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      Extracellular ATP elicits DORN1-mediated RBOHD phosphorylation to regulate stomatal aperture

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          Abstract

          In addition to acting as a cellular energy source, ATP can also act as a damage-associated molecular pattern in both animals and plants. Stomata are leaf pores that control gas exchange and, therefore, impact critical functions such as photosynthesis, drought tolerance, and also are the preferred entry point for pathogens. Here we show the addition of ATP leads to the rapid closure of leaf stomata and enhanced resistance to the bacterial pathogen Psuedomonas syringae. This response is mediated by ATP recognition by the receptor DORN1, followed by direct phosphorylation of the NADPH oxidase RBOHD, resulting in elevated production of reactive oxygen species and stomatal closure. Mutation of DORN1 phosphorylation sites on RBOHD eliminates the ability of ATP to induce stomatal closure. The data implicate purinergic signaling via DORN1 in the control of stomatal aperture with important implications for the control of plant photosynthesis, water homeostasis, pathogen resistance, and ultimately yield.

          Abstract

          Extracellular ATP acts as a damage-associated molecular pattern that triggers signaling responses to wounding and environmental stimuli in plants. Here Chen et al. show that ATP perception by DORN1 can trigger stomatal closure mediated via RBOHD phosphorylation and ROS production.

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          Most cited references46

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          Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response.

          Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica. We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica. Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.
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            CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis.

            Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis. The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola, was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.
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              NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis.

              Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca(2+) increases and ABA- activation of plasma membrane Ca(2+)-permeable channels in guard cells. Exogenous H(2)O(2) rescues both Ca(2+) channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.
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                Author and article information

                Contributors
                staceyg@missouri.edu
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                22 December 2017
                22 December 2017
                2017
                : 8
                : 2265
                Affiliations
                [1 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Division of Plant Science, C.S. Bond Life Science Center, , University of Missouri, ; Columbia, MO 65211 USA
                [2 ]ISNI 0000 0004 1790 4137, GRID grid.35155.37, State Key Lab of Agricultural Microbiology, College of Life Science and Technology, , Huazhong Agricultural University, ; Wuhan, 430070 China
                [3 ]ISNI 0000 0001 2162 3504, GRID grid.134936.a, Division of Biochemistry, C.S. Bond Life Science Center, , University of Missouri, ; Columbia, MO 65211 USA
                [4 ]Present Address: Center for Cancer Research Development, Proteomics Core Facility, Rhode Island Hospital,, Providence, RI 02903 USA
                Author information
                http://orcid.org/0000-0002-4715-4491
                http://orcid.org/0000-0002-2252-6551
                Article
                2340
                10.1038/s41467-017-02340-3
                5741621
                29273780
                7c165c91-331e-496e-8ae1-d5d569c25096
                © The Author(s) 2017

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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                : 11 April 2017
                : 21 November 2017
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