Title: Single‐cell damage elicits regional, nematode‐restricting ethylene responses in roots
Abstract: Article6 May 2019free access Transparent process Single-cell damage elicits regional, nematode-restricting ethylene responses in roots Peter Marhavý orcid.org/0000-0002-0178-2230 Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Andrzej Kurenda Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Shahid Siddique Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author Valerie Dénervaud Tendon Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Feng Zhou Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Julia Holbein Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author M Shamim Hasan Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author Florian MW Grundler Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author Edward E Farmer Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Niko Geldner Corresponding Author [email protected] orcid.org/0000-0002-2300-9644 Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Peter Marhavý orcid.org/0000-0002-0178-2230 Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Andrzej Kurenda Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Shahid Siddique Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author Valerie Dénervaud Tendon Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Feng Zhou Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Julia Holbein Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author M Shamim Hasan Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author Florian MW Grundler Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany Search for more papers by this author Edward E Farmer Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Niko Geldner Corresponding Author [email protected] orcid.org/0000-0002-2300-9644 Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland Search for more papers by this author Author Information Peter Marhavý1, Andrzej Kurenda1, Shahid Siddique2,†, Valerie Dénervaud Tendon1, Feng Zhou1, Julia Holbein2, M Shamim Hasan2, Florian MW Grundler2, Edward E Farmer1 and Niko Geldner *,1 1Department of Plant Molecular Biology, Biophore, UNIL-Sorge, University of Lausanne, Lausanne, Switzerland 2Department of Molecular Phytomedizin, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany †Present address: Department of Nematology and Entomology, University of California, Davis, Davis, CA, USA *Corresponding author. Tel: +41 21 692 4191; E-mail: [email protected] *Corresponding author. Tel: +41 21 692 4192; E-mail: [email protected] EMBO J (2019)38:e100972https://doi.org/10.15252/embj.2018100972 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Plants are exposed to cellular damage by mechanical stresses, herbivore feeding, or invading microbes. Primary wound responses are communicated to neighboring and distal tissues by mobile signals. In leaves, crushing of large cell populations activates a long-distance signal, causing jasmonate production in distal organs. This is mediated by a cation channel-mediated depolarization wave and is associated with cytosolic Ca2+ transient currents. Here, we report that much more restricted, single-cell wounding in roots by laser ablation elicits non-systemic, regional surface potential changes, calcium waves, and reactive oxygen species (ROS) production. Surprisingly, laser ablation does not induce a robust jasmonate response, but regionally activates ethylene production and ethylene-response markers. This ethylene activation depends on calcium channel activities distinct from those in leaves, as well as a specific set of NADPH oxidases. Intriguingly, nematode attack elicits very similar responses, including membrane depolarization and regional upregulation of ethylene markers. Moreover, ethylene signaling antagonizes nematode feeding, delaying initial syncytial-phase establishment. Regional signals caused by single-cell wounding thus appear to constitute a relevant root immune response against small invaders. Synopsis Damage affecting large cell populations is communicated to neighboring and distal tissues by mobile signals in plants. This study reveals that single cell tissue damage in roots triggers a regional, non-systemic ethylene response that can act as a defense mechanism against microscopic invaders such as nematodes. Single root-cell wounding by laser ablation causes regional surface potential changes, calcium waves, and production of reactive oxygen species. Laser ablation in roots does not induce jasmonate responses, but regional ethylene production and signaling. Activation of ethylene signaling requires a distinct set of calcium channels and NADPH oxidases. Nematode attack elicits analogous responses to laser ablation. Ethylene signaling limits nematode feeding and delays initial syncytial phase establishment. Introduction Dissecting the molecular events occurring during actual attack of an invading organism is highly challenging because of the complex interplay of pattern-recognition events and damage perception, as well as the manipulation of the host's cellular physiology and immune response by the invader. Understanding plant responses to physical damage in isolation is therefore of crucial importance. Molecules reporting damage to plant cells, such as cell wall fragments and release of strictly intracellular agents from cells (e.g., ATP, or cytosolic peptides), are thought to be indicators of damage to the plant's cellular integrity (Choi et al, 2014; Duran-Flores & Heil, 2016). Yet, such isolated agents are often not sufficient to reproduce the responses occurring upon actual physical damage, although recent data suggest that extracellular glutamate might be a central agent reporting cellular damage (Toyota et al, 2018). Current ways to induce physical damage generally involve destruction of large populations of cells, affecting most or all of the cell types in a given organ (Mousavi et al, 2013; Toyota et al, 2018). While this has been powerful to elicit and study the systemic alarm signals to other organs, it has the shortcoming of confounding responses from many different cell types. In addition, it is only an appropriate simulation of damage caused by big insects or vertebrate herbivores, but might not be a good reflection of the more restricted and precise damage caused by a plethora of other attackers, such as small insects, nematodes, or necrotrophic microbes. We therefore pioneered single-cell laser ablations as a potentially powerful addition to the currently used techniques in order to understand the fundamental mechanisms of damage perception in plants. Arabidopsis seedling roots allow live observations and manipulations at exquisite resolution and precision. While they are popular for investigations in cell biology, development, and hormone perception (Benfey & Scheres, 2000), their use in investigating basic aspects of plant–pathogen interactions and plant defense responses has been more limited. This not only leads to a severe lack of understanding of the specificities of root versus leaf defense responses, but also represents a lost opportunity to harness the specific advantages of roots as models for understanding plant defense responses. In recent years, breakthroughs in root microbiome research have led to an increased interest in understanding root defense responses in the larger context of root biotic interactions (Hacquard et al, 2017). In a recent collaborative effort, we have generated a set of defense response marker lines, consisting of promoters responsive to major stress and defense response pathways driving a NLS triple mVenus fusion that allows for highly sensitive and cell type-specific readouts (Poncini et al, 2017). Combining these with our ability for single-cell laser ablation in roots (Marhavý et al, 2016), pioneered by van den Berg et al (1997), we set out to explore the specific responses of roots to precise cellular damage. Results Single-cell damage induces regional ethylene, but not jasmonate or salicylic acid production and response In leaves, mechanical crushing of large cell populations leads to membrane depolarization and jasmonate production in damaged as well as undamaged tissues (Mousavi et al, 2013). We investigated whether precise wounding of single cells using an infrared (IR) laser in the root would cause similar physiological responses. At first, we analyzed the jasmonic acid marker lines pJAZ10::NLS-3xVenus and pAOS::NLS-3xVenus (Park et al, 2002; Poncini et al, 2017), the JAS9-YFP jasmonate biosensor (Larrieu et al, 2015), and the salicylic acid marker line pPR1::NLS-3xVenus (Poncini et al, 2017). After ablation of cortex cells in 5-day-old roots, neither jasmonate nor salicylic acid reporter lines showed any consistent responses within 10 h (Fig 1A–L; Appendix Fig S1A–D). We confirmed functionality of jasmonate and salicylic acid marker lines by treating roots with 1 μM methyl jasmonate (MeJA) and 1 μM salicylic acid (SA). All jasmonate marker lines respond to MeJA treatment in roots (Fig 1A–L). Interestingly, the pPR1::NLS-3xVenus did not show any increase of signal in roots after treatment with 1 μM SA (Appendix Fig S1A–D). Yet, strong induction of pPR1::NLS-3xVenus expression could be observed in the cotyledons, confirming functionality of the line and suggesting that SA elicits a root response different from that in leaves (Appendix Fig S1E and F). In order to investigate whether the lack of jasmonate response observed is due to the fact that only single cells are damaged in our experiments, we mechanically crushed large population of root cells, similar to standard wounding done on leaves. Crushing of root tips (Appendix Fig S1G) induced jasmonate production and response genes to some degree, as visualized by our JAZ10, AOS reporter, and an additional LOX6::GUS reporter. LIPOXYGENASE 6 has been shown to be the major LOX enzyme for jasmonate production in roots (Grebner et al, 2013; Gasperini et al, 2015a,b). However, none of the markers showed a consistent, robust induction; i.e., only a fraction of the roots responded and with an amplitude much lower (Appendix Fig S1H–L) than seen upon jasmonate treatment (Fig 1A–H). The non-transcriptional, normalized intensometric JAS9 jasmonate sensor did not show any measurable response upon ablation (Fig 1I). Contrasting this, ablation of single epidermal cells of cotyledons did lead to a consistent induction of the pJAZ10::NLS-3xVenus (Fig 1M and N, Movie EV7). Thus, the paradigmatic jasmonate induction upon wounding observed in aerial tissues does not appear to play a similarly predominant role in roots. We then analyzed two markers reporting ethylene synthesis and signaling, pACS6::NLS-3xVenus and pPR4::NLS-3xVenus (Liu & Zhang, 2004; Proietti et al, 2011), both of which we confirmed to be expressed and functional in roots (Appendix Fig S2A–F). Here, both lines responded to cortex cell ablations in the root (Fig 2A–D; Appendix Figs S2A–F and S3C–J). Consistent with previously reported expression and response patterns (Tsuchisaka & Theologis, 2004), ACS6 responses were not exclusive to, but very much biased toward, stele tissues, while PR4 responses were largely confined to the endodermis. ACS6 was also robustly induced by crushing of root tips, contrasting with the inconsistent induction of jasmonate markers described above (Appendix Fig S2G–I). Time-lapse imaging revealed that 2–3 h after cortex ablation, both pACS6::NLS-3xVenus signal intensity and the number of cells with mVenus signal increased significantly compared to control (Appendix Fig S3). However, time-lapse imaging itself causes significant induction of the ethylene-response genes in controls, partially leveling out the ablation response (Appendix Fig S3C–J). For unknown reasons, propidium iodide (PI) cell wall staining during imaging alleviated this problem (compare Appendix Fig S3C and D). We nevertheless avoided both long-term time-lapse imaging and use of PI in most experiments and confined ourselves to one time point, thus keeping background induction to a minimum. Interestingly, when measuring the spatial extent of the response, we found that our single-cell ablations upregulated ACS6 and PR4 in a regional, but non-systemic, fashion, encompassing a region of about 500 μm for ACS6 (Appendix Fig S4A and B). Cell ablation of a single cortical cell thus appears to be able to induce stress hormone production and response in a considerable number of neighboring endodermis and stele cells and to extend bi-directionally along the root axis over a number of cellular distances, begging the question as to the nature of the mobile signal that could mediate this effect. Figure 1. Jasmonate does not respond to single-cell laser ablation A–H. Propidium iodide (PI) staining of roots (red) allowed visualization of cell death (white arrowhead) by increase in PI fluorescence. (A–C, E–G) Real-time monitoring of 4D (xyzt) maximum projection images of jasmonate response marker lines JAZ10::NLS-3xVenus (A–C) and AOS::NLS-3xVenus (E–G) in the Arabidopsis root after laser ablation of cortex cells (C, G) on 1 μM methyl jasmonate (MeJA) treatment (B, F). Time-lapse images of representative movies are shown. (A, C) JAZ10::NLS-3xVenus showed no response, either in control roots or after ablation (n = 20 roots each). (E, G) AOS::NLS-3xVenus showed weak signals in 5 roots (n = 20) after ablation (G), similar to numbers in non-ablated controls, where 5 roots (n = 17) showed signals. (B, F) Both jasmonate markers showed responses in roots upon 1 μM MeJA treatment (n = 20). (D, H) Graphical representation of quantification of movies shown in (A–C, E–G), respectively. I–L. Ratiometric 35S::JAS9-Venus H2B-RFP biosensor in the Arabidopsis root before/after laser ablation of cortex cells (I) or treated with 1 μM MeJA (L). No response was observed after 20 min, either in control roots or after ablation, while the sensor responded to treatment with 1 μM MeJA, but not to a water control (J). (K, L) Representative picture of experiments quantified in (J). Graphs combine data from 2 experiments with n = 10 roots (error bars indicate standard error; **P < 0.002, the significance was determined by t-test). M, N. XYZ maximum projection images show the expression of JAZ10::NLS-3xVenus in PI-stained Arabidopsis cotyledons before (M) and after ablation (N). Expression of JAZ10::NLS-3xVenus was observed in 8 cotyledons (n = 9) in ablated and 1 cotyledon (n = 7) in non-ablated control. A white arrow indicates an ablated epidermal cell, and yellow arrows indicate nuclear JAZ10::NLS-3xVenus signals. Time points are indicated at the top right corner of each frame. Data information: Scale bar, (A–C, E–G, K, L) 70 μm and (M, N) 100 μm. Download figure Download PowerPoint Figure 2. Single-cell ablation induces ethylene responses and local surface depolarization A, B. 3D tile-scan (xyz) maximum projection images of ACS6::NLS-3xVenus ethylene biosynthesis marker line in the Arabidopsis root. Time points at the top right corner of each frame. Non-ablated control root (A) and root with cortex cell ablation (B). White arrowhead indicates position of ablated cell. Representative pictures of experiments quantified in (C, D). C, D. Laser ablation of single cortex cells induces ethylene biosynthesis marker ACS6 (ACS6::NLS-3xVenus), visualized as increases of number of cells with detectable nuclear signal (C), or increase in average signal intensity (D) (**P < 0.001, the significance was determined by t-test, pool of three repeats with n = 5 roots). E. Schematic representation of experimental setup for detecting surface potential changes after laser ablation. F, G. Electrophysiological recording of surface potential changes measured with a non-invasive electrode placed on the root surface with or without laser ablation of cortex cells. Laser ablation-induced depolarization, amplitude in mV (G) quantification of depolarization amplitude (***P < 0.0005, the significance was determined by t-test, pool of three repeats with n = 21 roots). H. Surface potential changes over varying distances of different cortex cells within the same root. Arrowheads indicate the distance of electrode placement from the ablation site. I–L. Average lag time of maximum depolarization after laser ablation (n = 31 roots). (K) Representative read of experiments quantified in (J). Duration of laser pulse indicated by black lines; red lines indicate lag between maximum depolarization and the end of laser pulse. (I, L) Comparison of surface potential changes caused by laser ablation of cortex cells (black arrowheads) with effect of application of the same laser power on (I) media next to the root and (L) on the cortex cell with reduced laser power (red arrowhead). Data information: Error bars in C, D, G, and K indicate 95% confidence interval (CI) around mean. Scale bar: (A, B) 100 μm. See also Fig 4, Appendix Fig S2, and Appendix Fig S3. Download figure Download PowerPoint Single-cell ablation causes a regional surface depolarization of roots Wound signals eliciting stress responses in unwounded cells have been described for decades, yet the nature of the mobile agent and the mechanism of signal propagation have often remained hotly debated. One common feature that has emerged to be associated with—and required for—transmission of diverse stress signals is a propagating increase of intracellular calcium, ROS production, and membrane depolarization (Gilroy et al, 2014). Often, such signals are of systemic nature, i.e., propagating through an entire organ or plant. Yet, their elicitation also involves exposure of a large group of cells to a stress, such as parts of one leaf or an entire root meristem. We wondered whether the exquisitely localized damage of one cell and the more restricted ethylene response we observe would also be associated with the same trio of molecular events. We therefore placed a non-invasive electrode on the root surface (epidermis), opposite to the side of cell ablation (Fig 2E). Upon ablation, we measured surface potential changes of −79.5 ± 6.4 mV (Fig 2F and G). Interestingly, these depolarization spikes triggered by laser ablation of single cells are of comparable amplitude to those measured in aerial tissues (Mousavi et al, 2013), although of much shorter duration. We also mapped the distance over which the electrical signal would be transmitted and found that it was still detectable at 200 μm, but absent beyond 400 μm (Fig 2H), strongly differing from the centimeter distances measured in aerial parts of the plants (Mousavi et al, 2013). In order to exclude that these surface potential changes were simply a result of the heat load applied by the two-photon laser, we performed “mock ablations” on the growth medium in close proximity to the root (Fig 2I, red arrowhead). Media heating by the laser only caused small changes (Fig 2I, red arrowhead), not comparable to those induced by cell ablation (Fig 2I, black arrowhead). The same was true for applying IR laser at doses that only heated, but did not ablate cortex cells (Fig 2J). The lag between the end of the 2-s laser pulse and the depolarization peak was 1.5 ± 0.6 s, translating into a maximal estimated signal speed of 85 μm/s, assuming a distance of 128 μm between ablated cortex cell and electrode, and a minimal speed of 37 μm/s, assuming immediate ablation of the cortex at the beginning of the pulse (Fig 2K and L). The short-distance electric signaling depends on multiple ion channel activities Changes in ion channel and pump activities are the major determinants of cell membrane electrical changes in plants (Pickard & Ding, 1993; Véry & Sentenac, 2002; Shomer et al, 2003; Kinraide, 2006; Mishra et al, 2013; Lim et al, 2015; Catterall et al, 2017; Flucher & Tuluc, 2017; Perez Garcia et al, 2017). Indeed, calcium channel inhibitors, such as methoxyverapamil or GdCl3, efficiently reduced or blocked depolarization after cell ablation, as did inhibitors of chloride and potassium channels and proton pumps (Fig 3A and B). Fusicoccin, by contrast, a well-described activator of plant plasma membrane proton pumps (Würtele et al, 2003), did not reduce, but rather enhanced the depolarization amplitude after ablation (Fig 3C). These results suggest that the known major ions underlying plant cell transmembrane potentials are also required for the ablation-induced depolarization that we observe here. Interestingly, the glutamate receptor-like channels GLR3.3 and GLR3.6, shown to be necessary for transmitting surface potential changes to distal leaves after leaf wounding (Mousavi et al, 2013), were not involved in mediating the regional depolarization after single-cell ablation observed here (Appendix Fig S5A), suggesting that other GLR family members might mediate this calcium inhibitor-sensitive depolarization. Figure 3. Ion channel inhibitors affect ablation-induced depolarization and cytosolic calcium increases A–C. Recording and quantification of surface depolarization amplitudes in 5-day-old Arabidopsis roots after cortex cell ablation under ion channel inhibitor and fusicoccin treatments. Anthracene-9-carboxylic acid (A9C, 50 μM): chloride channel blocker; GdCl3 (50 μM): calcium channel blocker; vanadate (50 μM): non-specific pump inhibitor; N,N-dicyclohexylcarbodiimide (DCCD, 50 μM): proton channel blocker, proton pump inhibitor; tetraethylammonium (TEA, 50 μM): potassium channel blocker; (C) fusicoccin (5 μM): proton pump activator; (B, C) quantification of read examples shown in (A) (**P < 0.005, ***P < 0.0005; the significance was determined by t-test, n = 15–20 roots, repeated three times; error bars indicate mean value with 95% CI). D–I. Real-time monitoring and quantification (D) of calcium wave propagation after cortex cell ablation using a R-GECO1 reporter line (repeated two times, each with n = 20 roots); error bars indicate standard error, and time-lapse images of representative movies are shown. (E, F) Comparison of calcium wave caused by laser ablation of cortex cells (F) with effect of reduced laser power (heated) application on the cortex cell (G). (E–I) Time points in seconds (″) at the top right corner of each frame. Signal increases after ablation at opposite root side show slight lag compared to ablated root side. In non-ablated control roots, no increases of signal were observed; the same applies for 50 μM GdCl3-treated roots after ablation. White arrowheads indicate ablation position, green arrowhead indicates heated cell, yellow arrowheads indicate calcium wave propagation, and red frame indicates region of signal quantification in (D). Data information: Scale bar: (E–I) 100 μm. See also Figure 5, Appendix Fig S5, and Movie EV1. Download figure Download PowerPoint Single-cell ablation induces regional calcium waves Stress stimuli such as PAMPs, salt, or mechanical damage have been shown to induce a Ca2+ wave that travels through the root for long distances (Steinhorst & Kudla, 2014; Choi et al, 2016; Gilroy et al, 2016). A number of genetically encoded live-imaging probes for intracellular calcium are available (Albrecht et al, 2003; Pandey et al, 2004; Monshausen et al, 2008; Matzke & Matzke, 2015), and we chose the widely used, intensometric R-GECO1 (Keinath et al, 2015) sensor as well as the intensity-based concentration sensor Case12 (Matzke & Matzke, 2015) in order to test whether we could observe a local Ca2+ wave upon single-cell laser ablation. We performed ablation of cortical cells in R-GECO lines (Fig 3D–I and Movie EV1). Interestingly, we could observe Ca2+ wave propagation starting at the ablation side, arriving with a slight delay at the opposite cortex side (Fig 3E, Movie EV1). When the cortical signals have already dissipated, a slower, more persistent propagation of the calcium signal was observed within the stele (Movie EV1). Increases of calcium signal could also be observed upon crushing of entire root tips (Appendix Fig S6A and B). Upon mere heating of single cells, the Ca2+ signal was weaker and stayed confined to the heated cell (Fig 3G, Movie EV6), similar to touch-induced calcium responses (Monshausen et al, 2009). Both basal and ablation-induced R-GECO signals were abrogated upon GdCl3 treatment of roots (Fig 3H–I). Using the Case12 marker (Appendix Fig S5B–E) gave very similar results than those using R-GECO. Calcium and ROS both contribute to local, damage-induced ethylene responses We next tested whether inhibiting cytosolic calcium increases would suppress induction of ethylene-response markers upon cell ablation. Indeed, after ablation of cortex cells, pACS6::NLS-3xVenus response was attenuated in GdCl3-treated seedlings (Fig 4A–F), indicating that cytosolic calcium increases contribute to, but are not fully required for induction of ethylene responses in neighboring cells. Similar to calcium, increases in reactive oxygen species (ROS) can be generated in response to wounding, pathogen attack, or a local abiotic stress and be transmitted through the entire plant, often in an intricate, not fully understood, feedback loop with calcium (Miller et al, 2009). We investigated the contribution of ROS to the surface potential changes by analyzing Arabidopsis lines affected in ROS production by plasma membrane localized NADPH oxidases: rbohA, rbohD, rbohF, and rbohDF double mutants (Torres et al, 2002; Lee et al, 2013). We observed a reduced depolarization amplitude in rbohD and F mutants, but not in rbohA mutants (Fig 5A). In line with the effects of the mutants, depolarization amplitude was also reduced by diphenyleneiodonium (DPI), an NAD(P)H oxidase inhibitor, as well as potassium iodide (KI), an H2O2 scavenger (Lee et al, 2013; Fig 5B). We visualized ROS production after laser ablation, by taking advantage of H2DCFDA, a fluorescent indicator of ROS accumulation (Shin et al, 2005). After ablation, H2DCFDA signals increased significantly in wild-type roots (Fig 5C–E; Appendix Fig S5F and G), while a much-reduced response was observed already in single rboh mutants, as well as double mutants (Fig 5E; Appendix Fig S5L–Q). DPI and KI treatment completely abrogated H2DCFDA signals (Fig 5C and D; Appendix Fig S5H–K). Since calcium increases were only partially required for induction of ethylene production and response, we wondered whether ROS might be required. Indeed, the ablation-induced increases in calcium signals were blocked by DPI treatment (Fig 6A) and we could observe block in pACS6 induction upon DPI and KI treatment (Fig 6B–D), as well as a decrease in pACS6::NLS-3xVenus signals in rbohF compared to wild-type roots (Fig 6E–F). Our data suggest that laser ablation of single root cells induces a similar, albeit more localized, trio of depolarization, calcium influx, and ROS production, as is observed upon other stresses. However, in our case, both ROS production and calcium increases appear to feed into the induction of ethylene production and response. Figure 4. Laser ablation-induced ethylene responses are partially dependent on calcium increases A–D. XYZ maximum projection images of ACS6::NLS-3xVenus ethylene biosynthesis marker line in the Arabidopsis root after laser ablation with or without GdCl3 (50 μM). Time points in hours (h) at the top right corner of each frame. White arrowheads indicate position of cortex cell ablation. Representative pictures are shown. E, F. Signal intensity quantification and number of cells with positive nuclear (NLS-3xVenus) signal increases after ablation in control, but increases are reduced upon GdCl3 (50 μM) inhibitor treatment (*P < 0.05, the significance was determined by t-test, data pooled from three independent experiments with n = 10 roots each; error bars indicate mean value with 95% CI). Data information: Scale bar, (A–D) 100 μm.