Abstract
SNF1-related protein kinase 2 s (SnRK2s) are major regulators of plant growth, development and responses to environmental stresses. Together with clade A protein phosphatases of type 2C (PP2C) and REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR also known as PYRABACTIN RESISTANCE1 (PYR1) or PYR1-LIKE (PYL)) soluble abscisic acid (ABA) receptors they form the core of ABA-signaling. Clade A PP2Cs play a negative role in ABA signaling, primarily by inhibiting SnRK2 activity, through direct interaction and dephosphorylation of SnRK2s. Here, we describe two methods, which can be used for monitoring inhibition of the SnRK2 activity by PP2C phosphatases. One of them is an in vitro dephosphorylation assay using SnRK2 as the substrate followed by a classical in-gel kinase-activity assay and the other is immunocomplex kinase-activity assay, which can be applied for analysis of the SnRK2 activity in plant material.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fuchs S, Grill E, Meskiene I, Schweighofer A (2013) Type 2C protein phosphatases in plants. FEBS J 280(2):681–693
Schweighofer A, Hirt H, Meskiene I (2004) Plant PP2C phosphatases: emerging functions in stress signaling. Trends Plant Sci 9(5):236–243
Bhaskara GB, Wong MM, Verslues PE (2019) The flip side of phospho-signalling: regulation of protein dephosphorylation and the protein phosphatase 2Cs. Plant Cell Environ 42(10):2913–2930
Hsu PK, Dubeaux G, Takahashi Y, Schroeder JI (2021) Signaling mechanisms in abscisic acid-mediated stomatal closure. Plant J 105(2):307–321
Chen K, Li GJ, Bressan RA, Song CP, Zhu JK, Zhao Y (2020) Abscisic acid dynamics, signaling, and functions in plants. J Integr Plant Biol 62(1):25–54
Yoshida T, Christmann A, Yamaguchi-Shinozaki K, Grill E, Fernie AR (2019) Revisiting the basal role of ABA - roles outside of stress. Trends Plant Sci 24(7):625–635
Mao X, Li Y, Rehman SU, Miao L, Zhang Y, Chen X, Yu C, Wang J, Li C, Jing R (2020) The sucrose non-fermenting 1-related protein kinase 2 (SnRK2) genes are multifaceted players in plant growth, development and response to environmental stimuli. Plant Cell Physiol 61(2):225–242
Fàbregas N, Yoshida T, Fernie AR (2020) Role of Raf-like kinases in SnRK2 activation and osmotic stress response in plants. Nat Commun 11(1):6184
Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, Grill E (2009) Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 324(5930):1064–1068
Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S, Santiago J, Rodrigues A, Chow TF, Alfred SE, Bonetta D, Finkelstein R, Provart NJ, Desveaux D, Rodriguez PL, McCourt P, Zhu JK, Schroeder JI, Volkman BF, Cutler SR (2009) Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324(5930):1068–1071
Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, Cutler SR, Sheen J, Rodriguez PL, Zhu JK (2009) In vitro reconstitution of an abscisic acid signalling pathway. Nature 462(7273):660–664
Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol 61:651–679
Vlad F, Rubio S, Rodrigues A, Sirichandra C, Belin C, Robert N, Leung J, Rodriguez PL, Laurière C, Merlot S (2009) Protein phosphatases 2C regulate the activation of the Snf1-related kinase OST1 by abscisic acid in Arabidopsis. Plant Cell 10:3170–3184
Umezawa T, Sugiyama N, Mizoguchi M, Hayashi S, Myouga F, Yamaguchi-Shinozaki K, Ishihama Y, Hirayama T, Shinozaki K (2009) Type 2C protein phosphatases directly regulate abscisic acid-activated protein kinases in Arabidopsis. Proc Natl Acad Sci U S A 106(41):17588–17593
Soon FF, Ng LM, Zhou XE, West GM, Kovach A, Tan MH, Suino-Powell KM, He Y, Xu Y, Chalmers MJ, Brunzelle JS, Zhang H, Yang H, Jiang H, Li J, Yong EL, Cutler S, Zhu JK, Griffin PR, Melcher K, Xu HE (2012) Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases. Science 335(6064):85–88
Krzywińska E, Bucholc M, Kulik A, Ciesielski A, Lichocka M, Dębski J, Ludwików A, Dadlez M, Rodriguez PL, Dobrowolska G (2016) Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase. BMC Plant Biol 16(1):136
Krzywińska E, Kulik A, Bucholc M, Fernandez MA, Rodriguez PL, Dobrowolska G (2016) Protein phosphatase type 2C PP2CA together with ABI1 inhibits SnRK2.4 activity and regulates plant responses to salinity. Plant Signal Behav 12:e1253647
Frangioni JV, Neel BG (1993) Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210(1):179–187
Wang P, Zhu JK (2016) Assessing kinase activity in plants with in-gel kinase assays. Methods Mol Biol 1363:189–197
Acknowledgments
This work was supported by National Science Centre (grant 2011/03/B/NZ3/00297).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Krzywinska, E., Szymanska, K.P., Dobrowolska, G. (2022). Inhibition of SnRK2 Kinases by Type 2C Protein Phosphatases. In: Yoshida, T. (eds) Abscisic Acid. Methods in Molecular Biology, vol 2462. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2156-1_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2156-1_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2155-4
Online ISBN: 978-1-0716-2156-1
eBook Packages: Springer Protocols