Nitric Oxide Negatively Regulates Abscisic Acid Signaling in Guard Cells by S-Nitrosylation of OST1

Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1

Pengcheng Wanga,1, Yanyan Dua,b,1, Yueh-Ju Houa, Yang Zhaoa,c, Chuan-Chih Hsud, Feijuan Yuanb, Xiaohong Zhua, W. Andy Taod, Chun-Peng Songb, and Jian-Kang Zhua,c,2

aDepartment of Horticulture and Landscape Architecture and dDepartment of Biochemistry, Purdue University, West Lafayette, IN 47907; bDepartment of Biology, Institute of Plant Stress Biology, State Key Laboratory of Cotton Biology, Henan University, Kaifeng 475001, China; and cShanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China

Contributed by Jian-Kang Zhu, December 8, 2014 (sent for review October 23, 2014) + The phytohormone abscisic acid (ABA) plays important roles in channel KAT1 (K channel in Arabidopsis thaliana 1) to cause plant development and adaptation to environmental stress. ABA stomatal closure (12, 13). induces the production of nitric oxide (NO) in guard cells, but how ABA also triggers the generation of several second-messenger NO regulates ABA signaling is not understood. Here, we show that molecules, such as calcium, inositol phospholipids, and nitric NO negatively regulates ABA signaling in guard cells by inhibiting oxide (NO) and reactive oxygen species (ROS) (14–17). These open stomata 1 (OST1)/sucrose nonfermenting 1 (SNF1)-related second messengers are involved in ABA regulation of stomatal protein kinase 2.6 (SnRK2.6) through S-nitrosylation. We found closure and other physiological processes (15, 18–20). Among that SnRK2.6 is S-nitrosylated at cysteine 137, a residue adjacent to these second messengers, NO has been well documented to have the kinase catalytic site. Dysfunction in the S-nitrosoglutathione important roles in ABA signal transduction. ABA induces NO (GSNO) reductase (GSNOR) gene in the gsnor1-3 mutant causes NO generation in roots and guard cells (15, 21). Exogenous application overaccumulation in guard cells, constitutive S-nitrosylation of of NO can trigger stomatal closure, whereas application of the SnRK2.6, and impairment of ABA-induced stomatal closure. Intro- NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- duction of the Cys137 to Ser mutated SnRK2.6 into the gsnor1-3/ 1-oxyl-3-oxide (c-PTIO) inhibits stomatal closure (22), sug- ost1-3 double-mutant partially suppressed the effect of gsnor1-3 gesting a positive role of exogenous NO in stomatal closure. on ABA-induced stomatal closure. A cysteine residue correspond- Several studies have suggested that exogenous NO may affect ing to Cys137 of SnRK2.6 is present in several yeast and human + stomatal responses to ABA by regulating an inward K channel protein kinases and can be S-nitrosylated, suggesting that the (20), an anion channel (23), and the generation of nitrated S-nitrosylation may be an evolutionarily conserved mechanism cGMP (24), although the direct target of NO in ABA signaling for protein kinase regulation. remains unclear. In plants, NO is produced by the nitrite-dependent nitrate re- NO | ABA | drought | GSNOR | stomata ductase pathway (15, 25) and a pathway dependent on the nitric

oxide associated 1 (NOA1) protein (21), although NOA1 is not PLANT BIOLOGY bscisic acid (ABA) plays critical roles in seed dormancy and an NO synthase (26). NO regulates many physiological processes Agermination, plant growth, and adaptation to environmental in plants, including responses to phytohormones, such as ABA, challenges (1, 2). Stresses, such as drought and high salt con- cytokinin, auxin, gibberellins, and salicylic acid, immunity against ditions, increase ABA concentration in plants as a result of ABA pathogens, senescence, and flowering (15, 27–31). The deficiency biosynthesis or ABA release from its inactive, conjugated forms in NO generation in the nia1nia2noa1 triple mutant results in (3). In the presence of ABA, the ABA receptors in the PYR1 ABA-hypersensitive stomatal closure (21), suggesting a negative (Pyrabactin Resistance 1)/PYL (PYR1-Like)/RCAR (Regula- tory Component of ABA receptor) protein family bind to and Significance inhibit the activity of clade A protein phosphatase 2Cs (PP2Cs), which are considered as coreceptors and negative regulators of – Drought stress induces the accumulation of the plant stress ABA signaling (4 6). This process then results in the release hormone abscisic acid (ABA). ABA then quickly activates the of sucrose nonfermenting 1 (SNF1)-related protein kinase 2s protein kinase OST1/SnRK2.6 to phosphorylate a number of (SnRK2s) from suppression by the PP2Cs. As central components proteins in guard cells, resulting in stomatal closure to reduce of the ABA signaling pathway, the activated SnRK2s phosphory- transpirational water loss. How SnRK2.6 is deactivated and late dozens of downstream effectors to regulate various physio- how ABA signaling may be desensitized are unclear. This study logical processes, including stomatal closure, root growth and found that nitric oxide (NO) resulting from ABA signaling development, seed dormancy, seed germination, and flowering (7). causes S-nitrosylation of SnRK2.6 at a cysteine residue close As the gateway for photosynthetic CO2 uptake and transpi- to the kinase catalytic site, which blocks the kinase activity. rational water loss, stomata are critical for plant growth and Dysfunction of S-nitrosoglutathione (GSNO) reductase causes physiology (8). ABA regulates stomatal movement and muta- GSNO overaccumulation in guard cells and ABA insensitivity in tions in ABA biosynthesis genes (9), or in the PYL or SnRK2.6 stomatal regulation. This work thus reveals how ABA-induced (also known as OST1) genes cause open-stomata phenotypes NO functions in guard cells to inactivate SnRK2.6 to negatively (10). On the other hand, dysfunction of the PP2Cs or over- feedback regulate ABA signaling. expression of RCAR1/PYL9 causes stomatal closure (5). Among Author contributions: P.W. and J.-K.Z. designed research; P.W., Y.D., Y.-J.H., Y.Z., C.-C.H., the three SnRK2s, SnRK2.2, -2.3, and -2.6, which are most im- F.Y., and X.Z. performed research; P.W., Y.D., W.A.T., C.-P.S., and J.-K.Z. analyzed data; portant for ABA signaling, SnRK2.6 is preferentially expressed and P.W., X.Z., and J.-K.Z. wrote the paper. in guard cells and plays a critical role in stomatal regulation, The authors declare no conflict of interest. whereas SnRK2.2 and -2.3 are mainly expressed in seeds and 1P.W. and Y.D. contributed equally to this work. young seedlings and are thus more important for seed germi- 2To whom correspondence should be addressed. Email: [email protected]. nation and seedling growth (4, 11). SnRK2.6 phosphorylates the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. slow (S-type) anion channel associated 1 and inward potassium 1073/pnas.1423481112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1423481112 PNAS | January 13, 2015 | vol. 112 | no. 2 | 613–618 Downloaded by guest on September 24, 2021 role of endogenous NO in ABA signaling. NO overaccumulates GSNO Cys-NO GSNO A 0 50 100 250 500 500 0 50100 250 500500 (μM) B 0 50100250500500 (μM) in Arabidopsis gsnor1(S-nitrosoglutathione reductase 1)/hot5 (sen- DTT - - - - - + ---- - + DTT - - - -- + sitive to hot temperatures 5)/par2 (paraquat resistant 2) mutant SNO plants that are impaired in the GSNO reductase gene (32–34). Autoradiograph Autoradiograph anti-biotin The gsnor1-3 mutant is hypersensitive to heat stress (33) and Total bacterial pathogen (28, 31), but is more resistant to oxidative Coomassie blue Coomassie blue anti-MBP stress (34). Characterization of gsnor1 mutant plants suggested C D that GSNOR regulates multiple developmental and metabolic GSNO - + - + - + - + - + - + - + GSNO - + - + - + - - - programs in Arabidopsis (35). In cytokinin signaling, NO causes DTT + - + - + - + - + - + - + - DTT + + +

S-nitrosylation and inhibition of the histidine phosphotransfer Autoradiograph Autoradiograph protein AHP1 (Arabidopsis histidine phosphotransfer protein 1) (27). In salicylic acid signaling, the S-nitrosylation of the salicylic Coomassie blue Coomassie blue acid receptor NPR1 facilitates its oligomerization (31). In addi- E tion, NO regulates cell death in plant immunity by S-nitrosylation αC αC of the NADPH oxidase AtRBOHD (28). H69 L72 H69 L72 E65 αE E65 αE Here, we show that GSNO and Cys-NO (S-nitrosocysteine) D160 C137 D160 F161 F161 can inhibit SnRK2.6 by S-nitrosylation. The S-nitrosylation of W137 SnRK2.6 occurs at Cys137, which is adjacent to the catalytic loop K142 D140 K142 D140 ATP binding loop ATP binding loop of the kinase. A Cys137 to Ser mutation causes the kinase to be catalytic loop catalytic loop

resistant to inhibition by GSNO in vitro, whereas a Cys137 to Trp SnRK2.6(WT) SnRK2.6(C137W) mutation results in an inactive kinase in vitro and in vivo. In- troduction of the Cys137 to Ser mutated form of SnRK2.6 into Fig. 1. S-nitrosylation at Cys-137 inhibits the activity of SnRK2.6. (A) Nitric gsnor1-3/ost1-3 (open stomata 1–3, a null allele of snrk2.6) par- oxide donors GSNO and Cys-NO inhibit the activity of SnRK2.6 in a dose- dependent manner. MBP–SnRK2.6 incubated with indicated concentration tially suppresses the defect in stomatal closure caused by over- γ 32 accumulation of SNOs because of the gsnor1-3 mutation. Our of GSNO (Left) and Cys-NO (Right) for 10 min and then [ - P]ATP was added to determine the autophosphorylation of SnRK2.6. In the rightmost lane results suggest that ABA induced S-nitrosylation of SnRK2.6 (DTT+), 1 mM DTT was added into the reaction before adding [γ-32P]ATP. functions to negatively feedback regulate ABA signaling in plants. (B) GSNO causes S-nitrosylation of SnRK2.6 as detected by the biotin-switch Moreover, the S-nitrosylation of cysteine137 is evolutionarily assay. (C) Effects of C-to-S site-directed mutation of the six cysteines on conserved in some AMPK/SNF1-related kinases and glycogen SnRK2.6 activity upon GSNO (50 μM) or DTT treatment. (D) Effects of C137S synthase kinase 3/SHAGGY-like kinases (SKs) in plants, yeast, and C137W mutations on the kinase activity of SnRK2.6. (E) Structure of and mammals, suggesting that S-nitrosylation–mediated inhi- SnRK2.6 showing the position of Cys-137 (Left) and Trp-137 (Right). Residues bition may be a general regulatory mechanism for these eukary- E65, H69, L72, K142, D160, F161, and C137 (W137) are shown by sticks. otic protein kinases. Results To further verify the S-nitrosylation of SnRK2.6 at Cys137, we GSNO Inhibits SnRK2.6 by S-Nitrosylation of Cys-137 in Vitro. To test subjected freshly purified SnRK2.6 to a biotin-switch assay, whether NO may regulate the activity of components in the core where free cysteines are blocked with S-methymethane thiosul- ABA signaling pathway, we tested the effects of the NO donors, fonate (MMTS) and nitrosylated cysteines are labeled with GSNO, Cys-NO, and SNAP (S-Nitroso-N-acetylpenicillamine), biotin. Mass spectrometry was performed immediately after the on the activity of SnRK2.6 in vitro because SnRK2.6 is a key labeling of biotin. The assay detected the S-nitrosylation of Cys137 player in the ABA regulation of guard cells and its activity can be in SnRK2.6 but none of the other cysteine was S-nitrosylated in readily assayed in vitro and in vivo. As shown in Fig. 1A and Fig. the presence of the blocking reagent MMTS (Fig. S2). In the S1, GSNO, Cys-NO, and SNAP inhibited the activity of SnRK2.6 absence of MMTS, Cys131, Cys159, Cys203, and Cys250 were in a dose-dependent manner, but such inhibitory effects were also labeled by biotin (Fig. S3). The peptide containing Cys107 reversed by DTT, indicating that the effects of NO donors rely was too short to be detected by mass spectrometry after trypsin on the thiol-based redox status. To determine whether SnRK2.6 digestion. These results suggested that S-nitrosylation occurred is S-nitrosylated in the presence of GSNO, we used a biotin- specifically at Cys137 after GSNO treatment. We also detected switch method in which the S-nitrosylated cysteine is labeled the dehydrogenation of Cys137 and Cys131 in the tryptic fragments with biotin and subsequently detected by antibiotin immuno- after the biotin-switch assay (Fig. S4), suggesting that a disulfide blot analysis (27, 31). As shown in Fig. 1B, GSNO induced bond may form between Cys137 and Cys131 in some SnRK2.6 S-nitrosylation of SnRK2.6 in a dose-dependent manner, and ap- molecules. Interestingly, the C131S mutation in SnRK2.6 (C131S) plication of DTT abolished the GSNO induced S-nitrosylation. appeared to increase the level of detectable S-nitrosylation relative Six cysteine residues in SnRK2.6 are putative target sites of to the wild-type SnRK2.6 (WT) (Fig. S5). When Cys137 was mu- S-nitrosylation. To determine which cysteine is required for tated, virtually no S-nitrosylated SnRK2.6 was detected in the GSNO-mediated inhibition, we mutated each cysteine (C) to presence of GSNO (Fig. S5). Collectively, these results show that serine (S) separately and tested the effect of GSNO on the SnRK2.6 is S-nitrosylated at Cys137 in the presence of NO donors mutated SnRK2.6. Of the six mutations, only the Cys137Ser and that the S-nitrosylation of SnRK2.6 blocks its kinase ac- (C137S) mutation eliminated the inhibitory effect of GNSO (Fig. tivity in vitro. 1C). The C137S mutation also caused a slight reduction in SnRK2.6 kinase activity (Fig. 1 C and D). The SnRK2.6C137W ABA Enhances the S-Nitrosylation of SnRK2.6 in Vivo. To further with a Trp substitution at Cys137 that may mimic S-nitrosylation confirm that the Cys137 is modified by S-nitrosylation in vivo, we (27, 36) did not show any detectable kinase activity (Fig. 1D). subjected total protein extracts from ost1-3 mutant plants com- Based on the crystal structure of SnRK2.6 resolved by a recent plemented with native promoter driven GFP-tagged wild-type study (37), the side-chain of C137 is exposed on the surface and SnRK2.6 (ost1-3/SnRK2.6WT-GFP) to biotin-switch analysis. Biotin- has only 8 Å distance from D140 in the catalytic loop and D160 labeled SnRK2.6-GFP was purified with streptavidin beads and in the Mg-binding loop in the catalytic cleft of SnRK2.6 (Fig. 1E, detected by anti-GFP immunoblot analysis. As shown in Fig. 2A, Left). The C137W mutation likely interferes directly with the a low level of S-nitrosylation of SnRK2.6 could be detected kinase catalytic activity. under control conditions and exogenous GSNO treatment on the

614 | www.pnas.org/cgi/doi/10.1073/pnas.1423481112 Wang et al. Downloaded by guest on September 24, 2021 ost1-3/ ost1-3/ 70 Consistent with a previous report (15), we found that ABA A B ost1-3/2.6WT SnRK2.6 60 2.6C137S ost1-3/2.6C137S increased NO generation in guard cells (Fig. 2E). To test

- + - + GSNO 50 ost1-3/2.6C137W

40 * whether ABA affects the S-nitrosylation of SnRK2.6, we exam-

SNO-SnRK2.6 * 30 * ined the S-nitrosylation of SnRK2.6-GFP upon ABA treatment. 20 Total Treatment with 50 μM ABA for 15 min did not increase the Water loss (%) Water 10 Col-0 ost1-3 anti-GFP 0 S-nitrosylation of SnRK2.6. However, ABA treatment for 30 or 020406080100 Time (min) 60 min increased the level of SnRK2.6 S-nitrosylation (Fig. 2F). 5 C control D As expected, preincubation with the NO-scavenger c-PTIO blocked μ m) 4 ABA S-nitrosylation of SnRK2.6 (Fig. 2F). These results indicate that 3 * prolonged treatment with ABA enhances the S-nitrosylation * * 2 ABA - + - + - + - + - + of SnRK2.6. 1 0 The NO Overaccumulation Mutant gsnor1-3 Is Insensitive to ABA- Stomatal aperture( Induced Stomatal Closure, a Phenotype That Is Partially Suppressed by the Cys137S Mutation in SnRK2.6. We hypothesized that NO in-gel kinase assay production inside guard cells may negatively regulate ABA signaling through SnRK2.6 S-nitrosylation. To test this hy-

E 100 anti-GFP control * pothesis, we investigated ABA responses in gsnor1-3 mutant

80 ABA + 50 μM ABA (min) plants, which overproduce SNOs because of a deficiency of the

* * F 60 * 0 15 30 60 c-PTIO GSNO reductase in Arabidopsis (32). S-nitrosylation of proteins 40 SNO-SnRK2.6 such as AHP1 (27) and SABP3 (salicylic acid-binding protein 3) 20 (Arbitrary unit) (38), is increased in gsnor1-3 mutant plants. In gsnor1-3 leaves, Total 0 Fluorescence intensity 0 15 30 60 NOoveraccumulatedinguardcells,wheretheGSNOR1 is anti-GFP Time (min) expressed (Fig. S6 A and B). Without ABA treatment, the level Fig. 2. ABA up-regulates the S-nitrosylation of Cys-137 of SnRK2.6 in vivo. of S-nitrosylation of SnRK2.6 was higher in gsnor1-3/ost1-3/ (A) GSNO-induced S-nitrosylation of wild-type but not Cys173Ser mutated SnRK2.6 than ost1-3/SnRK2.6 plants (Fig. 3A). After ABA form of SnRK2.6-GFP from plants. Twelve-day-old seedlings were treated treatment, the S-nitrosylation levels were similar. In the gsnor1-3 with or without 250 μM GSNO for 30 min. The biotin-switch and immunoblot mutant, ABA was substantially less effective in inducing stomatal assays were performed as described in SI Materials and Methods.(B and C) closure than in the Col-0 wild-type plants (Fig. 3B). However, Analysis of water loss (B) and stomata responses to 10 μM ABA (C) in the wild- exogenous H2O2, but not SNP or GSNO, could still induce type, ost1-3 mutant, and ost1-3 transgenic lines expressing SnRK2.6WT-GFP, C137S C137W stomatal closure in gsnor1-3 (Fig. S7A). The stomatal in- SnRK2.6 -GFP, and SnRK2.6 -GFP. Treatment with water was used as sensitivity of gnsor1-3 to ABA is similar to that of the ost1-3, control. Error bars indicate SD. n = 3–5 independent experiments. More than 20 stomata were measured for each treatment in each experiment in abi1-1, and abi2-1 mutants (Fig. 3B) (39, 40). Like these single C. Student’s t test, *P < 0.05 (significantly different from ost1-3). (D) In-gel mutants, gsnor1-3/abi1-1 and gsnor1-3/ost1-3 double-mutant kinase assay showing SnRK2.6 activity before (−) and after (+)50μM ABA plants are equally insensitive to ABA in stomatal closure (Fig.

treatment in the wild-type, the ost1-3 mutant, and ost1-3 transgenic lines 3B). Consistent with the impaired ABA response in guard cells, PLANT BIOLOGY expressing SnRK2.6WT-GFP, SnRK2.6C137S-GFP, and SnRK2.6C137W-GFP. The the excised leaves of gsnor1-3 showed a higher water-loss rate positions of SnRK2.6 and GFP-fused SnRK2.6 are indicated by open and closed than wild-type leaves (Fig. 3C). Two other T-DNA insertion triangles, respectively. (E) Average FDA-FM fluorescence intensity indicating mutants, gsnor1-4 and hot5-4 in the WS (Wassilewskija) back- μ NO levels in guard cells upon ABA (10 M) treatment in Col wild-type. ground, also exhibited higher water loss than their wild-type Treatment with water was used as control. Error bars indicate SD (n > 6 from 3 independent experiments). Student’s t test, *P < 0.01. (F) ABA regulation of control plants (Fig. 3C). However, hot5-1, which is another S-nitrosylation of SnRK2.6 in ost1-3/SnRK2.6WT-GFP transgenic plants. Twelve- missense allele of GSNOR1 in the Col-0 background (33), day-old seedlings were treated with or without 50 μM ABA for the indicated showed a similar water loss rate as the Col-0 wild-type (Fig. times. The seedlings treated with 200 μM c-PTIO were used as a control. The 3C). Immunoblot and GSNOR activity assays revealed that biotin-switch and immunoblot assays were performed as described in SI gsnor1-3, gsnor1-4,andhot5-4 were virtually null alleles (Fig. Materials and Methods. S7 D and E) (33), whereas hot5-1 plants contained a similar amount of GSNOR protein as the wild-type and retained about half of the GSNOR activity (Fig. S7 D and E) (33). seedlings increased the level of S-nitrosylated SnRK2.6 in ost1-3/ WT C137S Therefore, the transpirational water-loss phenotypes are con- SnRK2.6 -GFP transgenic plants. In ost1-3/SnRK2.6 -GFP sistent with the GSNOR protein and activity levels. Consistent plants, in which the Cys137 is mutated to Serine, S-nitrosylated with its higher transpirational water loss, the leaf surface SnRK2.6 was undetectable. These results suggest that S-nitrosylation temperature of gsnor1-3 was significantly lower than that of the of SnRK2.6 at Cys137 occurs in vivo. Col-0 wild-type, especially under drought stress (Fig. S8). Our We compared the water loss and ABA-induced stomatal clo- results suggest that increased NO accumulation in the gsnor1 sure in Col, ost1-3, and transgenic plants expressing wild-type mutants causes elevated S-nitrosylation of SnRK2.6, resulting WT and mutated forms of SnRK2.6. Only SnRK2.6 -GFP com- in ABA insensitivity in guard cells and higher transpirational plemented the water loss (Fig. 2B) and stomatal closure water loss. (Fig. 2C) phenotypes of ost1-3. The transgenic plant ost1-3/ We examined the kinase activity of SnRK2.6 in gsnor1 mu- C137W SnRK2.6 -GFP, which contains the S-nitrosylation–mimicking tant plants using an in-gel kinase assay (11, 41), which involves mutation at Cys137, was insensitive to ABA-induced stomatal clo- separating plant proteins in SDS-polyacrylamide gels contain- sure (Fig. 2C) and had a higher rate of water loss than the wild-type ing ABF2 fragments as a kinase substrate and renaturation of (Fig. 2B). SnRK2.6C137S-GFP partially complemented the pheno- the proteins with DTT after removal of SDS from the gels before types of ost1-3 (Fig. 2 B and C). Consistent with these phenotypes, detecting protein bands with kinase activities by incubation with in-gel kinase assays showed that SnRK2.6C137S-GFP had a reduced [λ-32P]ATP. The assay revealed higher SnRK2.6 kinase activities in kinase activity and that SnRK2.6C137W-GFP is a “dead” kinase hot5-4 and gsnor1-4 mutant plants (Fig. S7F). Because the DTT in vivo (Fig. 2D). Taken together, our in vitro and in vivo data used for protein renaturation in the assay would reverse suggest that S-nitrosylation at Cys137 of SnRK2.6 abolishes its S-nitrosylation and possibly some other modifications on Cys kinase activity. residues of SnRK2.6, the results suggest that S-nitrosylated

Wang et al. PNAS | January 13, 2015 | vol. 112 | no. 2 | 615 Downloaded by guest on September 24, 2021 members in the SnRK2 subfamily and SnRK3.1 in the SnRK3 A gsnor1-3/ost1-3 ost1-3/ B 5

μ m) control SnRK2.6 ABA /SnRK2.6 4 subfamily have the conserved cysteine (cystein-137 in SnRK2.6) ABA - + - + * * * * 3 * * that can be potentially S-nitrosylated. The conserved cysteine is SNO-SnRK2.6 2 also present in SnRK2 orthologs from other plants, including

Total 1 various crop plants (Fig. S9A). None of the other 24 members in 0 the SnRK3 subfamily and none of the three members in the C anti-GFP Stomatal aperture( 60 WS-4 SnRK1 subfamily in Arabidopsis has the conserved cysteine (Fig. hot5-4 * 4A and Fig. S9B). The proteins with the highest sequence ho- gsnor1-4 40 * * mologies to the Arabidopsis SnRK2s, AMPKs in mammals and SNF1 in yeasts have a hydrophobic amino acid rather than cys- 20 D 55 Water loss (%) Water Col-0 μ m) control ABA teine at the corresponding position. Interestingly, two AMPK- gsnor1-3 4 * * 0 hot5-1 * * related protein kinases in mammals, brain-specific kinases (BRSK) 33 0 20 40 60 80 100 120 *** 1 and 2, which are required for neuronal polarization (43), have Time (min) 2 E 60 the corresponding cysteine near their catalytic sites, similar to the gsnor1-3 * 1 ost1-3 * Arabidopsis SnRK2s. The yeast SNF1-related kinases Hsl1 and Stomatal aperture( 0 40 Col-0 *** Hal4, but not the SNF1 itself, also have the corresponding cysteine (Fig. 4A). Another family of protein kinases that contains the 20

Water loss (%) Water gsnor1-3/ost1-3 conserved cysteine is the glycogen synthase kinase 3(GSK)/SKs. gsnor1-3/ost1-3/SnRK2.6 0 gsnor1-3/ost1-3/2.6C137S The conserved cysteine residue is present in 9 of 10 GSKs in 0 20 40 60 80 100 120 Arabidopsis, in the human GSK3α/β, and in the yeast Mck1, Time (min) Rim11, and Mrk1 (Fig. 4A). To test whether the conserved cys- Fig. 3. Overaccumulation of SNOs in the gnsor1 mutant impairs the sto- teine residues in the AMPK1/SNF1-related kinases and SKs may matal response to ABA and partial suppression of the stomatal defect by also be modified by S-nitrosylation, we examined the effects of C137S mutated form of SnRK2.6. (A) ABA enhances S-nitrosylation of GSNO on their kinase activities. Of these protein kinases, we were SnRK2.6 in gsnor1-3/ost1-3/SnRK2.6-GFP and ost1-3/SnRK2.6-GFP plants as able to express the following four in Escherichia coli and the pu- revealed by a biotin-switch assay. Twelve-day-old seedlings were treated rified recombinant proteins had detectable autophosphorylation μ with or without 50 M ABA for 30 min. The biotin-switch and immunoblot activities: HsBRSK1, AtBIN2, HsGSK3b, and ScHsl1p (Fig. S10). assays were performed as described in SI Materials and Methods.(B) Sto- matal responses to exogenous ABA in wild-type and mutant plants. Stomatal All four kinases were inhibited by GSNO in a dose-dependent apertures were measured in epidermal strips peeled from rosette leaves of manner (Fig. S10). We mutated the conserved cysteines, Cys162 in 4- to 6-wk-old seedlings of Col-0 wild-type and the indicated mutants after AtBIN2 and Cys178 in HsGSK3b, to alanine (A) or tryptophan the strips were incubated for 2 h in a buffer without (control) or with 10 μM (W). GSNO abolished the kinase activities of wild-type BIN2 ABA. n = 3–5 independent experiments. More than 20 stomata were mea- (brassinosteroid insensitive 2) and GSK3b (Fig. 4 B and C,lanes1 sured for each treatment in each experiment. Error bars represent ± SD. and 2). The C-to-A mutated forms of BIN2 and GSK3b still Student’s t test, *P < 0.05 (significantly different from wild-type). (C)Water retained some kinase activities after the GSNO treatment (Fig. 4 loss (percentage of initial fresh weight) in the detached rosette leaves of B and C, lanes 3 and 4). The C-to-W mutated forms of BIN2 and = – gsnor1 mutant and wild-type plants. Error bars indicate SD. n 3 5in- GSK3b totally lost their kinase activities (Fig. 4 B and C,lanes5 dependent experiments. Student’s t test, *P < 0.05. (D) Stomatal responses to exogenous ABA (10 μM) in wild-type and mutant plants. n = 3in- and 6). As was the case with SnRK2.6, the S-nitrosylation of these dependent experiments. More than 20 stomata were used for each treat- kinases was induced by GSNO, and the mutation of the conserved ment in each experiment, Error bars represent ± SD. Note that stomatal apertures at the control condition were similar for all genotypes because the epidermal strips were placed under light and high humidity conditions and incubated in stomata-opening solution to maximize stomatal opening be- A fore ABA treatment. (E) Water loss (percentage of initial fresh weight) in the

detached leaves of Col-0 wild-type and mutant plants. Error bars indicate SD. GSK n = 3–5 independent experiments. For D and E, Student’s t tests show sig- nificant difference from Col-0 (*P < 0.05), and between gsnor1-3/ost1-3 and gsnor1-3/ost1-3 (**P < 0.05).

and other Cys-modified SnRK2.6 in the gsnor1 mutants are AMP/SNF1/SnRK not further subjected to any irreversible and inhibitory mod- B WT C162A C162W WT C162A C162W D WT C162A WT C162A GSNO - + - + - + - + - + - + GSNO - + - + - + - + ifications. DTT + - + - + - + - + - + - DTT + - + - + - + - To confirm that the ABA insensitivity in stomatal closure in AtBIN2 AtBIN2 gsnor1 mutant plants is a result of SnRK2.6 S-nitrosylation, we in- Autoradiograph Coomassie blue anti-Biotin anti-MBP WT C137S troduced native promoter-driven SnRK2.6 and SnRK2.6 into C WT C178A C178W WT C178A C178W E WT C178A WT C178A GSNO - + - + - + - + - + - + GSNO - + - + - + - + the gsnor1-3 mutant by crossing with ost1-3 mutant plants expressing DTT + - + - + - + - + - + - DTT + - + - + - + - the SnRK2.6 constructs, and analyzed the water loss and stomatal HsGSK3b HsGSK3b responses in the resulting gsnor1-3/ost1-3/SnRK2.6WT and gsnor1-3/ Autoradiograph Coomassie blue anti-Biotin anti-MBP C137S C173S ost1-3/SnRK2.6 plants. The SnRK2.6 mutant but not the Fig. 4. S-nitrosylation of Cys137 is evolutionarily conserved across king- wild-type SnRK2.6 partially restored ABA-induced stomatal closure doms. (A) Cys137 of SnRK2.6 is evolutionarily conserved in some AMPK/SNF1- in the gsnor1-3 mutant (Fig. 3D). The water-loss defect of the related kinases and GSKs in Arabidopsis, yeast, and mammals. The conserved gsnor1-3/ost1-3 double-mutant was also partially rescued by in- cysteine is indicated by the arrow. GSNO inhibits the kinase activity of Ara- troduction of SnRK2.6C173S but not the wild-type SnRK2.6 (Fig. 3E). bidopsis BIN2 (B) and human GSK3b (C). Recombinant wild-type and mutated MBP-AtBIN2 and MBP-HsGSK3b were incubated with DTT or GSNO 32 The S-Nitrosylated Cysteine Is Evolutionarily Conserved in Eukaryotes. (50 μM) for 10 min and then [γ- P]ATP was added to determine the autophosphorylation of recombinant kinases. GSNO induces the S-nitrosylation SNF1/AMPKs are conserved in all eukaryotes and play funda- of recombinant Arabidopsis MBP-BIN2 (D) and human MBP-GSK3b (E), as mental roles in cellular responses to metabolic stress (42). The 48 determined by the biotin-switch assay. All experiments were repeated at SnRKs in Arabidopsis are divided into three subfamilies. All 10 least twice with similar results.

616 | www.pnas.org/cgi/doi/10.1073/pnas.1423481112 Wang et al. Downloaded by guest on September 24, 2021 cysteine blocked the S-nitrosylation of the proteins (Fig. 4 D and redox homeostasis. Protein S-nitrosylation depends on the E). Our finding of the regulation of BIN2 by S-nitrosylation is proximity of the protein to NO sources, and on factors for consistent with the observation that brassinosteroid treatment transnitrosylation and denitrosylation, such as thioredoxin and induces NO accumulation in maize leaves (44). The conservation glutathione levels (50). The reversible inhibition of SnRK2 by ofthecysteineandtheS-nitrosylation–dependent regulation of the S-nitrosylation may fine-tune the strength or duration of SnRK2.6 activities of the kinases from diverse organisms suggest that activation in response to ABA. Such dynamic control may be S-nitrosylation–mediated inhibition is a general regulatory important for plants to balance stress resistance with growth that mechanism for these eukaryotic protein kinases. cannot happen if stomata are fully closed as a result of continued activation of SnRK2.6. Because NO originates from nitrogen Discussion sources, NO-mediated stomatal regulation through SnRK2.6 Our study has revealed a novel mechanism by which NO regu- S-nitrosylation may also help coordinate nitrogen supply with lates ABA signaling in Arabidopsis. We provided evidence that photosynthetic carbon availability. ABA enhances SnRK2.6 S-nitrosylation and the S-nitrosylation The negative effect of endogenous NO on ABA signaling in feedback inhibits ABA signaling. The evidence includes: (i) ex- guard cells is supported by genetic analysis using both NO de- ogenous NO donors caused S-nitrosylation of SnRK2.6 and ficient and overaccumulation mutants (Fig. 3) (21). On the other inhibited its activity in vitro, and an S-nitrosylation-mimicking hand, several studies have found a positive role of exogenous mutation abolished its kinase activity; (ii) ABA enhanced the application of NO in ABA-induced stomatal closure (15, 20, level of nitrosylated SnRK2.6 in planta; and (iii) NO-over- 22, 24). Exogenous NO might promote stomatal closure by producing mutant plants were less sensitive to ABA in stomatal regulating ion channels (20, 23), or by generating nitrated cGMP closure, but the defect was partially suppressed by ectopic ex- (24). Alternatively, the observed positive role of exogenous pression of C137S mutated form of SnRK2.6. Our study revealed NO on ABA responses in guard cells might be caused by sec- SnRK2.6 as a new S-nitrosylation target that bridges NO-medi- ondary effects, such as induction of ROS or other second mes- ated redox signaling and ABA signaling. sengers (24, 47). In the absence of ABA, PP2Cs inhibit SnRK2.6 activity by The activities of protein kinases are tightly controlled by multiple both dephosphorylation at Ser175 and direct binding (45). In the mechanisms. In most cases, dephosphorylation at the phosphory- presence of ABA, however, the binding of PYLs to PP2Cs lated sites in the “activation segment” causes kinase deactivation releases SnRK2.6 from inhibition. SnRK2.6 is activated very (51). For SnRK2.6, the phosphorylation of Ser175 is necessary for quickly by ABA because 2 min of ABA treatment is enough to its full activity (37). The negative regulators of ABA signaling, cause a strong activation of SnRK2.6 (41). In contrast to the PP2Cs, inhibit SnRK2.6 partly by dephosphorylation at Ser175. early activation of SnRK2.6, NO-mediated inhibition of SnRK2.6 Similarly, in brassinosteroid signaling, AtBIN2 is dephosphorylated is expected to happen later during ABA treatment because the and inactivated by BSU1 (bri1 SUPPRESSOR 1), a Kelch-repeat level of S-nitrosylated SnRK2.6 is not increased after 15 min of domain-containing protein phosphatase (52). In contrast to the ABA application (Fig. 2F). NO accumulation did not reach peak phosphorylation-mediated activation, the phosphorylation of levels until 30 min after ABA application (Fig. 2E) (46). The GSK3 at serine 9 by Akt kinase inhibits its activity (53), suggesting notion that NO accumulation and SnRK2.6 S-nitrosylation are a “pseudo-substrate” mechanism to reduce the kinase activity (51). slow or late events during ABA treatment is consistent with the PLANT BIOLOGY Our finding that GSNO-mediated S-nitrosylation of HsBRSK1, observation that ABA-induced NO generation is dependent on ScHsl1p, GSKs, and AtSnRK2s suggests a third way to deactivate the quick burst of ROS (47). The accumulation of endogenous these protein kinases. The cysteine in the catalytic cleft exists only NO may function as one of the negative feedback mechanisms to in certain members of the SnRK/GSK family. In most protein prevent overactivation of ABA signaling in guard cells. This feedback regulation is achieved by S-nitrosylation of SnRK2.6. kinases, the conserved cysteine is substituted by a hydrophobic ABA-activated SnRK2.6 phosphorylates NADPH oxidases to amino acid. Other cysteines not in the position near the catalytic loop could still be modified by S-nitrosylation, leading to the in- cause ROS production in guard cells (18, 48). The S-nitrosylation – of the NADPH oxidase AtRBOHD has been shown to cause hibition of some kinases (54 56). The S-nitrosylation of cysteine inhibition of the NADPH oxidase (28), and thus may also in the catalytic cleft reported here represents a unique mechanism contribute to the NO-mediated negative feedback regulation of in the regulation of some of the AMPK/SNF1-related and GSK ABA signaling. Although detailed dynamic changes in the levels family of protein kinases. of activated SnRK2.6, NO, and S-nitrosylated SnRK2.6 in guard Materials and Methods cells are not yet known, available evidence suggests the following Plant Materials and Growth Condition. scenario: ABA treatment causes very fast and strong activation The Arabidopsis wild-type Columbia-0 (Col-0), Wassilewskija-4 (WS-4), and Landsberg (Ler) plants were used in this of SnRK2.6 in guard cells. The activated SnRK2.6 phosphor- study. The gsnor1-3 (GABI_315D11), hot5-1 (CS66011), hot5-4 (FLAG_298F11), ylates many downstream effector proteins to cause stomatal gsnor1-4 (FLAG_220G07), and ost1-3 (Salk_008068) mutant seeds were ordered closure, and it also phosphorylates the NADPH oxidases from the Arabidopsis Biological Resource Center. Seeds were germinated on AtRBOHD and AtRBOHF to cause a burst of ROS, which in half-strength MS agar plates containing 1.5% sucrose. For the measurement turn causes NO accumulation in the guard cells. When NO of water loss or stomatal apertures, 10-d-old seedlings on MS plates were accumulates to high levels, it causes S-nitrosylation and inhibition transferred to soil and grown under short-day conditions (12 h light, 100– − − of SnRK2.6 and the NADPH oxidases. This inhibition serves to 120 μmol·m 2·s 1)at22°C. desensitize ABA signaling. The phenomenon of desensitization of ABA signaling was observed 20 y ago, when water deficit stress Epidermal Strip Bioassay and Water Loss Measurement. Stomatal bioassay was shown to reduce the stomatal sensitivity to ABA (49). experiments were performed as described previously (15). Epidermal strips In gsnor1 mutant plants, overaccumulated SNOs cause con- were peeled from the rosette leaves of 4- to 6-wk-old seedlings that were stitutive S-nitrosylation of SnRK2.6 such that the kinase is in- placed under the light and high humidity for 12 h and incubated in stomata- opening solution containing 50 mM KCl, 10 mM MES, pH 6.15 in a growth activated, and thus the stomata are insensitive to ABA (Fig. 3). chamber for 1.5 h to maximize stomatal opening before ABA was added. In support of our conclusion that endogenous NO has a negative Stomatal apertures were measured 2 h after 10 μM ABA was added. The role in ABA signaling in guard cells, a recent study demonstrated apertures of about 60 stomata were measured in three independent that the NO-deficient mutant nia1nia2noa1 is hypersensitive to experiments. ABA in stomatal closure (21). The kinase activity of S-nitro- For the measurement of water loss, detached rosette leaves of 4-wk-old sylated SnRK2.6 may be resumed quickly, depending on cellular plants were placed in weighing dishes and left on the laboratory bench with

Wang et al. PNAS | January 13, 2015 | vol. 112 | no. 2 | 617 Downloaded by guest on September 24, 2021 − − light (30–40 μmol·m 2·s 1). Fresh weight was monitored at the indicated Other Methods. Details for other methods are provided in SI Materials and times. Water loss was expressed as a percentage of initial fresh weight. Methods, including recombinant protein expression and site-directed mu- tagenesis, structure information of SnRK2.6, histochemical detection of GUS In Vitro Kinase Assay and In-Gel Kinase Assay. The in vitro kinase assay and in- activity, infrared thermography imaging, NO detection by confocal micros- gel kinase assay were performed as described previously (11); details are copy, and homology search and sequence alignment. The primers used in provided in SI Materials and Methods. this study are listed in Table S1.

In Vitro and in Vivo S-Nitrosylation Assays. In vitro and in vivo S-nitrosylation ACKNOWLEDGMENTS. This work was supported by NIH Grant R01GM059138 assays were performed as described (28, 31); details are provided in (to J.-K.Z.) and National Natural Science Foundation of China Grants 91017001 SI Materials and Methods. and 31171363 (to P.W.).

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