Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jun 13;16(1):136.
doi: 10.1186/s12870-016-0817-1.

Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase

Ewa Krzywińska  1   2 , Maria Bucholc  1 , Anna Kulik  1 , Arkadiusz Ciesielski  1   3 , Małgorzata Lichocka  1 , Janusz Dębski  1 , Agnieszka Ludwików  4 , Michał Dadlez  1   5 , Pedro L Rodriguez  6 , Grażyna Dobrowolska  7
Affiliations

Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase

Ewa Krzywińska et al. BMC Plant Biol. .

Abstract

Background: SNF1-related protein kinases 2 (SnRK2s) are key regulators of the plant response to osmotic stress. They are transiently activated in response to drought and salinity. Based on a phylogenetic analysis SnRK2s are divided into three groups. The classification correlates with their response to abscisic acid (ABA); group 1 consists SnRK2s non-activated in response to ABA, group 2, kinases non-activated or weakly activated (depending on the plant species) by ABA treatment, and group 3, ABA-activated kinases. The activity of all SnRK2s is regulated by phosphorylation. It is well established that clade A phosphoprotein phosphatases 2C (PP2Cs) are negative regulators of ABA-activated SnRK2s, whereas regulators of SnRK2s from group 1 remain unidentified.

Results: Here, we show that ABI1, a PP2C clade A phosphatase, interacts with SnRK2.4, member of group 1 of the SnRK2 family, dephosphorylates Ser158, whose phosphorylation is needed for the kinase activity, and inhibits the kinase, both in vitro and in vivo. Our data indicate that ABI1 and the kinase regulate primary root growth in response to salinity; the phenotype of ABI1 knockout mutant (abi1td) exposed to salt stress is opposite to that of the snrk2.4 mutant. Moreover, we show that the activity of SnRK2s from group 1 is additionally regulated by okadaic acid-sensitive phosphatase(s) from the phosphoprotein phosphatase (PPP) family.

Conclusions: Phosphatase ABI1 and okadaic acid-sensitive phosphatases of the PPP family are negative regulators of salt stress-activated SnRK2.4. The results show that ABI1 inhibits not only the ABA-activated SnRK2s but also at least one ABA-non-activated SnRK2, suggesting that the phosphatase is involved in the cross talk between ABA-dependent and ABA-independent stress signaling pathways in plants.

Keywords: ABI1; Arabidopsis thaliana; Osmotic stress signaling; PP2C; PPP; Phosphoprotein phosphatases; SNF1-related protein kinases 2; Salinity; SnRK2.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Analysis of interactions of selected members of the SnRK2 family with clade A PP2C phosphatases. a Yeast two-hybrid analysis; growth of yeast expressing the indicated constructs was monitored on selective media: without Leu and Trp (−LW); without Leu, Trp and His (−LWH) supplemented with different concentrations of aminotriazole (3-AT); without Leu, Trp and Ade (−LWA). AD, Gal4 activation domain; BD, Gal4 binding domain. Data represent one of three independent experiments showing similar results. b Pull down assays; GST-fused ABI1 or GST alone, bound to glutathione-sepharose beads, were incubated with recombinant SnRK2.4. Proteins bound to the resin were separated by SDS-PAGE, electroblotted to a membrane and visualized by immunoblotting using anti-SnRK2.4/SnRK2.10 antibodies. In parallel, visualization of proteins in samples used for the Western blotting was performed by staining with Coomassie Brilliant Blue, CBB. Data represent one of two independent experiments showing similar results
Fig. 2
Fig. 2
ABI1 interacts with SnRK2.4 in planta. Interaction of the proteins was analyzed by BiFC assay. Protoplasts isolated from Arabidopsis leaves were transiently co-transformed with pairs of plasmids encoding: ABI1-cEYFP and nEYFP-SnRK2.4 or ABI1-cEYFP and nEYFP-SnRK2.8 Positive control of the BiFC assay is provided by the ABI1-cEYFP/nEYFP-SnRK2.6 interaction. For negative control, cEYFP-ABI1 was co-expressed with nEYFP. Scale bar = 10 μm; BF, bright field. Data represent one of three independent experiments showing similar results
Fig. 3
Fig. 3
ABI1 inhibits kinases from all SnRK2 groups in vitro and in planta. a Recombinant SnRK2.4 (1 μg) was incubated with increasing amounts of PP2Cs for 30 min at 30 °C and kinase activity was analyzed by in-gel kinase assay using MBP as substrate. Dephosphorylation of SnRK2.4 was monitored by immunoblotting using specific anti-P-SnRK2 antibodies. Recombinant kinases SnRK2.8 and SnRK2.6 (as positive control) were incubated with increasing amounts of phosphatases for 30 min at 30 °C and the kinase activity was monitored by in–gel kinase assay with MBP as substrate. SnRK2 phosphorylation status was visualized by immunoblotting with specific anti-P-SnRK2 antibodies recognizing a specific phosphorylated residue in the kinase activation loop (Ser-158 in SnRK2.4, Ser-175 in SnRK2.6, and Thr-158 in SnRK2.8). In parallel, visualization of proteins in samples used for the Western blotting was performed by staining with Coomassie Brilliant Blue, CBB. Data represent one of two independent experiments showing similar results. b EGFP-SnRK2s were co-expressed with c-Myc-ABI1 in Arabidopsis protoplasts isolated from the T87 cell line. The protein level of kinases studied and ABI1 in extracts from protoplasts treated with 300 mM NaCl was monitored by immunoblotting; the activity of the kinases was analyzed by in-gel kinase activity assay using MBP as substrate. c Arabidopsis T87 cells and T87 cells expressing StrepTag-ABI1 were exposed to 500 mM NaCl. Activity of SnRK2.4/SnRK2.10 in the cell extracts was monitored by immunocomplex kinase activity assay, using MBP as substrate. The level of StrepTag-ABI1 and SnRK2.4/SnRK2.10 proteins was monitored by immunoblotting. Autorad, autoradiograph; CBB, Coomassie Brilliant Blue
Fig. 4
Fig. 4
ABI1 negatively regulates root elongation under salt stress. Seven-day-old seedlings grown vertically on ½ MS media were transferred into square Petri plates with control media or 115 mM NaCl and the increase in primary root length was measured. Dotted line shows the approximate length of roots just after transfer. The graphs present mean values (± SE), n = 7. Statistical analysis was done by t-test; bar on photographs = 0.5 cm
Fig. 5
Fig. 5
Pretreatment of T87 cells with OA enhances the salinity-induced activity of ABA-non-activated SnRK2s. Arabidopsis T87 cells were preincubated with indicated concentrations of OA for 2 h, followed by exposure to salinity stress. SnRK2.4/SnRK2.10 activity in cell extracts was monitored by immuno-in-gel kinase activity assay, using MBP as substrate and anti-SnRK2.4/SnRK2.10 antibodies. The level of IgG is presented as loading control. CBB, Coomassie Brilliant Blue. Data represent one of two independent experiments showing similar results
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Yamaguchi-Shinozaki K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol. 2006;57:781–803. doi: 10.1146/annurev.arplant.57.032905.105444. - DOI - PubMed
    1. Roychoudhury A, Paul S, Basu S. Cross-talk between abscisic acid-dependent and abscisic acid-independent pathways during abiotic stress. Plant Cell Rep. 2013;32(7):985–1006. doi: 10.1007/s00299-013-1414-5. - DOI - PubMed
    1. Yoshida T, Mogami J, Yamaguchi-Shinozaki K. ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. Curr Opin Plant Biol. 2014;21:133–139. doi: 10.1016/j.pbi.2014.07.009. - DOI - PubMed
    1. Fujii H, Zhu JK. Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc Natl Acad Sci U S A. 2009;106(20):8380–8385. doi: 10.1073/pnas.0903144106. - DOI - PMC - PubMed
    1. Nakashima K, Fujita Y, Kanamori N, Katagiri T, Umezawa T, Kidokoro S, Maruyama K, Yoshida T, Ishiyama K, Kobayashi M, et al. Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol. 2009;50(7):1345–1363. doi: 10.1093/pcp/pcp083. - DOI - PubMed

MeSH terms