Probing plant signal processing optogenetically by two channelrhodopsins

Abstract

Early plant responses to different stress situations often encompass cytosolic Ca²⁺ increases, plasma membrane depolarization and the generation of reactive oxygen species^1-3. However, the mechanisms by which these signalling elements are translated into defined physiological outcomes are poorly understood. Here, to study the basis for encoding of specificity in plant signal processing, we used light-gated ion channels (channelrhodopsins). We developed a genetically engineered channelrhodopsin variant called XXM 2.0 with high Ca²⁺ conductance that enabled triggering cytosolic Ca²⁺ elevations in planta. Plant responses to light-induced Ca²⁺ influx through XXM 2.0 were studied side by side with effects caused by an anion efflux through the light-gated anion channelrhodopsin ACR1 2.0⁴. Although both tools triggered membrane depolarizations, their activation led to distinct plant stress responses: XXM 2.0-induced Ca²⁺ signals stimulated production of reactive oxygen species and defence mechanisms; ACR1 2.0-mediated anion efflux triggered drought stress responses. Our findings imply that discrete Ca²⁺ signals and anion efflux serve as triggers for specific metabolic and transcriptional reprogramming enabling plants to adapt to particular stress situations. Our optogenetics approach unveiled that within plant leaves, distinct physiological responses are triggered by specific ion fluxes, which are accompanied by similar electrical signals.

Fig. 1. Functional characterization of XXM 2.0, a channelrhodopsin variant with enhanced Ca²⁺ conductance.

a,b, Blue-light (473 nm, 3 mW mm⁻²)-activated calcium current (a) and reversal potential (V_r) shift (b) of X. laevis oocytes expressing XXM and XXM 1.1. Error bars show s.e.m., n = 6 (a) and 5 (b) cells of 2 oocyte batches. Significance was determined by two-sided Student’s t-test. ***P ≤ 0.001. c, Blue-light-induced photocurrents of XXM variants in X. laevis oocytes. Error bars show s.e.m., n = 6 cells of 2 oocyte batches. Significance was determined by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. Different letters indicate significant differences among samples (capital letters: P ≤ 0.01 and lowercase letters: P ≤ 0.05). d–f, Confocal images of leaf epidermis (d), and mesophyll cell membrane voltage of transgenic Ret-eYFP #1, Ret-XXM 1.2 #1 and Ret-XXM 2.0 #1 (e) and WT (f) N. tabacum leaves. Scale bars, 20 μm (d). n = 6 leaves of 2 batches of N. tabacum plants. Green bars indicate green light application (532 nm, 180 μW mm⁻²). g, Membrane potential changes of WT or transgenic N. tabacum leaves during green light irradiation (532 nm, 180 μW mm⁻²). Error bars show s.e.m., n = 10, 8, 10 and 10 leaves from 2 batches of N. tabacum plants. One-way ANOVA followed by a Dunnett T3 post hoc test was used to determine significance. h, Aequorin-luminescence recordings in N. benthamiana leaves following green light (520 nm, 50 µW mm⁻²) illumination. Error bars show s.e.m., n = 9, 7, 8 and 10 leaves from 2 batches of N. benthamiana plants. i, R-GECO1-based [Ca²⁺]_cyt changes in N. benthamiana mesophyll cells transiently expressing the denoted constructs following local green light (532 nm, 180 µW mm⁻²) illumination. Error bars show s.e.m., n = 16, 25 leaves from 5 batches of N. benthamiana plants. Scale bars, 50 μm. Source Data

Fig. 2. Distinct plant stress responses induced by ACR1 2.0 and XXM 2.0 stimulation.

a, Mesophyll cell depolarization induced by 60 s global green light illumination (520 nm, 9 µW mm⁻²) in WT, a transgenic Ret-eYFP line or in two Ret-ACR1 2.0 and two Ret-XXM 2.0 transgene plant lines. Error bars show s.e.m., n = 6 leaves of 2 batches of N. tabacum plant. One-way ANOVA followed by a Dunnett T3 post hoc test was used to determine significance. b, Mean R-GECO1 fluorescence change in transgenic N. tabacum mesophyll cells following global green light illumination (green bar). Error bars show s.e.m., n = 8 and 7 leaves from 2 batches of N. tabacum plants. c, Phenotypes of transgenic N. tabacum leaves after 24 h global green light treatment. Scale bars, 5 cm. n = 6 leaves of 2 batches of N. tabacum plants. d, Relative ion leakage from leaf tissue at different time points following global green light treatment. Error bars show s.e.m., n = 6 leaves from 2 batches of N. tabacum plants. e, ROS detection in N. tabacum leaves by diaminobenzidine staining. N. tabacum leaves were collected at indicated time points after global green light illumination. Scale bar, 5 cm. n = 5 leaves from 2 batches of N. tabacum plants. f, Simultaneous amperometric quantification of hydrogen peroxide (H₂O₂) dynamics and membrane potential (V_m) in transgenic N. tabacum leaves following global green light illumination. Error bars show s.e.m., n = 7, 8 and 10 leaves from 2 batches of N. tabacum plants. Source Data

Fig. 3. XXM 2.0 and ACR1 2.0 activation trigger distinct metabolite and hormone patterns.

a, Quantification of ABA in Ret-eYFP #1 transgenic N. tabacum plants at indicated time points after watering with 35% PEG, spray inoculation with Pst or 10 mM MgCl₂ as control. Error bars show s.e.m., n = 5 and 6 leaves from 2 batches of N. tabacum plants. b–d, ABA content in WT or transgenic N. tabacum plants at indicated time points upon constant (b), 4 h (c) or 1 h (d) global green light illumination (520 nm, 9 μW mm⁻²). Error bars show s.e.m., n = 4, 5, 6 and 7 leaves from 2 batches of N. tabacum plants. e, JA-Ile content in Ret-eYFP #1 transgenic N. tabacum plants following PEG, Pst or MgCl₂ treatment. Error bars show s.e.m., n = 4, 5 and 6 leaves from 2 batches of N. tabacum plants. f–h, JA-Ile content in WT or transgenic N. tabacum plants at different time points in response to constant (f), 4 h (g) or 1 h (h) global green light illumination. Error bars show s.e.m., n = 4, 5, 6 and 7 leaves from 2 batches of N. tabacum plants. i, SA content in Ret-eYFP #1 transgenic N. tabacum plants following PEG, Pst or MgCl₂ treatment. Error bars show s.e.m., n = 4, 5 and 6 leaves from 2 batches of N. tabacum plants. j–l, SA content in WT or transgenic N. tabacum plants at indicated time points in response to constant (j), 4 h (k) or 1 h (l) global green light illumination, with the inset in j showing a magnified view of the indicated time points. Error bars show s.e.m., n = 4, 5, 6 and 7 leaves from 2 batches of N. tabacum plants. The exact numbers of samples in a–l are listed in Supplementary Table 1. Source Data

Fig. 4. Rapid, reversible and divergent transcriptional reprogramming by optogenetic activation of ACR1 2.0 and XXM 2.0.

a–d, Time course of DEGs at 1, 4 and 8 h after 1 h or 4 h of green light stimulation (520 nm, 9 μW mm⁻²) in Ret-ACR1 2.0 (a) and Ret-XXM 2.0 (b) transgenic plants, or PEG-watered (c) and Pst-sprayed (d) control plants. e–m, Heat maps showing representative transcripts upregulated (red) or downregulated (blue) when ACR1 2.0 plants (upper heat map) or XXM 2.0 plants (lower heat map) were stimulated with green light. ACR1 2.0 and XXM 2.0 plants not stimulated with green light, at the 1 h time point (ACRC1h or XXMC1h), PEG8h or Pst8h served as biological controls. Representative DEGs associated with responses or pathways to ABA (e), water stress (f), photosynthesis (g), auxin (h), JA (i), SA (j), ethylene (k), ROS (l) and PCD (m) are shown. The expression levels of the DEGs are represented by z-score-normalized colour scales from blue (low expression) to red (high expression). Source Data

Extended Data Fig. 1. Improved channelrhodopsin variant XXM 2.0 with enhanced expression and plasma membrane targeting.

a, Scheme of DNA constructs used in this study. ChR2(DH), ChR2 D156H single mutant; eYFP, enhanced yellow fluorescent protein; ChR2(HQ/DH), ChR2 H134Q and D156H double mutant (XXM 1.1 for short in this study); LR, the cleavable N-terminal signal peptide Lucy-Rho; T, the plasma membrane trafficking signal from Kir2.1; E, the endoplasmic reticulum (ER) export signal from Kir2.1; RC2, a synthetic chloroplast transit peptide; MbDio, a β-carotene 15,15′-dioxygenase from a marine bacterium; P2A, the self-cleaving peptide from porcine teschovirus; ChR2(HQ/DH)-(N)11aa, a truncated ChR2(HQ/DH) mutant lacking 11 amino acids at the N terminus named XXM 2.0 when the LR, T and E sequences were added. Sequences of XXM 1.1 and XXM 2.0 are provided in the Supplementary File 1. b, Structure of ChR2 (PDB ID: 6EID) with highlighted retinal chromophore and molecular gates (red for extracellular molecular gates (ECG), magenta for center molecular gates (CG), orange for intracellular molecular gates (ICG), blue for ‘DC gate’). Note that D156 was covered by the calculated internal cavity (in grey) of ChR2. Residues in these gates with strong influence on ion selectivity of ChR2 were marked by asterisk*. Side-chain highlighted residues were selected for mutagenesis analysis. c, Photocurrent comparison of ChR2 variants triggered by blue light (473 nm, 3 mW mm⁻²) in Xenopus laevis oocytes. Photocurrent was normalized to the mean photocurrent of XXM 1.1. Error bars = s.e.m., n = 7, 7, 9, 6, 6, 8, 7, 8, 7, 7 cells from three batches of oocytes. Significance analysis was analyzed by One-way ANOVA followed by a Games-howell post hoc test. d, Reversal potential shift of ChR2 variants-expressing Xenopus laevis oocytes during blue light (473 nm, 3 mW mm⁻²) illumination. Error bars = s.e.m., n = 3 cells from two batches of oocytes. Significance analysis was performed by One-way ANOVA followed by a Tukey post hoc test. e, Representative confocal images of Xenopus laevis oocytes expressing different eYFP fused channelrhodopsin XXM variants as indicated above the images. Scale bar = 100 μm. n = 6 cells from two batches of oocytes. f, Photocurrents of XXM 2.0-expressing Xenopus laevis oocytes following light illumination of different wavelengths (399.3, 422, 440.7, 456, 479.5, 496, 516, 541, 562, 595 nm). Equal photon flux was set for each wavelength. The action spectrum of XXM 2.0 was normalized to photo-stimulation at 456 nm. Error bars = s.e.m., n = 4 from two batches of oocytes. Source Data

Extended Data Fig. 2. Transgenic N. tabacum plants grow like wild-type plants in red light condition.

a, WT, Ret-eYFP (#1 and #2), Ret-ACR1 2.0 (#1 and #2) and Ret-XXM 2.0 (#1 and #2) N. tabacum plants grown in constant red light (30 µW mm⁻²) for 45 days. Scale bar = 5 cm. n = 6 plants from two batches of N. tabacum plants. b, Transcript level of Ret-ACR1 2.0 (line #1, line #2) and Ret-XXM 2.0 (line #1, line #2) in transgenic plants. The transcript numbers of genes were normalized to 10,000 actin molecules. Error bars = s.e.m., n = 6 leaves from two batches of N. tabacum plants. c, Retinal concentrations measured in extracts from WT plants, Ret-eYFP, Ret-ACR1 2.0 and Ret-XXM 2.0-expressing N. tabacum lines; leaves were collected after 45 days’ grown in red light. Error bars = s.e.m., n = 10 leaves from two batches of N. tabacum plants. d, Carotenoid concentrations measured from the same batches of plants as in c. Error bars = s.e.m., n = 10 leaves from two batches of N. tabacum plants. One-way ANOVA followed by Dunnett T3 post hoc test was performed for significance analysis. e, Maximum quantum yield of energy conversion in leaves of WT, Ret-eYFP (line #1, line #2), Ret-ACR1 2.0 (line #1, line #2) and Ret-XXM 2.0 (line #1, line #2) N. tabacum plants grown under red light condition. Error bars = s.e.m., n = 10 leaves from three batches of N. tabacum plants. Significance analysis was analyzed by one-way ANOVA following a Tukey post hoc test. f, Dry weight of N. tabacum plants’ aboveground part when grown in red light for 45 days. Error bars = s.e.m., n = 7 plants from two N. tabacum plants batches. Significance analysis was analyzed by one-way ANOVA following a Tukey post hoc test. g, Representative confocal images of N. benthamiana leaf epidermal cells transiently expressing Ret-eYFP, Ret-XXM 1.2, Ret-XXM 1.3 and Ret-XXM 2.0. Images were taken 3 days post Agrobacterium infiltration, scale bar = 20 μm. n = 7 leaves from two batches of N. benthamiana plants. h, Representative confocal images of transgenic N. tabacum leaf epidermal cells expressing Ret-eYFP (line #2), Ret-XXM 1.2 (line #2), Ret-XXM 2.0 (line #2). Scale bar = 20 μm. n = 6 leaves from two batches of N. tabacum plants. i, Mesophyll cell plasma membrane depolarizations induced by 5 s green light (532 nm, 180 μW mm⁻²) were compared for WT, Ret-eYFP, Ret-XXM 1.2 and Ret-XXM 2.0-expressing transgenic N. tabacum plants (line #2 for all). Error bars = s.e.m., n = 9, 10, 11 leaves from two batches of N. tabacum. One-way ANOVA followed by a Games-Howell post hoc test was performed for the significance analysis. Source Data

Extended Data Fig. 3. Variations in cytosolic pH and Ca²⁺ by XXM 2.0 stimulation.

a, R-GECO1 fluorescence in N. benthamiana mesophyll protoplasts following green light (532 nm, 180 µW mm⁻²) illumination. Error bars = s.e.m., n = 12 samples of protoplast from 6 N. benthamiana leaves transiently expressing R-GECO1. n = 14 samples of protoplast from 7 N. benthamiana leaves transiently co-expressing Ret-XXM 2.0 and R-GECO1. Two batches of N. benthamiana plants were used. Scale bar = 50 μm. b, Mean pHuji fluorescence in pHuji and Ret-XXM 2.0 with pHuji transgenic N. tabacum mesophyll cells following global green light (520 nm, 9 µW mm⁻²) illumination as indicated by the green bar. A decrease of pHuji fluorescence indicates an increase in cytosolic H⁺ concentration. Error bars = s.e.m., n = 10 for Ret-XXM 2.0 with pHuji transgenic N. tabacum plants and n = 6 for pHuji transgenic N. tabacum plants. Three batches of N. tabacum plants were used. Note the identical steady-state pHuji fluorescence values in pHuji and Ret-XXM 2.0 with pHuji transgenic N. tabacum mesophyll cells during global green light (520 nm, 9 µW mm⁻²) illumination. c, Simultaneous recording of cytosolic Ca²⁺ levels ([Ca²⁺]_cyt) (R-GECO1 fluorescence, top) and plasma membrane potential (V_m) by intracellular microelectrodes (bottom) in Ret-XXM 2.0 with R-GECO1 transgenic N. tabacum mesophyll cell. Three technical replicates of local green light (2 s, 532 nm, 100 µW mm⁻²) illumination were applied to stimulate XXM 2.0 as indicated by the green bars below the traces. d, Mean changes in [Ca²⁺]_cyt (R-GECO1 fluorescence change, red) and V_m changes (black) following 3 technical replicates of local green light (2 s, 532 nm, 100 µW mm⁻²) irradiation. Error bars = s.e.m., n = 7 leaves from two batches of N. tabacum plants. One-way ANOVA followed by a Tukey post hoc test was performed for significance analysis. e, Representative traces of a simultaneous recording of [Ca²⁺]_cyt levels (R-GECO1 fluorescence, top) and V_m (bottom) in Ret-XXM 2.0 with R-GECO1 transgenic N. tabacum mesophyll cells following XXM 2.0 stimulation. Green light (2 s, 532 nm) with different light intensities (300, 150, 100, 50, 25 µW mm⁻²) was applied to stimulate XXM 2.0 as indicated by the green bars. f, Mean V_m change and mean [Ca²⁺]_cyt change (R-GECO1 fluorescence change) plotted against the green light intensities used in e. Error bars = s.e.m., n = 11 for R-GECO1 (Control in figure) and n = 15 for Ret-XXM 2.0 with R-GECO1 (Ret-XXM 2.0 in figure) transgenic plants. Three batches of N. tabacum plants were used. g, Relationship between V_m change and R-GECO1 fluorescence change, fitted with a linear function (data obtained from f). Error bars = s.e.m., n = 15 plants from three batches of N. tabacum plants. h, R-GECO1 fluorescence recordings in R-GECO1 and Ret-XXM 2.0 with R-GECO1 transgenic N. tabacum mesophyll cells following green light (532 nm, 50 µW mm⁻²) treatment with different durations (2 s, 4 s, 10 s, 60 s). Error bars = s.e.m., n = 11 for R-GECO1 and Ret-XXM 2.0 with R-GECO1 transgenic plants. Two batches of N. tabacum plants were used. i, R-GECO1 fluorescence change of Ret-XXM 2.0 with R-GECO1 transgenic N. tabacum mesophyll cells shown in h. Error bars = s.e.m., n = 11 plants. Two batches of N. tabacum plants were used. One-way ANOVA followed by a Dunnett T3 post hoc test was performed for significance analysis. Source Data

Extended Data Fig. 4. Plasma membrane potential and R-GECO1 fluorescence recording in N. tabacum plants.

a, Simultaneous recording of [Ca²⁺]_cyt (R-GECO1 fluorescence) and V_m in Ret-ACR1 2.0 with R-GECO1 transgenic N. tabacum mesophyll cells, global green light (520 nm, 9 μW mm⁻²) illumination was indicated by the green bar. Enlargement of the marked segment is shown in the blue box in the middle. A marked V_m change (~36 mV) was induced in Ret-ACR1 2.0 with R-GECO1 expressing mesophyll cell by a 50 ms 570 nm excitation light pulse which was used for R-GECO1 Ca²⁺-imaging. In contract, V_m recordings in Ret-XXM 2.0 with R-GECO1 transgenic N. tabacum mesophyll cells, shown in the lower blue box, does not induce considerable V_m changes (~2 mV) by 50 ms 570 nm excitation light during the R-GECO1 fluorescence Ca²⁺ imaging routine. Thus, it should be noted that combining Ca²⁺-imaging and optogenetic activation of Ret-ACR1 2.0 suffers from spectral overlap due to the red-shifted absorption spectrum of ACR1, which is not the case for Ret-XXM 2.0. b, Representative mesophyll membrane voltage traces in leaves of WT and transgenic Ret-eYFP (line #1), Ret-ACR1 2.0 (line #1) and Ret-XXM 2.0 (line #1) N. tabacum plants, 1 min global green light illumination was indicated by the green bar.

Extended Data Fig. 5. Phenotypes caused by ACR1 2.0 and XXM 2.0 activation or PEG and Pst treatment in transgenic N. tabacum plants.

a, Phenotype of Ret-eYFP #1 transgenic N. tabacum plants at different time points after the treatment with 10 mM MgCl₂ or Pst. Scale bar = 3 cm. Enlarged images of leaf areas in the red boxes are presented in the right-most figures. The blue arrows point to necrotic leaf parts. Scale bar = 3 cm. n = 6 plants from two batches of N. tabacum plants. b, Phenotype of Ret-XXM 2.0 #1 or Ret-XXM 2.0 #2 transgenic N. tabacum lines at the indicated time points at the bottom when exposed to global continuous green light (520 nm, 9 µW mm⁻²). Scale bar = 3 cm. Enlarged images of leaf areas in the red boxes are presented in the right-most figures. The blue arrows point to necrotic leaf parts. Scale bar = 3 cm. n = 6 plants from two batches of N. tabacum plants. c, d, Phenotypes of leaf discs in response to global continuous green light illumination. Leaf discs of (c) Ret-eYFP #1 and (d) Ret-XXM 2.0 #1 transgenic lines were floated either in 1 mM CaCl₂, 10 mM CaCl₂, 5 mM EGTA (pH 7.0, KOH), 5 mM K₄BAPTA or 5 mM K₄BAPTA + 10 mM NaCl solution in the dark for 1 h prior to the movement to global continuous green light condition. Pictures were captured after 24 h green light treatment. n = 7 leaves from two batches of N. tabacum plants. e−h Phenotype of (e) WT, (f) Ret-eYFP #1, (g) Ret-ACR1 2.0 #1, and (h) Ret-ACR1 2.0 #2 N. tabacum plants after different durations of constant global green light illumination, as indicated in the figures. Plants were grown in red light (650 nm, 30 μW mm⁻²) condition for 45 days before additional green light (520 nm, 9 μW mm⁻²) was applied at t = 0 h for the experimental group. Scale bar = 5 cm. n = 6 plants from two batches of N. tabacum plants. i, Phenotype of N. tabacum Ret-eYFP #1 control plants at different time points after watering with 35% PEG. Scale bar = 5 cm. n = 6 plants from two batches of N. tabacum plants. j, The fifth leaves of N. tabacum plants after global continuous green light or 35% PEG treatment. Photos were taken after 24 h treatment with green light or PEG. Scale bar = 5 cm. n = 6 plants from two batches of N. tabacum plants.

Extended Data Fig. 6. Ion leakage, voltage, [Ca²⁺]_cyt and ROS dynamics in N. tabacum leaves.

a, Relative ion leakage from N. tabacum leaf tissues at different time points after global green light (520 nm, 9 μW mm⁻²) illumination, spraying of 10 mM MgCl₂, Pst, or watering with 35% PEG. Error bars = s.e.m., n = 6 leaves from two batches of N. tabacum plants. b, c, R-GECO1 fluorescence change in R-GECO1 transgenic N. tabacum leaves in the (b) presence or (c) absence of Pst treatment, as indicated by the bar above the trace. Bath solution without Pst was used as the control solution (control). Error bars = s.e.m., n = 6 leaves from two batches of N. tabacum plants. d, ROS detection in N. tabacum leaves by diaminobenzidine staining. N. tabacum leaves were collected at different time points after 10 mM MgCl₂, Pst, or 35% PEG treatment. Scale bar = 5 cm. n = 5 leaves from two batches of N. tabacum plants. e, Mean staining intensities of whole leaves after the diaminobenzidine staining for ROS detection in d and Fig. 2e at the indicated time points. Error bars = s.e.m., n = 5 leaves from two batches of N. tabacum plants. Significance analysis was analyzed by One-way ANOVA following a Dunnett T3 post hoc or Tukey post hoc test. f, Amperometric quantification of hydrogen peroxide (H₂O₂) dynamics in transgenic N. tabacum leaves, global green light illumination was indicated by the green bar. Error bars = s.e.m., n = 6 for Ret-XXM 2.0 (line #1), n = 14 for Ret-ACR1 2.0 (line #1) and n = 4 for Ret-eYFP (line #1) plants. Two batches of N. tabacum plants were used. g, ROS production following 5 min global green light illumination detected by luminol chemiluminescence assay. The luminescence was normalized to the mean value of Ret-XXM 2.0 samples. Error bars = s.e.m., n = 5 leaves from two batches of N. tabacum plants. h, i, Example of simultaneous (h) V_m recording and (i) H₂O₂ recoding in Ret-XXM 2.0 transgenic N. tabacum leaves, global green light illumination was indicated by the green bar. j, R-GECO1 fluorescence in Ret-XXM 2.0 with R-GECO1-expressing N. tabacum mesophyll cells indicating cytosolic Ca²⁺ levels in response to global green light illumination as indicated by the green bar. Source Data

Extended Data Fig. 7. Distinct patterns of metabolites in N. tabacum plants following different treatments.

a, Proline content in WT and transgenic N. tabacum leaves during constant global green light (520 nm, 9 µW mm⁻²) illumination. Error bars = s.e.m., n = 4, 5 leaves from two batches of N. tabacum plants. b, Proline content in Ret-eYFP #1 transgenic N. tabacum leaves following watering 35% PEG as well as spraying 10 mM MgCl₂ or Pst at t = 0 h on the whole plants. Error bars = s.e.m., n = 5, 6 leaves from two batches of N. tabacum plants. c−g, Quantification of (c) ABA, (d) JA-Ile, (e) SA, (f) OPDA, and (g) JA in transgenic N. tabacum leaves in the absence (0 h) of green light treatment. Error bars = s.e.m., n = 5, 6 leaves from two batches of N. tabacum plants. h, Quantification of OPDA in Ret-eYFP #1 transgenic N. tabacum leaves at different time points after the treatment with 35% PEG, spray-inoculation with Pst or 10 mM MgCl₂ as control. Error bars = s.e.m., n = 5, 6 leaves from two batches of N. tabacum plants. i−k, OPDA content in WT or transgenic N. tabacum leaves at different time points with (i) constant, (j) 4 h or (k) 1 h global green light illumination. Error bars = s.e.m., n = 4, 5, 6, 7 leaves from two batches of N. tabacum plants. l, JA in leaves of Ret-eYFP #1 upon watering with 35% PEG as well as spraying 10 mM MgCl₂ or Pst. Error bars = s.e.m., n = 5, 6 leaves from two batches of N. tabacum plants. m−o, JA content in transgenic N. tabacum leaves with (m) constant, (n) 4 h, (o) 1 h global green light illumination. Error bars = s.e.m., n = 4, 5, 6, 7 leaves from two batches of N. tabacum plants. N. tabacum plants were grown in red light (650 nm, 30 μW mm⁻²) for 45 days and additional constant global green light (520 nm, 9 μW mm⁻²) was applied from t = 0 h. The exact sample size (n) for each experimental group was listed in Supplementary Table 1. Source Data

Extended Data Fig. 8. Venn diagram and gene ontology analysis showing the overlap of DEGs following different stimulation.

a, Illustration of the experimental design for the transcriptomic analysis in Ret-XXM 2.0 #1, Ret-ACR1 2.0 #1 and Ret-eYFP #1 control plants subjected to red light only, or illumination with an additional 1 or 4 h of green light (520 nm, 9 μW mm⁻²) followed by light shut-off. As physiological control to the ACR1 2.0 or XXM 2.0 light stimulation, Ret-eYFP #1 control plants were watered with 35% PEG or sprayed with Pst, respectively. As a negative control experiment to the Pst spraying treatment, Ret-eYFP #1 control plants were sprayed with buffer solution (10 mM MgCl₂) at t = 0 h. n = 3 plants from two batches of N. tabacum plants were collected at 0, 1, 4 and 8 h. b, c, Venn diagrams showing the overlap of (b) DEGs (differentially expressed genes) from Ret-eYFP #1 plants 8 h after PEG-treatment (PEG8h) with those from 1 or 4 h global green light treated Ret-ACR1 2.0 #1 plants (ACR1GL1h, ACR4GL4h) or (c) DEGs from Ret-eYFP #1 plants 8 h after Pst treatment (Pst8h) with DEGs from 1 or 4 h global green light treated Ret-XXM 2.0 #1 plants (XXM1GL1h, XXM4GL4h). Numbers highlighted in red represent up-regulated DEGs, numbers highlighted in blue denote down-regulated DEGs, and those highlighted in yellow exhibit inverse regulation. d, e, Venn diagrams showing the overlap of (d) gene ontology (GO) terms from PEG8h, ACR1GL1h and ACR4GL4h or (e) GO terms from Pst8h with XXM1GL1h and XXM4GL4h. f, g, Venn diagrams showing the overlap of (f) DEGs from ACR1GL1h with those from PEG8h or (g) DEGs from XXM1GL1h with those from Pst8h. Numbers highlighted in red represent up-regulated DEGs, numbers highlighted in blue denote down-regulated DEGs, and those highlighted in yellow exhibit inverse regulation. h, Bubble plot depicting prevalent significantly enriched GO terms of ACR1GL1h. P-values were indicated by color. The size of the bubble plot represents the numbers of DEGs mapped in the represented GO terms. i, Bubble plots of prevalent enriched GO terms shared between ACR1GL1h and PEG8h. P-values were indicated by color. The size of the bubble plot represents the numbers of DEGs mapped in the represented GO terms. j, Bubble plot depicting prevalent significantly enriched GO terms of XXM1GL1h plants. P-values were indicated by color. The size of the bubble plot represents the numbers of DEGs mapped in the represented GO terms. k, Bubble plots of prevalent enriched GO terms shared between XXM1GL1h and Pst8h. P-values were indicated by color. The size of the bubble plot represents the numbers of DEGs mapped in the represented GO terms. The image of the tobacco plant in a is adapted from TurboSquid.

Extended Data Fig. 9. Distinct transcription profiles and impaired photosynthesis induced by different treatments.

a, Maximum quantum yield of photosystem II energy conversion in Ret-eYFP #1, Ret-ACR1 2.0 #1 and Ret-XXM 2.0 #1 N. tabacum leaves following global green light (520 nm, 9 μW mm⁻²) treatment or in Ret-eYFP #1 leaves when plants were watered with 35% PEG or leaves were sprayed with 10 mM MgCl₂ or Pst. Error bars = s.e.m., n = 5, 6, 7, 9 leaves from two batches of N. tabacum plants. N. tabacum plants were grown in red light (650 nm, 30 μW mm⁻²) for 45 days and additional global green light, 35% PEG, 10 mM MgCl₂ or Pst treatment was applied from t = 0 h. b, c, Venn diagrams showing the (b) overlap of DEGs from ACR1GL1h and XXM1GL1h or (c) overlap between DEGs from ACR4GL4h and XXM4GL4h. Numbers highlighted in red represent up-regulated DEGs, numbers highlighted in blue denote down-regulated DEGs, and those highlighted in yellow exhibit inverse regulation. d, e, Bubble plots of prevalent enriched GO terms (d) shared between ACR1GL1h and XXM1GL1h or (e) shared between ACR4GL4h and XXM4GL4h. P-values were indicated by color. The size of the bubble plot represents the numbers of DEGs mapped in the represented GO terms. f-m, Relative expression levels of (f) stress related genes NtP5CS1B, (g) ABA synthesis gene NtNCED3, (h) ABA signaling pathway gene NtPP2C24, (i) JA synthesis genes NtLOX3, (j) NtAOS, (k) the specific ROS-controlled transcription factor NtZAT12, (l) SA synthesis gene NtPAL1A and (m) the hypersensitive maker HSR203J for Ret-eYFP (line #1), Ret-ACR1 2.0 (line #1) and Ret-XXM 2.0 (line #1) transgenic N. tabacum leaves after the indicated durations of global green light illumination. The transcript numbers of genes were normalized to 10,000 molecules of actin. Error bars = s.e.m., n = 4, 5 plants from two batches of N. tabacum plants. The exact sample size (n) for each experimental group was listed in Supplementary Table 1. Source Data

Extended Data Fig. 10. Local green light stimulation triggers a long distance electrical signal traveling in XXM plants.

a, Diagram of the experimental design for detecting long-distance traveling electrical signal induced by local green light (532 nm, 5.3 mW mm⁻²) illumination on Ret-eYFP #1 or Ret-XXM 2.0 #1 transgenic N. tabacum leaves. Green light illumination, indicated by the green spot, was applied through a black covered optical fiber directly on the main vein of the leaves. Surface potential was recorded at a distance of 5 cm from the local illumination site. b, c, Surface potential recordings of electrical waves in (b) Ret-eYFP #1 or (c) Ret-XXM 2.0 #1 transgenic N. tabacum leaves by applying 600 ms local green light illumination. Green arrows indicate the time when the 600 ms green light illumination applied. During green light treatment, no obvious electrical signal was detectable at the unilluminated region (see the electrical trace where the green arrow pointed). A depolarization event at a distance of 5 cm from the illuminated region was only detected in Ret-XXM 2.0 #1 transgenic N. tabacum about 30 s after green light treatment. d, e, The mean values of (d) amplitude and (e) duration of electrical waves triggered in Ret-XXM 2.0 #1 transgenic N. tabacum plants. Error bars = s.e.m., n = 18 leaves from 4 batches of N. tabacum plants. Source Data