Abstract
Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.
| Original language | English |
|---|---|
| Pages (from-to) | 19187-19192 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 116 |
| Issue number | 38 |
| DOIs | |
| Publication status | Published - Sep 17 2019 |
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Autophagy controls reactive oxygen species homeostasis in guard cells that is essential for stomatal opening. / Yamauchi, Shota; Mano, Shoji; Oikawa, Kazusato; Hikino, Kazumi; Teshima, Kosuke M.; Kimori, Yoshitaka; Nishimura, Mikio; Shimazaki, Ken ichiro; Takemiya, Atsushi.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 38, 17.09.2019, p. 19187-19192.Research output: Contribution to journal › Article › peer-review
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TY - JOUR
T1 - Autophagy controls reactive oxygen species homeostasis in guard cells that is essential for stomatal opening
AU - Yamauchi, Shota
AU - Mano, Shoji
AU - Oikawa, Kazusato
AU - Hikino, Kazumi
AU - Teshima, Kosuke M.
AU - Kimori, Yoshitaka
AU - Nishimura, Mikio
AU - Shimazaki, Ken ichiro
AU - Takemiya, Atsushi
N1 - Funding Information: ACKNOWLEDGMENTS. We thank Jyunichi Mano, Masaru Shibata, Tsuneaki Takami, Koichi Sugimoto, Shino Goto-Yamada, and Noriyuki Suetsugu for valuable discussion; Michitaro Shibata for technical information; Michito Tsuyama for equipment support; Ann Cuypers and Takahiro Ishikawa for providing vtc1-1, cad2-1, and vtc1-1 cad2-1 mutants; the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants; the Arabidopsis Biological Resource Center and NASC for providing seeds; the Model Plant Research Facility, National Institute for Basic Biology BioResource Center, and Spectrography and Bioimaging Facility, NIBB Core Research Facilities, for technical support; and the Iwate Biotechnology Research Center (IBRC) for providing MutMap pipeline. This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant numbers: 18H02468, 26711019, 15K14552 [to A.T.]; 19K16171 [to S.Y.]; 17K07457 [to S.M.]); the Japan Foundation for Applied Enzymology (to A.T.); the Cooperative Research Grant of the Plant Transgenic Design Initiative (PTraD) at Gene Research Center, University of Tsukuba (to A.T.); the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University (to A.T.); and NIBB Collaborative Research Program Number 17-518 (to K.O.). Funding Information: We thank Jyunichi Mano, Masaru Shibata, Tsuneaki Takami, Koichi Sugimoto, Shino Goto-Yamada, and Noriyuki Suetsugu for valuable discussion; Michitaro Shibata for technical information; Michito Tsuyama for equipment support; Ann Cuypers and Takahiro Ishikawa for providing vtc1-1, cad2-1, and vtc1-1 cad2-1 mutants; the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants; the Arabidopsis Biological Resource Center and NASC for providing seeds; the Model Plant Research Facility, National Institute for Basic Biology BioResource Center, and Spectrography and Bioimaging Facility, NIBB Core Research Facilities, for technical support; and the Iwate Biotechnology Research Center (IBRC) for providing MutMap pipeline. This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant numbers: 18H02468, 26711019, 15K14552 [to A.T.]; 19K16171 [to S.Y.]; 17K07457 [to S.M.]); the Japan Foundation for Applied Enzymology (to A.T.); the Cooperative Research Grant of the Plant Transgenic Design Initiative (PTraD) at Gene Research Center, University of Tsukuba (to A.T.); the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University (to A.T.); and NIBB Collaborative Research Program Number 17-518 (to K.O.). Funding Information: 26. Y. Liu, D. C. Bassham, Autophagy: Pathways for self-eating in plant cells. Annu. Rev. Plant Biol. 63, 215–237 (2012). Materials and Methods Plant materials and growth conditions, measurements of stomatal opening, isolation of guard cell protoplasts, measurement of H+ pumping, immunoblot analysis, identification of ATG2 gene, construction of transgenic plants, detection of ROS, detection of H2O2 in peroxisomes, confocal microscopy, measurement of CAT activity, preparation of recombinant proteins, and measurement of GOX activity are described in SI Appendix, SI Methods. ACKNOWLEDGMENTS. We thank Jyunichi Mano, Masaru Shibata, Tsuneaki Takami, Koichi Sugimoto, Shino Goto-Yamada, and Noriyuki Suetsugu for valuable discussion; Michitaro Shibata for technical information; Michito Tsuyama for equipment support; Ann Cuypers and Takahiro Ishikawa for providing vtc1-1, cad2-1, and vtc1-1 cad2-1 mutants; the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants; the Arabidopsis Biological Resource Center and NASC for providing seeds; the Model Plant Research Facility, National Institute for Basic Biology BioResource Center, and Spectrography and Bioimaging Facility, NIBB Core Research Facilities, for technical support; and the Iwate Biotechnology Research Center (IBRC) for providing MutMap pipeline. This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant numbers: 18H02468, 26711019, 15K14552 [to A.T.]; 19K16171 [to S.Y.]; 17K07457 [to S.M.]); the Japan Foundation for Applied Enzymology (to A.T.); the Cooperative Research Grant of the Plant Transgenic Design Initiative (PTraD) at Gene Research Center, University of Tsukuba (to A.T.); the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University (to A.T.); and NIBB Collaborative Research Program Number 17-518 (to K.O.). 27. Y. Wang, M. T. Nishimura, T. Zhao, D. Tang, ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis. Plant J. 68, 74–87 (2011). 28. K. Yoshimoto et al., Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell 21, 2914–2927 (2009). 29. M. S. Jahan et al., Deficient glutathione in guard cells facilitates abscisic acid-induced stomatal closure but does not affect light-induced stomatal opening. Biosci. Bio-technol. Biochem. 72, 2795–2798 (2008). 30. L. A. Del Río, E. López-Huertas, ROS generation in peroxisomes and its role in cell signaling. Plant Cell Physiol. 57, 1364–1376 (2016). 31. K. Yoshimoto, Y. Ohsumi, Unveiling the molecular mechanisms of plant autophagy-from autophagosomes to vacuoles in plants. Plant Cell Physiol. 59, 1337–1344 (2018). 32. S. Mano et al., Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: Dynamic morphology and actin-dependent movement. Plant Cell Physiol. 43, 331–341 (2002). 33. K. Yoshimoto et al., Organ-specific quality control of plant peroxisomes is mediated by autophagy. J. Cell Sci. 127, 1161–1168 (2014). 34. T. Hackenberg et al., Catalase and NO CATALASE ACTIVITY1 promote autophagy-dependent cell death in Arabidopsis. Plant Cell 25, 4616–4626 (2013). 35. J. Li et al., A chaperone function of NO CATALASE ACTIVITY1 is required to maintain cat-alase activity and for multiple stress responses in Arabidopsis. Plant Cell 27, 908–925 (2015). 36. A. Costa et al., H2O2 in plant peroxisomes: An in vivo analysis uncovers a Ca(2+)-dependent scavenging system. Plant J. 62, 760–772 (2010). 37. D. Winter et al., An “Electronic Fluorescent Pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS One 2, e718 (2007). 38. Y. Dellero et al., Decreased glycolate oxidase activity leads to altered carbon alloca-tion and leaf senescence after a transfer from high CO2 to ambient air in Arabidopsis thaliana. J. Exp. Bot. 67, 3149–3163 (2016). 39. P. J. Jewess, M. W. Kerr, D. P. Whitaker, Inhibition of glycollate oxidase from pea leaves. FEBS Lett. 53, 292–296 (1975). 40. Y. Dellero, M. Jossier, J. Schmitz, V. G. Maurino, M. Hodges, Photorespiratory glycolate-glyoxylate metabolism. J. Exp. Bot. 67, 3041–3052 (2016). 41. P. Christen, A. Gasser, Production of glycolate by oxidation of the 1,2-dihydroxyethyl-thamin-diphosphate intermediate of transketolase with hexacyanoferrate(III) or H2O2. Eur. J. Biochem. 107, 73–77 (1980). 42. D. H. McLachlan et al., The breakdown of stored triacylglycerols is required during light-induced stomatal opening. Curr. Biol. 26, 707–712 (2016). 43. J. Kim et al., Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth. Plant Cell 25, 4956–4966 (2013). 44. T. Jiang, X. F. Zhang, X. F. Wang, D. P. Zhang, Arabidopsis 3-ketoacyl-CoA thiolase-2 (KAT2), an enzyme of fatty acid β-oxidation, is involved in ABA signal transduction. Plant Cell Physiol. 52, 528–538 (2011). 45. S. Wada et al., Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol. 149, 885–893 (2009). 46. M. Izumi, H. Ishida, S. Nakamura, J. Hidema, Entire photodamaged chloroplasts are transported to the central vacuole by autophagy. Plant Cell 29, 377–394 (2017). 47. Q. Xie, S. Michaeli, H. Peled-Zehavi, G. Galili, Chloroplast degradation: One organelle, multiple degradation pathways. Trends Plant Sci. 20, 264–265 (2015). 48. J. M. Kwak et al., NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J. 22, 2623–2633 (2003). 49. I. C. Mori, R. Pinontoan, T. Kawano, S. Muto, Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba. Plant Cell Physiol. 42, 1383– 1388 (2001). Publisher Copyright: © 2019 National Academy of Sciences. All rights reserved.
PY - 2019/9/17
Y1 - 2019/9/17
N2 - Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.
AB - Reactive oxygen species (ROS) function as key signaling molecules to inhibit stomatal opening and promote stomatal closure in response to diverse environmental stresses. However, how guard cells maintain basal intracellular ROS levels is not yet known. This study aimed to determine the role of autophagy in the maintenance of basal ROS levels in guard cells. We isolated the Arabidopsis autophagy-related 2 (atg2) mutant, which is impaired in stomatal opening in response to light and low CO2 concentrations. Disruption of other autophagy genes, including ATG5, ATG7, ATG10, and ATG12, also caused similar stomatal defects. The atg mutants constitutively accumulated high levels of ROS in guard cells, and antioxidants such as ascorbate and glutathione rescued ROS accumulation and stomatal opening. Furthermore, the atg mutations increased the number and aggregation of peroxisomes in guard cells, and these peroxisomes exhibited reduced activity of the ROS scavenger catalase and elevated hydrogen peroxide (H2O2) as visualized using the peroxisome-targeted H2O2 sensor HyPer. Moreover, such ROS accumulation decreased by the application of 2-hydroxy-3-butynoate, an inhibitor of peroxisomal H2O2-producing glycolate oxidase. Our results showed that autophagy controls guard cell ROS homeostasis by eliminating oxidized peroxisomes, thereby allowing stomatal opening.
UR - http://www.scopus.com/inward/record.url?scp=85072275693&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85072275693&partnerID=8YFLogxK
U2 - 10.1073/pnas.1910886116
DO - 10.1073/pnas.1910886116
M3 - Article
C2 - 31484757
AN - SCOPUS:85072275693
VL - 116
SP - 19187
EP - 19192
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 38
ER -