Stomatal regulation: Role of H2S-induced persulfidation in ABA signaling

Abstract

Hydrogen sulfide (H2S) is an important signaling molecule in plants. It is a small gasotransmitter with high permeativity through lipid membranes. H2S can convert the mercapto groups (Cys–SH) of the amino acid cysteine into hydropersulfide groups (–Cys–SSH). This process is called persulfidation. Post-translational modifications such as persulfidation can play an important role in protein functionality, altering protein conformation and/or activity. In plants, H2S can be generated within guard cells by CYSTEINE-DESULFHYDRASES, such as DES1, by degradation of cysteines into H2S, ammonia, and pyruvate (Zhang et al., 2020Zhang J. Zhou M. Ge Z. Shen J. Zhou C. Gotor C. Romero L.C. Duan X. Liu X. Wu D. et al.Abscisic acid-triggered guard cell l-cysteine desulfhydrase function and in situ hydrogen sulfide production contributes to heme oxygenase-modulated stomatal closure.Plant Cell Environ. 2020; 43: 624-636https://doi.org/10.1111/pce.13685Crossref PubMed Scopus (31) Google Scholar). H2S was reported to take part in physiological processes, such as stress response, and in the regulation of the functions of phytohormones such as salicylic acid and abscisic acid (ABA) (Pandey and Gautam, 2020Pandey A.K. Gautam A. Stress responsive gene regulation in relation to hydrogen sulfide in plants under abiotic stress.Physiol. Plant. 2020; 168: 511-525https://doi.org/10.1111/ppl.13064Crossref PubMed Scopus (37) Google Scholar). Although the mechanism of how H2S affects ABA signaling pathways is not well understood, two recent studies by Chen et al., 2020Chen S. Jia H. Wang X. Shi C. Wang X. Ma P. Wang J. Ren M. Li J. Hydrogen sulfide positively regulates abscisic acid signaling through persulfidation of SnRK2.6 in guard cells.Mol. Plant. 2020; 13: 732-744https://doi.org/10.1016/j.molp.2020.01.004Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar and Zhou et al., 2021Zhou M. Zhang J. Shen J. Zhou H. Zhao D. Gotor C. Romero L.C. Fu L. Li Z. Yang J. et al.Hydrogen sulfide-linked persulfidation of ABSCISIC INSENSITIVE 4 controls Arabidopsis ABA responses through the transactivation of mitogen-activated protein kinase kinase kinase 18.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.007Abstract Full Text Full Text PDF Scopus (18) Google Scholar shed light on this topic, reporting how H2S-induced persulfidation regulates the stomatal ABA signaling pathway. Chen et al., 2020Chen S. Jia H. Wang X. Shi C. Wang X. Ma P. Wang J. Ren M. Li J. Hydrogen sulfide positively regulates abscisic acid signaling through persulfidation of SnRK2.6 in guard cells.Mol. Plant. 2020; 13: 732-744https://doi.org/10.1016/j.molp.2020.01.004Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar identified the effect of cysteine persulfidation on ABA-mediated stomatal closure. SnRK2.6 (SUCROSE NONFERMENTING 1-RELATED PROTEIN KINASE 2.6), which is a major player in the regulation of stomatal movement, was identified as a persulfidation target. Signaling by the phytohormone ABA is of paramount importance in plants, and stomatal closure is one of the important processes governed by ABA. In the presence of ABA, the ABA receptors PYRABACTIN RESISTANCE/PYR-LIKE/REGULATORY COMPONENT OF ABA RECEPTOR (PYR/PYL/RCAR) bind to the negative regulator PP2C (protein phosphatase type 2C), thereby removing the inhibition of SnRK2.6. As a result, SnRK2.6, also known as Open Stomata 1 (OST1), is phosphorylated and activated by PYR/PYL/RCAR. Activated SnRK2.6 induces reactive oxygen species (ROS) production and Ca2+ influx into guard cells, thereby triggering stomatal closure (Figure 1). This new study identified persulfidation of SnRK2.6 at two Cys residues, Cys131 and Cys137. Persulfidation resulted in elevated SnRK2.6 kinase activity and its ability to interact with ABA response element-binding factor 2 (ABF2). Treatment of wild-type SnRK2.6 with H2S in vitro and in vivo resulted in increased phosphorylation and binding affinity toward its substrate ABF2, whereas mutation of both Cys sites resulted in loss of phosphorylation and ABF2 binding activity. Transgenic ost1-3/SnRK2.6C131SC137S plants showed also decreased drought tolerance due to the lack of SnRK2.6-induced Ca2+ influx into guard cells and therefore their inability to close stomata. Interestingly, Cys137 of SnRK2.6 was described previously as an S-nitrosylation site, which inhibited kinase activity (Wang et al., 2015Wang P. Du Y. Hou Y.J. Zhao Y. Hsu C.C. Yuan F. Zhu X. Tao W.A. Song C.P. Zhu J.K. Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1.Proc. Natl. Acad. Sci. U S A. 2015; 112: 613https://doi.org/10.1073/pnas.1423481112Crossref PubMed Scopus (237) Google Scholar). This finding implies that persulfidation and nitrosylation on Cys137 affect SnRK2.6 activity in competitive and opposite manners. Taken together, the results reported by Chen et al. (2020) provide new insights into the link between H2S and the ABA signaling pathway (Figure 1). Zhou et al., 2021Zhou M. Zhang J. Shen J. Zhou H. Zhao D. Gotor C. Romero L.C. Fu L. Li Z. Yang J. et al.Hydrogen sulfide-linked persulfidation of ABSCISIC INSENSITIVE 4 controls Arabidopsis ABA responses through the transactivation of mitogen-activated protein kinase kinase kinase 18.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.007Abstract Full Text Full Text PDF Scopus (18) Google Scholar revealed recently another example how H2S links with ABA signaling. Persulfidation of abscisic acid insensitive 4 (ABI4) was observed to affect ABA-mediated responses in plants. The transcription factor ABI4 regulates a variety of stimuli and their signaling pathways. It acts as inhibitor as well as activator of the expression of its downstream targets (Wind et al., 2013Wind J.J. Peviani A. Snel B. Hanson J. Smeekens S.C. ABI4: versatile activator and repressor.Trends Plant Sci. 2013; 18: 125-132https://doi.org/10.1016/j.tplants.2012.10.004Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). ABI4 harbors several cysteines. Zhou et al., 2021Zhou M. Zhang J. Shen J. Zhou H. Zhao D. Gotor C. Romero L.C. Fu L. Li Z. Yang J. et al.Hydrogen sulfide-linked persulfidation of ABSCISIC INSENSITIVE 4 controls Arabidopsis ABA responses through the transactivation of mitogen-activated protein kinase kinase kinase 18.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.007Abstract Full Text Full Text PDF Scopus (18) Google Scholar report the reversible persulfidation of ABI4 in vivo and in vitro upon ABA and NaHS treatments. Wild-type ABI4 was necessary for NaHS-induced phenotypes, like inhibition of seed germination, seedling establishment, primary root growth, and stomatal closure. Similar results were obtained with ABI4Cys250Ala mutants, which performed like abi4 knockout lines and could not rescue ABA-responsive plant phenotypes. Non-modified Cys250 was needed for ABI4 autoactivation activity, explaining the observed mutant phenotype. Persulfidation of ABI4 also affected the expression of its downstream target MAPKKK18, which regulates the ABA-mediating MKK3-MPK1/2/7/14 MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) pathway (de Zelicourt et al., 2016de Zelicourt A. Colcombet J. Hirt H. The role of MAPK modules and ABA during abiotic stress signaling.Trends Plant Sci. 2016; 21: 677-685https://doi.org/10.1016/j.tplants.2016.04.004Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). MAPKKK18 expression is reduced in abi4 plants or ABI4Cys250Ala plants in the abi4 background, indicating that ABI4 is an upstream regulator of MAPKKK18 (Zhou et al., 2021Zhou M. Zhang J. Shen J. Zhou H. Zhao D. Gotor C. Romero L.C. Fu L. Li Z. Yang J. et al.Hydrogen sulfide-linked persulfidation of ABSCISIC INSENSITIVE 4 controls Arabidopsis ABA responses through the transactivation of mitogen-activated protein kinase kinase kinase 18.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.007Abstract Full Text Full Text PDF Scopus (18) Google Scholar). Moreover, SnRK2 induces RAV1 phosphorylation and thereby inhibits ABI4 expression (Feng et al., 2014Feng C.Z. Chen Y. Wang C. Kong Y.H. Wu WH W.H. Chen Y.F. Arabidopsis RAV1 transcription factor, phosphorylated by SnRK2 kinases, regulates the expression of ABI3, ABI4, and ABI5 during seed germination and early seedling development.Plant J. 2014; 80: 654-668https://doi.org/10.1111/tpj.12670Crossref PubMed Scopus (154) Google Scholar). Analysis of the ABI4 binding site revealed its high affinity toward the CACCG motif, which is present in the MAPKKK18 promoter, and its interaction was proven in vivo and in vitro. The binding ability of ABI4 was enhanced by NaHS and ABA, while mutation of Cys250 abolished the binding and ABI4Cys150Ala was unable to bind to the promoter of MAPKKK18. Further work also identified DES1 as an ABI4 target that also harbors a CACCG motif in its promoter and was observed to be bound by ABI4. ABI4 and DES1 seem to be subject to a mutual regulation, in which DES1 expression is regulated by ABI4 and ABA-induced ABI4 expression is DES1 dependent. Des1 mutants showed impaired enhancement of ABI4 transcripts after ABA treatment, whereas NaHS induced ABI4 expression. As an upstream factor, DES1 not only affected ABI4 expression but also induced ABA-triggered persulfidation of ABI4. To summarize, these results reveal an interesting regulation of ABI4 activity by DES1-regulated persulfidation on Cys250 and its effect on downstream transcription targets (Figure 1). Cysteines are not only persulfidation targets. They are also subjected to oxidation by ROS. In fact, oxidation of Cys–thiol groups into sulfenic acid by H2O2 is a requirement for H2S-induced persulfidation (Zhang et al., 2021Zhang J. Zhou M. Zhou H. Zhao D. Gotor C. Romero L.C. Shen J. Ge Z. Zhang Z. Shen W. et al.Hydrogen sulfide, a signaling molecule in plant stress responses.J. Integr. Plant Biol. 2021; 63: 146-160https://doi.org/10.1111/jipb.13022Crossref PubMed Scopus (40) Google Scholar). The NADPH oxidases RESPIRATORY BURST OXIDASE (RbohF, RbohD) produce ROS upon ABA signaling, inducing stomatal closure (Postiglione and Muday, 2020Postiglione A.E. Muday G.K. The role of ROS homeostasis in ABA-induced guard cell signaling.Front. Plant Sci. 2020; 11https://doi.org/10.3389/fpls.2020.00968Crossref PubMed Scopus (31) Google Scholar). Evidence was reported that ROS can affect ABA signaling in a regulatory feedback mechanism. HAB1, RbohD, and DES1 are examples of ABA signaling molecules controlled by ROS on Cys side chains, oxidation of which induces ABA desensitivity, implying a feedback inhibition (Sridharamurthy et al., 2014Sridharamurthy M. Kovach A. Zhao Y. Zhu J.-K. Xu H.E. Swaminathan K. Melcher K. H2O2 inhibits ABA-signaling protein phosphatase HAB1.PLoS One. 2014; 9: e113643https://doi.org/10.1371/journal.pone.0113643Crossref PubMed Scopus (18) Google Scholar; Shen et al., 2020Shen J. Zhang J. Zhou M. Zhou H. Cui B. Gotor C. Romero L.C. Fu L. Yang J. Foyer C.H. et al.Persulfidation-based modification of cysteine desulfhydrase and the NADPH oxidase RBOHD controls guard cell abscisic acid signaling.Plant Cell. 2020; 32: 1000-1017https://doi.org/10.1105/tpc.19.00826Crossref PubMed Scopus (78) Google Scholar). These findings emphasize the strong correlation between ROS and H2S in ABA signaling. It remains to be elucidated how ROS and H2S interact and compete for cysteines, thereby regulating ABA signaling. So far, research has been focused on the molecular mechanisms underlying the responses to plant-derived H2S. However, H2S is also produced by plant-associated bacteria, such as the recently reported beneficial bacterial Cronobacter muytjensii strain JZ38, which secretes H2S and induces plant resistance to abiotic and biotic stress (Eida et al., 2020Eida A.A. Bougouffa S. L'Haridon F. Alam I. Weisskopf L. Bajic V.B. Saad M.M. Hirt H. Genome insights of the plant-growth promoting bacterium cronobacter muytjensii JZ38 with volatile-mediated antagonistic activity against phytophthora infestans.Front. Microbiol. 2020; 11https://doi.org/10.3389/fmicb.2020.00369Crossref PubMed Scopus (14) Google Scholar). These findings raise the possibility that H2S of both plant and microbial origin might be involved in regulating plant signaling pathways, promising further insights into plant stress signaling.