Chloroplast-Derived Photo-Oxidative Stress Causes Changes in H2O2 And

bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>12Short title: Transmission of ROS signals from chloroplasts 3456789Corresponding authors: Andreas J. Meyer Institute of Crop Science and Resource Conservation (INRES), Chemical Signalling, University of Bonn Friedrich-Ebert-Allee 144, D-53113 Bonn, Germany Phone: +49 228 73 60353 Email: <a href="mailto:[email protected]" target="_blank">[email protected] </a>1bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>10 Chloroplast-derived photo-oxidative stress causes changes in H2O2 and 11 EGSH in other subcellular compartments 12 Authors: 13 José Manuel Ugalde1, Philippe Fuchs1,2, Thomas Nietzel2, Edoardo A. Cutolo4, Ute C. 14 Vothknecht4, Loreto Holuigue3, Markus Schwarzländer2, Stefanie J. Müller-Schüssele1, 15 Andreas J. Meyer1,* 116 Institute of Crop Science and Resource Conservation (INRES), University of Bonn, 17 Friedrich-Ebert-Allee 144, D-53113 Bonn, Germany 218 Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, D- 19 48143 Münster, Germany 320 Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, 21 Pontificia Universidad Católica de Chile, Avda. Libertador Bernardo O’Higgins 340, 22 Santiago 8331150, Chile 23 4 Institute of Cellular and Molecular Botany (IZMB), University of Bonn, Kirschallee 1, D- 24 53115 Bonn, Germany 25 *Corresponding author: [email protected] 26 One sentence summary: Methyl viologen-induced photooxidative stress causes an increase 27 of H2O2 and oxidation of glutathione in chloroplasts, cytosol and mitochondria as well as 28 autonomous oxidation in mitochondria. 29 Keywords: Arabidopsis thaliana, Methyl viologen, Glutathione redox potential, H2O2, 30 Chloroplast, Mitochondria, Retrograde signaling, Interorganellar communication, Grx1- 31 roGFP2, roGFP2-Orp1, Reactive oxygen species. 2bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>32 Footnotes: 33 Author contributions: J.M.U., U.C.V., L.H., S.J.M-S. and A.J.M. conceived and designed 34 the experiments; J.M.U. performed the experiments; T.N. and E.A.C. contributed new 35 constructs and plant lines; J.M.U., P.F., M.S., S.J.M.-S. and A.J.M. analyzed the data; 36 J.M.U., S.J.M-S., and A.J.M. wrote the manuscript with contributions from all authors. 37 Funding information: 38 This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the 39 Research Training Group GRK 2064 “Water use efficiency and drought stress responses: 40 From Arabidopsis to Barley“ (A.J.M.; M.S.; S.J.M.-S.; U.C.V.), grants ME1567/9-1/2 and 41 SCHW719/7-1 within the Priority Program SPP1710 “Dynamics of thiol-based redox 42 switches in cellular physiology“ (A.J.M.; M.S.), the MSC-ITN project no. 607607 43 ‘CALIPSO’ (U.C.V.; E.A.C.), grant MU 4137/1-1 (S.J.M.-S.) and the Emmy-Noether 44 programme (SCHW1719/1-1; M.S.). 45 Present address: E.A.C.: Laboratory of Photosynthesis and Bioenergy, Department of 46 Biotechnology, University of Verona, Verona, Strada le Grazie 15, 37134 Italy 47 Author for correspondence: <a href="mailto:[email protected]" target="_blank">[email protected] </a>3bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>48 ABSTRACT 49 Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals 50 to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen 51 species (ROS), which can operate protein redox switches by oxidation of specific cysteine 52 residues. Redox buffers such as the highly reduced glutathione pool serve as reservoirs of 53 reducing power for several ROS scavenging and ROS-induced damage repair pathways. 54 Formation of glutathione disulfide (GSSG) and a shift of the glutathione redox potential 55 (EGSH) towards less negative values is considered a hallmark of several stress conditions. 56 Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of 57 intact Arabidopsis seedlings and recorded dynamic changes in EGSH and H2O2 levels with the 58 genetically-encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) 59 targeted to chloroplasts, the cytosol or mitochondria. Treatment of seedlings with MV caused 60 a rapid oxidation in chloroplasts and subsequently also in the cytosol and mitochondria. The 61 MV-induced oxidation was significantly boosted by illumination with actinic light and 62 largely abolished by inhibitors of photosynthetic electron transport. In addition, MV also 63 induced an autonomous oxidation in the mitochondrial matrix in an electron transport chain 64 activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the 65 combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics 66 of compartmental responses to local ROS generation and provide a basis for understanding 67 how compartment-specific redox dynamics may operate in retrograde signaling and stress 68 acclimation in plants. 4bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>69 INTRODUCTION 70 Communication between different subcellular compartments of plant cells is 71 fundamental to establish and sustain cooperative functioning and to acclimate to diverse 72 environmental conditions. Since most plastidial and mitochondrial proteins are encoded in 73 the nuclear genome, retrograde signals from the organelles to the nucleus are essential to 74 adjust organelle function by coordinating the expression of nuclear and organellar genomes 75 (Van Aken et al., 2016; de Souza et al., 2017; Dietz et al., 2019). Communication between 76 the endosymbiotic organelles and the nucleus is likely to involve the cytosol as the 77 intermediate compartment. However, chloroplasts can also make direct physical contact with 78 the nuclear envelope via stromules, which has been suggested to mediate signaling (Caplan et 79 al., 2015; Erickson et al., 2017; Exposito-Rodriguez et al., 2017). Physical interaction also 80 occurs between different organelles and may facilitate efficient exchange of metabolites and 81 information (Pérez-Sancho et al., 2016; Perico and Sparkes, 2018). 82 Reactive oxygen species (ROS), such as H2O2, have emerged as signaling molecules 83 in plants and their roles in early signaling events initiated by cellular metabolic perturbation 84 and environmental stimuli are established (Waszczak et al., 2018; Smirnoff and Arnaud, -85 2019). During unfavorable environmental conditions, superoxide (O2 ) is produced at an -86 increased rate by the electron transport chains (ETCs) in chloroplasts and mitochondria. O2 87 is rapidly converted to hydrogen peroxide (H2O2) and molecular oxygen (O2) by superoxide 88 dismutases (SODs). H2O2 can be further detoxified through a set of peroxidases, including 89 peroxiredoxins (PRX) (Liebthal et al., 2018)) several glutathione S-transferases (GST) 90 (Sylvestre-Gonon et al., 2019; Ugalde et al., 2020), glutathione peroxidase-like enzymes 91 (GPXL) (Attacha et al., 2017), and ascorbate peroxidases (APX). The latter operate as part of 92 the glutathione–ascorbate cycle in the plastid stroma, mitochondrial matrix, peroxisomes and 93 cytosol (Foyer and Noctor, 2005; Narendra et al., 2006). The transient drain of electrons from 94 the local glutathione redox buffer causes a concomitant increase in glutathione disulfide 95 (GSSG) and hence a change in the glutathione redox potential (EGSH) (Marty et al., 2009; 96 Bangash et al., 2019; Nietzel et al., 2019; Wagner et al., 2019). Intracellular H2O2 levels 97 reached under stress conditions can affect cellular redox regulation leading to the oxidation of 98 protein thiols (Dietz et al., 2016). H2O2 was shown to diffuse across the chloroplast envelope 99 even at low concentrations, and it has been estimated that about 5% of the total ROS 100 produced in high light leave the chloroplast (Mubarakshina et al., 2010). Those properties 5bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>101 contributed to the suggestion of H2O2 to operate as a messenger in signaling processes arising 102 from the organelles. Moreover, direct transfer of H2O2 from a subpopulation of chloroplasts 103 localized in close proximity to the nucleus itself was recently found to mediate 104 photosynthetic control over gene expression in tobacco leaves (Caplan et al., 2015; Exposito105 Rodriguez et al., 2017). 106 ROS production at specific sites of the photosynthetic ETC (pETC) can be artificially 107 enhanced by using inhibitors and redox catalysts. Among these, the herbicide methyl 108 viologen (MV) acts by re-directing electrons from photosystem I (PSI) to O2 and thereby 109 enhancing the production of O2 - (Scarpeci et al., 2008). Based on its mechanism, MV is also 110 useful as an experimental cue to induce photo-oxidative stress in photosynthetic organisms. -111 In mammals and other non-photosynthetic organisms, MV induces the generation of O2 by 112 re-directing electrons from complex I of the mitochondrial ETC (mETC) to O2 (Cochemé and 113 Murphy, 2008), suggesting that current models to study retrograde signaling are likely to be 114 more complex than previously expected and involve additional subcellular sites (Cui et al., 115 2019; Shapiguzov et al., 2019). Steady-state measurements in cotyledons of Arabidopsis 116 seedlings have previously shown that MV can induce oxidation in both cytosol and 117 mitochondria in the absence of illumination (Schwarzländer et al., 2009). 118 Chemical probes for detecting ROS in living systems, such as 2’,7’- 119 dihydrodichlorofluorescein diacetate are typically converted to a fluorescent product through 120 reaction with ROS and accumulate in tissues with different specificities for distinct forms of 121 ROS (Fichman et al., 2019). While these dyes provide evidence for redox processes and ROS 122 formation, a potential drawback is that those probes act irreversibly by generating an 123 accumulative signal rather than a reversible, dynamic response. Further, their lack of 124 unambiguous subcellular localization and chemical specificity make it frequently difficult to 125 draw mechanistic conclusions. During the last decade, genetically encoded biosensors have 126 revolutionized the field of cell physiology by being targetable to specific subcellular 127 compartments and enabling dynamic measurements (Morgan et al., 2016). Among them, 128 Grx1-roGFP2 for sensing EGSH (Marty et al., 2009) and roGFP2-Orp1 for sensing transient 129 changes in H2O2 (Nietzel et al., 2019); the latter being based on a redox relay between the 130 H2O2-sensitive Gpx3 peroxidase from yeast (Orp1) and roGFP2 (Gutscher et al., 2009). 131 These sensors have become instrumental to monitor the dynamics of oxidative signals in real132 time in a wide range of organisms, including plants. Similarly, probes of the HyPer family, 6bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>133 which exploit the H2O2-sensitive bacterial transcription factor OxyR for their response, can 134 report on local alterations in H2O2 concentrations (Bilan and Belousov, 2018; Pak et al., 135 2020). 136 Despite compelling evidence for the signaling functions of H2O2, it is neither known 137 how H2O2 concentrations and the redox buffers dynamically respond to increased ROS 138 production in chloroplasts nor how much other organelles contribute to a cumulative 139 oxidation in the cytosol. Here, we targeted two different roGFP2-based biosensors to the 140 stroma of the chloroplasts, the cytosol and the matrix of the mitochondria to live monitor the 141 local EGSH and H2O2 dynamics specifically in those three compartments. We investigated the 142 dynamic subcellular responses to primary oxidative events triggered by MV, light or a 143 combination of both. To dissect the contribution of chloroplasts and mitochondria in the MV- 144 induced overall oxidation, the respective ETCs were blocked using ETC-specific inhibitors 145 acting at early steps of electron transport. 146 7bioRxiv preprint <a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">doi: </a><a href="/goto?url=https://doi.org/10.1101/2020.07.20.212670" target="_blank">https://doi.org/10.1101/2020.07.20.212670</a>; this version posted July 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made <a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">available under a</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">CC-BY-NC-ND 4.0 International license</a><a href="/goto?url=http://creativecommons.org/licenses/by-nc-nd/4.0/" target="_blank">. </a>147 RESULTS 148 Spectral properties of roGFP2-based probes in planta 149 To visualize changes of EGSH or H2O2 levels in chloroplasts, cytosol and 150 mitochondria, we selected previously published Arabidopsis reporter lines with roGFP2 151 linked to Grx1 or Orp1, respectively (Marty et al., 2009; Park et al., 2013; Albrecht et al., 152 2014; Nietzel et al., 2019). Since no roGFP2-Orp1 reporter line for H2O2 sensing in the 153 plastid was available, we generated this line de novo (Supplemental Fig. S1). Subcellular 154 localization of all reporter constructs was verified side-by-side in 7-day-old seedlings by 155 confocal microscopy (Fig. 1A–F, left panels and Supplemental Fig. S1). Plants of the same 156 age were used to systematically corroborate the in vivo excitation spectra of both redox 157 sensors in all three compartments of intact seedlings. Sensor response and the dynamic 158 spectroscopic response range was assessed by recording the fluorescence of seedlings 159 immersed in imaging buffer using a fluorescence multiwell plate reader. Fluorescence spectra 160 were collected for non-treated seedlings and seedlings incubated with either 20 mM DTT for 161 complete reduction or with 100 mM H2O2 for complete oxidation of the sensors in situ (Fig. 162 1A–F, right panels). Sensor fluorescence intensities were sufficiently high to be clearly 163 distinguishable from background fluorescence with a suitable signal-to-noise ratio for in situ 164 readings (Supplemental Fig. S2). Fully reduced Grx1-roGFP2 (roGFP2-Grx1 in the case of 165 the mitochondria) or roGFP2-Orp1 showed low excitation at 400 nm and a pronounced 166 excitation peak close to 488 nm in all compartments. Probe oxidation led to the appearance of 167 a second distinct excitation peak close to 400 nm, while excitation at 488 nm was decreased 168 (Fig. 1A–F, right panels, Supplemental Fig. S2). The spectral behavior of both probes in 169 planta was consistent with the spectra of the purified roGFP2 in vitro (Fig. 1H). These data 170 validate that changes in the redox state of both roGFP2-based sensor variants can be reliably 171 visualized and recorded in chloroplast stroma, cytosol and mitochondrial matrix using plate 172 reader-based fluorimetry (Nietzel et al., 2019; Wagner et al., 2019). 173 Real-time monitoring of EGSH and H2O2 dynamics in Arabidopsis in response to 174 externally imposed oxidative stress 175 To further validate the responsiveness of both probes in planta, 7-day-old Arabidopsis 176 seedlings expressing cytosol-targeted Grx1-roGFP2 or roGFP2-Orp1 were exposed to 177 different concentrations of H2O2 <ul style="display: flex;"><li style="flex:1">(Fig. 2A, B) Changes in the </li><li style="flex:1">.</li><li style="flex:1">to impose oxidative stress </li></ul>

Chloroplast-Derived Photo-Oxidative Stress Causes Changes in H2O2 And

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