among these differentially expressed genes (DEGs), only 674 genes were significantly induced (>= 3-fold, p < 0.05) and 399 genes were significantly repressed (>= 3-fold, p < 0.05) by ABA in the OK100-oct seedlings (Fig. 5a, Supplementary Data 3). The heatmap indicated that most ABA-induced and -repressed genes in wild type were less responsive in the OK100-oct mutant (Fig. 5b). Quantitative RT-PCR analysis of several ABA-inducible genes, including RESPONSIVE TO ABA 18 (RAB18), COLD-REGULATED 15A (COR15A), KINASE 1 (KIN1), and RESPONSIVE TO DESICCATION 29B (RD29B), also showed that the induction of these genes by ABA was dramatically impaired in both the OK100-oct and OK100-nonu mutants (Fig. 5c).

      a Venn diagrams showing the overlaps of ABA-induced and ABA-repressed genes in the wild type and OK100-oct seedlings. b Heat map showing the expression levels of ABA-responsive genes in wild type and OK100-oct seedlings. c Expression of the ABA-inducible marker genes in wild type, OK100-oct, and OK100-nonu seedlings after 6 h of ABA treatment. Error bars, SEM (n = 3 biological replicates). Two-tailed paired t-tests, *p < 0.05, **p < 0.01, ***p < 0.001. d The activation assay of the ABA-responsive RD29B-LUC reporter gene in wild type and mutants. The protoplasts were transformed with the reporter plasmid and incubated with or without 5 µM ABA for 5 h under light. Error bars, SEM (n = 3 individual transfections). e Add-back assay testing RAF1 to RAF12 in activating the reporter gene in the protoplasts of OK100-oct. Error bars, SEM (n = 4 individual transfections). f Activation of the reporter gene by the combinations of RAFs with SnRK2.2, SnRK2.3, or SnRK2.6 in the protoplasts of OK100-oct. The ratio of RD29B-LUC expression in the protoplasts with 5 µM ABA relative to that without ABA treatment was used to indicate the activation activity of RAF-SnRK2 pairs. Error bars, SEM (n = 4 individual transfections). g Activation of the reporter gene by RAF3 or RAF7 in the protoplasts of wild type, pyl112458, or snrk2-triple. Error bars, SEM (n = 3 individual transfections). Source data are provided as Source Data files.

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      Among the DEGs between wild type and the mutant, 67 genes showed significantly higher expression (>= 3-fold, p < 0.05) in the OK100-oct mutant under control conditions. Gene Ontology (GO) analysis indicated that these genes are enriched in plant response to chitin, fungus, bacterium, and oxidative stress, suggesting a potential role of the B2 and B3 RAFs in these biotic stress responses (Supplementary Data 4).

      To further evaluate the role of B2 and B3 RAFs in ABA-induced gene expression, we used transient activation assays. We used the LUCIFERASE (LUC) reporter gene driven by the ABA-responsive RD29B promoter as an indicator of ABA response8. In the wild type, ABA clearly induced the expression of RD29B-LUC in the mesophyll cell protoplasts, while ABA-induced RD29B-LUC expression was completely abolished in the protoplasts of OK100-oct, snrk2-triple, pyl112458, and abi1-1 mutants (Fig. 5d). Co-expression of RAF3, RAF5, or RAF11 fully rescued the ABA-induced expression of RD29B-LUC, and co-expression of RAF4, RAF6, RAF7, RAF9, or RAF10 partially rescued the ABA-induced expression of RD29B-LUC in the protoplasts of OK100-oct mutant (Fig. 5e). No rescue was seen with co-expression of RAF1, RAF2, RAF8, or RAF12 (Fig. 5e).

      We then co-transfected SnRK2.2, SnRK2.3, or SnRK2.6 in the transient activation assays with each of RAF1 to RAF12 to evaluate the specificity of different RAFs for different SnRK2 activation (Fig. 5f). Protoplasts co-expressing RAF4, RAF5, RAF6, or RAF9 with SnRK2.2 had higher ABA-induced RD29B-LUC expression, while the combination of RAF3, RAF7, RAF8, or RAF10 with SnRK2.6 showed higher ABA-induced RD29B-LUC expression in the OK100-oct protoplasts. These results suggest that different RAFs exhibit activation specificity for SnRK2.2, SnRK2.3, or SnRK2.6. In addition, RAF3 and RAF7 could not rescue ABA-induced RD29B-LUC expression in the pyl112458 protoplasts (Fig. 5g), which suggests that activation of SnRK2s by RAFs requires ABA-induced release of SnRK2s from PP2C inhibition.

      ABA does not activate B2 and B3 RAFs in plants

      Unlike the strong activation of RAFs by hyperosmolarity, the kinase activity of RAFs was not enhanced by ABA treatment (Fig. 4f). Consistently, the phosphorylation of pSTAGTPEWMAPEVLR, a conserved peptide located in the activation loop of RAF2/EDR1 and RAF3, was not affected by ABA treatment but highly induced by osmotic stress caused by mannitol treatment (Fig. 6a, b, Supplementary Fig. 8a). Multiple phosphosites in this region also could be detected without ABA treatment27,36 (Fig. 6b, Supplementary Fig. 8a, b, highlighted in Supplementary Data 5). SnRK2.6 showed clearly induced phosphorylation in the peptide containing the phosphorylation site Ser175 by both ABA and mannitol treatments (Supplementary Fig. 8c). We further tested whether RAF3 phosphorylation is required for its activity on SnRK2.6 by generating a non-phosphorylatable mutation of RAF3. Co-transfection of RAF3S763AS766AT770A, with Ser to Ala substitutions in the activation loop, did not rescue the ABA-induced RD29B-LUC expression in the protoplasts of OK100-oct (Fig. 6c). RAF3S763AS766AT770A completely lost its ability to phosphorylate SnRK2.6 in vitro (Fig. 6d, left panel). However, mutating these conserved residues of RAF10, producing RAF10T706AT709AT713A, hardly affected RAF10 activity in in vitro kinase or transient expression assays (Fig. 6d, right panel, Supplementary Fig. 8d). Therefore, the activation mechanism of RAF10 in the B2 RAF subgroup may differ from that of RAF3 in the B3 RAF subgroup. Taken together, these results indicate that B2 and B3 RAFs have basal levels of phosphorylation and activity under normal conditions and that application of ABA does not increase their phosphorylation.

      a The phosphorylation of the conserved phosphosite in RAF2 and RAF3 showing enhanced phosphorylation by mannitol but not ABA treatment. The relative intensity of the phosphopeptide was obtained from previous phosphoproteomics results (n = 3 biological replicates). b Sequence alignment showing the conserved phosphosites (indicated by arrows) in the activation loop of Arabidopsis B2, B3, B4 RAFs, and PpARK/PpCTR1 from Physcomitrella patens. The conserved serine residues corresponding to Ser1029 in PpARK/PpCTR1 are highlighted by the red arrow. c Activation of the reporter gene by wild type and the non-phosphorylatable mutants of Ser763, Ser766, and Thr770 in RAF3 in transient reporter gene expression in protoplasts of OK100-oct. RAF3K636R (RAF3KR), a kinase-dead form of RAF3, is used as a control. Error bars, SEM (n = 3 individual transfections). d Phosphorylation of SnRK2.6KR by recombinant kinase domain of RAF3 and RAF10. Wild type and mutated recombinant GST-RAF3-KD (left panel) and GST-RAF10-KD (right panel) was used to determine the phosphorylation of SnRK2.6KR expressed and purified from E. coli in the presence of [γ-32P]ATP. GST-RAF3K636R (RAF3KR) and GST-RAF10K515R (RAF10KR) were used as negative controls. Autoradiograph (upper) and Coomassie staining (lower) show phosphorylation and loading, respectively, of purified GST-RAF3-KD, GST-RAF10-KD, and HIS-SnRK2.6KR. Images shown are representative of at least two independent experiments. Source data are provided in Source Data.

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      Discussion

      Subgroup B RAFs belong to the MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE (MAPKKK) family due to their similarity with animal B-Raf protein kinases37,38,39. In a canonical MAPK cascade, MAPKKKs are activated by extracellular signals and phosphorylate and activate MAPK KINASEs (MAPKKs), which then phosphorylate and activate MAPKs to regulate various cellular processes. Instead of phosphorylating MAPKKs, after rapid activation by osmotic stresses, B2, B3, and B4 RAFs phosphorylate and activate SnRK2s27,28,29,30,31. Plants use this noncanonical RAF-SnRK2 cascade to relay early osmotic stress signaling. Here we show a unique initiation-amplification mechanism of RAF-SnRK2 in ABA signaling to ensure rapid activation of SnRK2s with a basal level of Raf kinase activity (Fig. 7). Unlike the B4 subgroup RAFs, which are quickly activated by hyperosmolality27, the application of exogenous ABA does not enhance the phosphorylation or activity of B2 and B3 subgroup RAFs, as indicated by in-gel kinase assays and phosphoproteomics (Figs. 4 and 6). The phosphoproteomics and the in-gel kinase assay result also revealed the existence of basal-level phosphorylation and activity of B2 and B3 RAFs even without ABA (Figs. 4f and 6a, Supplementary Data 5). This basal level activation of RAFs might be necessary and sufficient for maintaining the SnRK2 activity and ABA signaling required for normal growth and development6. The upstream kinases or other mechanisms for this basal activity of RAFs need to be determined in the future. In the presence of ABA, the ABA and PYR1/PYL/RCAR complex releases SnRK2s from PP2C-mediated inhibition, resulting in the accumulation of uninhibited forms of SnRK2s. The RAFs quickly trans-phosphorylate uninhibited SnRK2s to initiate SnRK2 activation. The activated SnRK2s then intermolecularly transphosphorylate and activate other SnRK2 molecules not yet activated by RAFs, to amplify the ABA signaling (Fig. 7). By this activation-amplification mechanism, the basal level activity of RAF kinases is sufficient to quickly activate SnRK2s to transduce the ABA signal. It would be interesting to apply the ATP analog-based method to evaluate whether this activation-amplification mechanism also exists in other kinase cascades in plants or animal cells, as autophosphorylation is a general feature of many protein kinases.

      Under unstressed conditions, PP2C binds to and inhibits SnRK2 protein to prevent the transphosphorylation by activated RAFs (left panel). In the presence of ABA, ABA receptor PYR/PYL/RCARs (PYL) complex binds to and inhibits PP2C and SnRK2 is released from PP2C-mediated inhibition. SnRK2 can then be quickly activated by RAFs. In the meantime, stress also activates RAFs by an unknown mechanism (middle panel). The activated SnRK2 can quickly trans-phosphorylate more SnRK2 proteins to amplify the activation and phosphorylate downstream substrates to mediate stress responses (right panel).

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      Several B3 subgroup Raf-like kinases, M3Kδ6/SIS8/RAF5, M3Kδ7/RAF4, M3Kδ1/RAF3, and RAF6, phosphorylate SnRK2.6, and are essential for ABA-induced SnRK2 activation27,28. In this study, we show that the B2 subgroup RAFs, together with B3 subgroup, have an essential role in the ABA core signaling pathway. OK100-nonu and OK100-oct show strong ABA-insensitivity in germination, leaf yellowing, and stomatal closure. The OK100-nonu seeds even germinate at an ABA concentration of up to 25 µM (Fig. 4). To our knowledge, OK100-oct and OK100-nonu are among the few mutants, including snrk2-triple, pyl112458 and pyl-duodec, that can germinate on such an extremely high concentration of ABA, further supporting the critical role of the B2 and B3 RAFs in ABA responses. However, ABA-insensitivity of OK100-nonu is still not identical with that of snrk2-triple and pyl high-order mutants. This suggests that, besides B2 and B3 RAFs, additional protein kinases also participate in ABA-induced SnRK2 activation. At least two protein kinases, BRASSINOSTEROID-INSENSITIVE2 (BIN2) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), can phosphorylate SnRK2s and are involved in ABA signaling40,41. BIN2 likely phosphorylates only SnRK2.2 and SnRK2.3, but not SnRK2.641, at the conserved threonine corresponding to Thr179 in SnRK2.6 (Supplementary Fig. 2). Thus, whether BIN2, or other members of the GSK family, cooperate with RAFs in amplifying ABA-triggered SnRK2 activation, needs to be further studied.

      Although both B2 and B3 RAFs are essential for ABA-induced SnRK2 activation, they might have distinct roles in some ABA-regulated biological processes. OK100-B3, but not OK100-B2, has arrested growth in soil, suggesting a unique role of B3 subgroup RAFs in growth regulation (Fig. 3). OK100-B2 shows similar ABA-insensitivity on medium with or without sucrose, whereas OK100-B3 only shows strong ABA-insensitivity with exogenous sucrose (Fig. 3). Supporting this notion, the miRNA-M3K was screened from the medium containing sucrose28,42. RAF3-5 and RAF7-9 might have more dominant roles in germination and seedling establishment, while RAF3, RAF5, RAF7, and RAF11 rescue the ABA-induced RD29B-LUC expression more robustly. Several key regulators in ABA signaling and synthesis are also involved in sugar responses43,44. Mutants raf1/ctr1/sis1, and raf5/sis8, are resistant to high concentrations of sugar45,46. Together with these findings, our results suggest a crucial role of B3 subgroup RAFs in sucrose signaling and/or in seed development (e.g., in the accumulation of energy reserve in the seeds). It is notable that the role of RAFs in rescuing ABA-induced RD29B-LUC transcription is not identical to their contribution in gemination and seedling establishment, further indicating that different RAFs have various contributions to different ABA-mediated biological processes. Such functional diversity is also observed in the 14 PYR1/PYL/RCAR ABA receptors. Only pyl112458, but not 3791112 (pyl3/7/9/11/12) shows arrested growth under normal condition33. By contrast, pyl112458 has more predominant roles in ABA-mediated regulation of germination, stomatal movement, etc.33. In guard cell, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential47. PYL8 directly binds to the transcription factor MYB77 to regulate auxin responsive gene expression48. PYR1 especially participates in cross-talk between salicylic acid and ethylene, thereby redirecting defense disease resistance towards fungal Plectosphaerella cucumerina49. In Arabidopsis, 14 PYLs, at least 8 PP2Cs, three SnRK2s, and 12 members of the B2 and B3 RAF subgroups comprise a complex network in ABA sensing and signaling, which may ensure that plants precisely respond to ever-changing environments. The engineering of ABA receptors is an efficient way to improve stress resistance in both Arabidopsis and crops50,51,52. Our findings regarding B2 and B3 RAFs in stress signaling provide additional targets (e.g., ectopic expression of stress-inducible or constitutively activated forms of RAFs in guard cells) for engineering crops resistant to harsh environmental conditions.

      Besides involvement in sugar and ABA signaling, CTR1/RAF1 is a crucial component in ethylene signaling. We excluded RAF1/CTR1 from the OK100 high-order mutants because the ctr1 mutant displays severe growth inhibition under normal conditions35. However, although the KD of RAF1/CTR1 strongly phosphorylates SnRK2.6 in vitro, neither the full-length RAF1/CTR1 nor RAF1/CTR1-KD rescued the ABA-induced expression of RD29B-LUC in the protoplasts of OK100-oct (Supplementary Fig. 8e). Thus, additional mechanisms may determine RAF1 specificity in vivo. Similarly, RAF2, RAF8, and RAF12 only show weak activities on the induction of RD29B-LUC expression in the protoplasts. The roles of these RAFs in ABA signaling therefore need to be further investigated.

      The phosphorylation of Ser1029 of the ABA AND ABIOTIC STRESS-RESPONSIVE RAF-LIKE KINASE (PpARK)/PpCTR1 in Physcomitrella patens is induced by exogenous ABA in P. patens53,54,55, which is inconsistent with our observations on RAF3 and RAF10 (Fig. 6). Therefore, P. patens and higher plants may adopt different machinery to relay ABA signaling. In addition, non-phosphorylatable mutations at Ser1029 in PpARK/PpCTR1, or Ser763Ser766AThr770 in RAF3, abolished their kinase activities, suggesting phosphorylation-dependent activation of PpARK/PpCTR1 and RAF3. By contrast, the activation of RAF10 might be independent of phosphorylation. In animal cells, RAF kinases can be activated through phosphorylation, dimerization, or by binding of small GTPases, scaffold protein, 14-3-3 proteins, etc.56,57,58. Future work will investigate the phosphorylation or other activation mechanisms of RAF-SnRK2 cascades in different plant species and their roles in plant adaptive plasticity.

      Methods

      Seed germination and plant growth assay

      Seeds were surface-sterilized in 70% ethanol for 10 min, followed by four times washing with sterile-deionized water. For the germination assay, seeds were sown on 1/2 Murashige and Skoog (MS) medium (0.75% agar, pH 5.7) with or without the indicated concentrations of ABA and 1% sucrose. Plates were kept at 4 °C for 3 days in darkness for stratification and then shifted to a plant growth chamber set at 23 °C and a 16 h light/8 h dark photoperiod. After 72 h of transfer, radical emergence was examined, and photographs of seedlings were taken at the times indicated. For growth assays, seeds were placed on 1/2 MS medium (0.75% agar, pH 5.7) and plates were placed vertically in a plant growth chamber after 3 days of stratification. After 3–4 days, the seedlings were transferred to medium with or without the indicated concentrations of ABA. Root length and fresh weight were measured at the indicated days. For seed dormancy assays, fresh seeds were harvested and sown on 1/2 MS medium (0.75% agar, pH 5.7) and plates were placed in a plant growth chamber. Radical emergence was measured 48 h after transferring.

      Generation of OK100 high-order mutants

      The clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9) and guide RNA fragment from pCAMBIA-2300-11RAFs27 was cloned into pCAMBIA-1300. The resulting vectors containing sgRNAs targeting B2, B3, or B2/B3 RAFs were used to transform wild type to generate OK100-B2, OK100-B3, OK100-oct, and OK100-nonu. The transgenic plants were screened for hygromycin resistance. The T1 transformants were identified by sequencing the fragments with the RAF target regions, which were amplified by PCR using primer pairs listed in Supplementary Data 6.

      In-gel kinase assay

      For in-gel kinase assays, 20 µg extract of total proteins were electrophoresed on 10% SDS/PAGE embedded with histone in the separating gel as a substrate for kinase. The gel was then washed three times at room temperature for 30 min each with washing buffer (25 mM Tris-Cl, pH 7.5, 0.5 mM Dithiothreitol (DTT), 0.1 mM Na3VO4, 5 mM NaF, 0.5 mg/mL BSA, and 0.1% Triton X-100). The kinase was allowed to renature in renaturing buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.1 mM Na3VO4, and 5 mM NaF) and incubated at 4 °C overnight with three changes of renaturing buffer. The gel was further incubated at room temperature in 30 mL reaction buffer (25 mM Tris-Cl, pH 7.5, 2 mM EGTA, 12 mM MgCl2, 1 mM DTT, and 0.1 mM Na3VO4) with 200 nM ATP plus 50 µCi of [γ-32P]ATP for 90 min. The reaction was stopped by transferring the gel into 5% (w/v) trichloroacetic acid and 1% (w/v) sodium pyrophosphate. The gel was then washed to remove unincorporated [γ-32P]ATP in the same solution for at least 5 h with five changes. Radioactivity was detected with a Personal Molecular Imager (Bio-Rad).

      RNA sequencing and data analysis

      Total RNA was isolated from two-week-old seedlings of Col-0 and OK100-oct mutant, with and without ABA treatment, using RNeasy Plant Mini Kit (Qiagen). Total RNA (1 µg) was used for library preparation with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, E7765) following the manufacturer’s instructions. Prepared libraries were assessed for fragment size using NGS High-Sensitivity kit on a Fragment Analyzer (AATI), and for quantity using Qubit 2.0 fluorometer (Thermo Fisher Scientific) and KAPA Library Quantification Kit (Kapa, KK4824). All libraries were sequenced in paired-end 150 bases protocol (PE150) on an Illumina Nova sequencer.

      The paired-end reads were cleaned by Trimmomatic59 (version 0.39). After trimming the adapter sequence, removing low quality bases, and filtering short reads, clear read pairs were retained for further analysis. The Arabidopsis thaliana reference genome sequence was downloaded from TAIR10. Clean reads were mapped to the genome sequence by HISAT (2.1.0)60 with default parameters. Number of reads that were mapped to each gene was calculated with the htseq-count script in HTSeq (0.11.2)61. EdgeR62 was used to identify genes that were differentially expressed. Genes with at least three-fold change in expression and with an FDR < 0.05 were considered differentially expressed genes (DEGs).

      Analysis of gene expression by qRT-PCR

      Total RNA was extracted from two-week-old wild-type, OK100-oct, andOK100-nonu seedlings with or without 50 µM ABA treatment for 6 h. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. Genomic DNA was removed using RNase-free DNase and subsequently, 1 µg of total RNA was reverse transcribed using the iScriptTM gDNA Clear cDNA Synthesis Kit (Bio-Rad) following the manufacturer’s instructions. The actin gene was used as an internal control. Quantification was performed using three independent biological replicates.

      Water loss measurement

      The water loss was estimated on detached rosette leaves of 4-week-old plants by weighing using a weighing dish. Leaves were then kept on the laboratory bench for at least 30 min. Fresh weight was monitored before and after the procedure and at the times indicated. Water loss was expressed as a percentage of initial fresh weight.

      Stomatal bioassay

      For stomatal aperture assay, rosette leaves of 4-week-old Arabidopsis seedlings were taken. Epidermal strips were peeled out and incubated in buffer containing 50 mM KCl, 10 mM MES, pH 6.15, in a plant growth chamber for 3 h before ABA treatment. Stomatal apertures were measured 2 h after the addition of 5 μM ABA. The apertures of about 60 stomata per sample were measured by quantifying the pore width of stomata using Image J software (1.51 K). All the experiments were repeated at least three times.

      Protein purification and in vitro kinase assay

      For in vitro kinase assays, full-length coding sequence of SnRK2.6 and kinase domains of RAFs were cloned into either pGEX-4T-1, pET28a or pET-SUMO vectors and transformed into BL21 or ArecticExpression cells. The recombinant proteins were expressed and purified using standard protocols. For the phosphorylation assay, recombinant kinase domains of RAFs (aa541-821 for RAF1-KD, aa650-933 for RAF2-KD, aa600-880 for RAF3-KD, aa710-992 for RAF4-KD, aa733-1030 for RAF5-KD, aa645-956 for RAF6-KD, aa470-773 for RAF7-KD, aa424-671 for RAF8-KD, aa436-730 for RAF9-KD, aa466-767 for RAF10-KD, aa472-765 for RAF11-KD, aa457-735 for RAF12-KD) were incubated with “kinase-dead” forms of SnRK2.6 with or without Ser to Ala mutations at Ser171 and Ser175 in reaction buffer (25 mM Tris HCl, pH 7.4, 12 mM MgCl2, 2 mM DTT), with 1 μM ATP plus 1 µCi of [γ-32P] ATP for 30 min at 30 °C. Reactions were stopped by boiling in SDS sample buffer and proteins were separated by 10% SDS-PAGE.

      For the dephosphorylation assay, SnRK2.6M94G coated on Glutathione Sepharose (Cytiva) were dephosphorylated with Lambda Protein Phosphatase (λPP, NEB, P0753S) for 30 min and the λPP was removed by washing three times with protein buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl). To detect the effects of RAF3-KD and RAF10-KD on SnRK2.6M94G, SnRK2.2M96G, and SnRK2.3M95G thiophosphorylation and activity, recombinant GST-RAF3/10-KD was incubated with pre-dephosphorylated SnRK2.6M94G, SnRK2.2M96G, or SnRK2.3M95G for 30 min in reaction buffer (25 mM Tris HCl, pH 7.4, 12 mM MgCl2, 2 mM MnCl2, 0.5 mM DTT, 50 μM ATP, 50 μM N6-Benzyl-ATPγS). Then ABF2 was added to the reaction and incubated for an additional 30 min. This phosphorylation reaction was stopped by adding EDTA to a final concentration of 25 μM. A final concentration of 2.5 mM p-nitrobenzyl mesylate (Abcam, ab138910) was added to proceed the alkylating reaction for 1 h at room temperature. Samples with SDS sample buffer were boiled and separated by SDS-PAGE, transferred to Polyvinylidene fluoride (PVDF) membrane, and immunoblotted with antibodies against thiophosphate ester (Abcam, ab92570). To pre-activate SnRK2.6, HIS-SUMO-RAF-KD proteins were coated on the Ni-NTA beads and incubated with SnRK2.6 (in solution) in the presence of ATP. HIS-SUMO-RAF-KD were removed by centrifuging after the reaction.

      Protoplast isolation and transactivation assay

      Protoplasts were isolated from leaves of 4-week-old plants grown under a short photoperiod (10 h light at 23 °C/14 h dark at 20 °C). Leaf strips were excised from the middle parts of young rosette leaves, dipped in enzyme solution containing cellulase R10 (Yakult Pharmaceutical Industry) and macerozyme R10 (Yakult Pharmaceutical Industry) and incubated at room temperature in the dark. The protoplast solution was diluted with an equal volume of W5 solution (2 mM MES, pH 5.7, 154 mM NaCl, 125 mM CaCl2, and 5 mM KCl) and filtered through a nylon mesh. The flow-through was centrifuged at 100 g for 2 min to pellet the protoplasts. Protoplasts were resuspended in W5 solution and incubated for 30 min. 100 μL of protoplasts suspended in MMG solution (4 mM MES, pH 5.7, 0.4 M mannitol, and 15 mM MgCl2) were mixed with the plasmid mix and added to 110 μL PEG solution (40% w/v PEG-4000, 0.2 M mannitol, and 100 mM CaCl2). The transfection mixture was mixed completely by gently tapping the tube followed by incubation at room temperature for 5 min. The protoplasts were washed twice with 1 mL W5 solution. After transfection, protoplasts were left for incubation for a further 5 h under light in washing and incubation solution (0.5 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7) with or without 5 μM ABA. The RD29B-LUC (7 μg of plasmid per transfection) and ZmUBQ-GUS (1 μg per transfection) were used as an ABA-responsive reporter gene and as an internal control, respectively. For wild-type and mutated RAF, SnRK2 plasmids, 3 μg per transfection were used. After transfection, protoplasts were incubated for 5 h under light in washing and incubation solution (0.5 M mannitol, 20 mM KCl, 4 mM MES, pH 5.7) with or without 5 μM ABA. The mutations were introduced into wild-type RAFs using the primers listed in Supplementary Data 6.

      Generation of anti-pS171-SnRK2.6 antibody

      The anti-pS171-SnRK2.6 antibody was generated by ABcloneal. Phosphopeptide C-KSSVLHpSQPK was synthesized and used as an antigen to immunize rabbit and generate the polyclonal anti-phosphorylation antibody. Phosphorylation-non-specific antibody was removed using peptide C-KSSVLHSQPK.

      Immunoblotting

      30 mg seedling samples of wild-type, OK100-oct, and OK100-nonu were ground into fine powder in liquid nitrogen. Total proteins were extracted in 100 μL protein extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF, 5 μg/mL leupeptin, 5 μg/mL antipain, 5 μg/mL aprotinin, and 5% glycerol). Cell debris was removed by centrifugation at 12,000 g at 4 °C for 40 min and supernatant was collected. 20 µg protein were separated by 10% SDS/PAGE, and the proteins were transferred to PVDF membrane. Blots were probed with primary antibodies against SnRK2.2/2.3/2.6 (Agrisera) at a dilution of 1:5000, p-S175-SnRK2.6 at a dilution of 1:500033, and p-S171-SnRK2.6 (ABclonal) at a dilution of 1:5000. Anti-actin antibody (ABclonal) was used as the loading control at a dilution of 1:10,000. Secondary anti-rabbit antibodies at a dilution of 1:20,000 were used to detect antibodies in conjugation with secondary horseradish peroxidase and enhanced chemoluminescence reagent (ShengEr).

      Reporting summary

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

      Data availability

      The RNA sequencing data were deposited to the GEO database with the dataset identifier GSE152691. Source data are provided with this paper.

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