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
. 2002 Dec;14(12):3089-99.
doi: 10.1105/tpc.007906.

Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production

Anna-Chiara Mustilli  1 , Sylvain Merlot, Alain Vavasseur, Francesca Fenzi, Jérôme Giraudat
Affiliations

Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production

Anna-Chiara Mustilli et al. Plant Cell. 2002 Dec.

Abstract

During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO(2), suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H(2)O(2) or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
ost1 Mutants Are Defective in the Regulation of Transpiration upon Water Stress. (A) False-color infrared image of drought-stressed plantlets. Twelve-day-old Ler wild-type (top row), ost1-1 (middle row), and ost1-2 (bottom row) plantlets were subjected to drought for 4 days. As calculated from the quantification of infrared images, the leaf temperatures were 24.7 ± 0.18°C for the wild type, 23.66 ± 0.24°C for ost1-1, and 23.21 ± 0.21°C for ost1-2 (means ± sd of measurements on ∼5000 square pixels). (B) Kinetics of water loss from detached leaves of the wild type (open circles), ost1-1 (closed diamonds), and ost1-2 (closed triangles). Water loss is expressed as the percentage of initial fresh weight. Values are means ± sd of four samples of three leaves each.
Figure 2.
Figure 2.
ost1 Mutations Impair the ABA Induction of Stomatal Closing and the ABA Inhibition of Stomatal Opening. Stomatal apertures were measured on epidermal peels of the wild type (white columns), ost1-1 (hatched columns), and ost1-2 (black columns). Values are means ± se (n = 60). (A) ABA-induced stomatal closing. Stomata were preopened in the light for 3 h and then incubated in the indicated concentrations of ABA for 3 h in the light. (B) Darkness-induced stomatal closing. Stomata were preopened in the light for 2.5 h and then incubated in darkness for 2.5 h. (C) ABA inhibition of light-induced stomatal opening. Stomata were preclosed in darkness and then incubated for 2.5 h in the light at the indicated concentrations of ABA. (D) Induction of stomatal opening by CO2-free air. Stomata were preclosed in darkness in normal air and then incubated for 3 h in CO2-free air in darkness.
Figure 3.
Figure 3.
Map-Based Cloning of the OST1 Gene. (A) Genetic mapping of 1024 chromosomes localized OST1 (white rectangle) to the 119-kb region between markers CER449763 on BAC F17I5 and CER451680 on BAC F28A23. (B) OST1 consists of 10 exons (rectangles). ost1-1 is a G-to-A change at nucleotide 1486 that disrupts the acceptor splice site of intron 6. ost1-2 is a G-to-A change at nucleotide 97 that converts Gly-33 to Arg. (C) False-color infrared image of detached leaves from the Ler wild type (wt), ost1-1 and ost1-2 mutants, and ost1-1 and ost1-2 mutants transformed with the wild-type At4g33950 gene. Functional complementation was observed in eight independent transgenic lines.
Figure 4.
Figure 4.
Structure of the OST1 Protein. (A) Alignment of the predicted amino acid sequences of the 10 members of the Arabidopsis OSKL family, V. faba AAPK, and barley PKABA. Residues conserved between OST1 and at least one other sequence are shaded black. Conserved subdomains of the protein kinase family are indicated by roman numerals. Positions of the ost1 mutations are shown. Dashed lines represent spaces that were introduced to maximize alignment. Arabidopsis Genome Initiative identification numbers of the OSKL family sequences are as follows: OST1 (At4g33950), OSKL2 (At5g66880), OSKL3 (At3g50500), OSKL4 (At1g78290), OSKL5 (At4g40010), OSKL6 (At1g60940), OSKL7 (At1g10940), OSKL8 (At5g08590), OSKL9 (At5g63650), and OSKL10 (At2g23030). (B) Relation tree of OSKL proteins from Arabidopsis and other plant species.
Figure 5.
Figure 5.
Expression Pattern of OST1. (A) RT-PCR analysis of OST1 mRNA expression in guard cell protoplasts (GCP), mesophyll cell protoplasts (MCP), leaf (L), flower (F), stem (S), and root (R) from 4-week-old plants and aerial parts of 2-week-old seedlings grown in vitro that were treated for 1 h with 10 μM ABA (+) or solvent control (−). EF1-α was used as an internal standard for cDNA amounts. (B) PCR amplification of OST1 (lane 1), OSKL2 (lane 2), and OSKL3 (lane 3) cDNAs with oligonucleotides specific for OST1 (A), OSKL2 (B), and OSKL3 (C). (C) Expression of pOST1::GUS in leaf tissue. (D) Expression of pOST1::GUS in leaf guard cells. (E) Expression of pOST1::GUS in root tissue.
Figure 6.
Figure 6.
OST1 Is an ABA-Activated Protein Kinase. (A) Protein kinase activities of root and guard cell protoplasts (GCP) from the Ler wild type (wt) and ost1-2 mutant were analyzed by an in-gel assay using Histone III-S as the substrate. Root extracts were obtained from 2-week-old seedlings treated for 3 h with 10 μM ABA (+) or solvent control (−). Guard cell protoplasts were treated for 20 min with 30 μM ABA (+) or solvent control (−). Sizes of molecular mass markers are shown at left. The arrow indicates the position of the ABA-activated 42-kD kinase. (B) Roots and guard cell protoplast extracts from the Ler wild type were analyzed by replacing CaCl2 with EGTA in the kinase buffer.
Figure 7.
Figure 7.
OST1 Acts Upstream of ROS Production. (A) ABA-induced ROS production. Changes in ROS levels were analyzed by measuring 2′,7′-dichlorofluorescein diacetate fluorescence levels in guard cells of the Ler wild type (wt) and ost1-2 after treatment with 50 μM ABA (black columns) or solvent control (white columns). Values are means ± se (n = 58). (B) H2O2-induced stomatal closing. Stomatal apertures were measured on epidermal peels of the wild type (white columns), ost1-1 (hatched columns), and ost1-2 (black columns). Stomata were preopened in the light for 2 h in the presence of 0.1 mM EGTA and then incubated for 2 h in 0.2 mM CaCl2, 0.1 mM EGTA, and the indicated concentrations of H2O2 (Pei et al., 2000). Values are means ± se (n = 60). (C) Extracellular Ca2+–induced stomatal closing. Stomatal apertures were measured on epidermal peels of the wild type (white columns), ost1-1 (hatched columns), and ost1-2 (black columns). Stomata were preopened in the light for 2 h and then incubated in the indicated concentrations of extracellular Ca2+ for 2 h. Values are means ± se (n = 60). (D) OST1 kinase activity. In-gel kinase assay of extracts from roots of the Ler wild type, ost1-2, and abi1-1 treated for 3 h with 10 μM ABA (+) or solvent control (−). The arrow indicates the position of the ABA-activated 42-kD kinase.
Figure 8.
Figure 8.
Model for the Position of OST1 in the ABA Signaling Cascade. This minimal linear model integrates the present results on ost1 mutants with previous data on gca2 (Pei et al., 2000) and abi1 and abi2 mutants (Murata et al., 2001). OST1 is an ABA-activated protein kinase. The protein phosphatases 2C (PP2C) ABI1 and ABI2 are negative regulators of ABA action. The GCA2 gene has not been cloned. The relative order of ABI2 and the gca2 mutation is purely speculative. ICa indicates cytosolic calcium increase.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allen, G.J., Kuchitsu, K., Chu, S.P., Murata, Y., and Schroeder, J.I. (1999). Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells. Plant Cell 11, 1785–1798. - PMC - PubMed
    1. Assmann, S.M., and Wang, X.-Q. (2001). From milliseconds to millions of years: Guard cells and environmental responses. Curr. Opin. Plant Biol. 4, 421–428. - PubMed
    1. Blatt, M.R. (2000). Cellular signaling and volume control in stomatal movements in plants. Annu. Rev. Cell Dev. Biol. 16, 221–241. - PubMed
    1. Blazquez, M.A., Soowal, L.N., Lee, I., and Weigel, D. (1997). LEAFY expression and flower initiation in Arabidopsis. Development 124, 3835–3844. - PubMed
    1. Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743. - PubMed

Publication types

MeSH terms

Associated data