Probing plant signal processing optogenetically by two channelrhodopsins

Meiqi Ding; Yang Zhou; Dirk Becker; Shang Yang; Markus Krischke; Sönke Scherzer; Jing Yu-Strzelczyk; Martin J Mueller; Rainer Hedrich; Georg Nagel; Shiqiang Gao; Kai R Konrad

doi:10.1038/s41586-024-07884-1

. 2024 Aug 28;633(8031):872–877. doi: 10.1038/s41586-024-07884-1

Probing plant signal processing optogenetically by two channelrhodopsins

Meiqi Ding ^1,^#, Yang Zhou ^2,^4,^#, Dirk Becker ^1,^#, Shang Yang ^2,^#, Markus Krischke ³, Sönke Scherzer ¹, Jing Yu-Strzelczyk ², Martin J Mueller ³, Rainer Hedrich ^1,^✉, Georg Nagel ^2,^✉, Shiqiang Gao ^2,^✉, Kai R Konrad ^1,^✉

PMCID: PMC11424491 PMID: 39198644

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.

Subject terms: Plant signalling, Plant physiology, Plant stress responses, Optical imaging

Using new optogenetic tools to induce distinct ion fluxes, a study shows that these discrete signals trigger different metabolic and transcriptional pathways that allow plants to respond to specific types of stress.

Main

When threatened, sessile plants respond rapidly with metabolic switches and genetic reprogramming to environmental cues^5–7. Electrical, cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and reactive oxygen species (ROS) signals are among the earliest plant reactions observed with stressors as diverse as wounding, pathogen attack, water and salt stress, and are suggested to be intertwined^1–3. After pathogen attack or wounding, jasmonic acid (JA) is rapidly synthesized, playing a crucial role in balancing plant growth and defence. In this trade-off scenario, JA and salicylic acid (SA) act antagonistically in the control of immunity and programmed cell death (PCD)^8,9. A ROS burst precedes PCD playing a crucial role in pathogen defence¹⁰. Conversely, abscisic acid (ABA) governs drought and salt stress by regulating the plant water status when turgor pressure declines. This simplified view of hormonal control is in fact much more complicated. Mutual control of the phytohormones and second messengers involved is challenging to dissect. This optogenetics study aimed to investigate the role of [Ca²⁺]_cyt- and anion-efflux-induced electrical signals in encoding specificity in plant processes by individually triggering them by means of light-gated ion channels.

Microbial rhodopsins are light-sensitive proteins and powerful tools for minimally invasive manipulation of cells by light (optogenetics)^11,12. These optogenetic tools have recently been made available for use in plants^4,13. ACR1 2.0 triggers defined membrane depolarizations by anion efflux and guides pollen tubes when stimulated locally⁴ or initiates stomatal closure through depolarization-synchronized anion and cation efflux¹⁴. So far, the role of [Ca²⁺]_cyt signals in plants has been studied with loss-of-function approaches of Ca²⁺-signalling elements or Ca²⁺-permeable channels^15,16. Here we established an approach equivalent to a gain-of-function strategy, a light-gated channel with on–off features allowing defined [Ca²⁺]_cyt modifications.

A light-gated Ca²⁺-permeable channel

Channelrhodopsins are light-gated cation channels with a broad selectivity for cations^17,18. Previously, we engineered a channelrhodopsin variant with extra expression and medium long open time (XXM)¹⁹ and pronounced Ca²⁺ permeability²⁰. Here we screened a set of XXM mutants and identified an extra H134Q substitution in XXM (XXM 1.1) that augments photocurrents and Ca²⁺ conductance (Fig. 1a–c and Extended Data Fig. 1a–d). Endoplasmic reticulum-export and plasma membrane-targeting signal peptides in XXM 1.2 and XXM 1.3 further improved membrane targeting and photocurrents in Xenopus laevis oocytes, and an N-terminal 11 amino acid truncation in XXM 1.3 led to the final XXM 2.0 version (Fig. 1c and Extended Data Fig. 1a,e).

Fig. 1 — 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

Extended Data Fig. 1 — 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

XXM 2.0 was cloned into a plant expression vector providing for in planta production of all-trans retinal, the essential chromophore of rhodopsin⁴ (Extended Data Fig. 1a). Retinal synthesis did not affect carotenoid content or plant growth under non-stimulating red light conditions when compared to wild-type (WT) plants (Extended Data Fig. 2a–f). The broad XXM 2.0 action spectrum (Extended Data Fig. 1f) permits green light stimulation, minimizing interference with plant photoreceptor signalling^21,22. Compared to Ret-XXM 1.2 or 1.3, Ret-XXM 2.0 exhibited improved plasma membrane targeting and stronger membrane depolarizations when stimulated with green light (Fig. 1d–g and Extended Data Fig. 2g–i). This probably resulted from pronounced cation influx and Ca²⁺-dependent alteration of endogenous ion transport²³. No notable light response was induced in WT or control plants expressing the retinal-producing enzyme and soluble enhanced yellow fluorescent protein (Ret-eYFP; Fig. 1e–g and Extended Data Fig. 1a).

Extended Data Fig. 2 — 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

In contrast to Ret-eYFP or Ret-ACR1 2.0, Ret-XXM 2.0 elicited a substantial increase in [Ca²⁺]_cyt in Nicotiana benthamiana leaves expressing the aequorin Ca²⁺ sensor²⁴ (Fig. 1h). Using the red fluorescent Ca²⁺ sensor R-GECO1²⁵ and the pH reporter pHuji²⁶, we observed sustained [Ca²⁺]_cyt elevations but only minor, short-lived pH deflections with Ret-XXM 2.0 at cellular resolution (Fig. 1i, Extended Data Fig. 3a,b and Supplementary Video 1). Simultaneous electrical recordings and [Ca²⁺]_cyt imaging revealed that light-induced Ret-XXM 2.0-dependent [Ca²⁺]_cyt signals were accompanied by reproducible membrane depolarizations and both could be fine-tuned by light intensity or duration (Extended Data Fig. 3c–i). Ret-ACR1 2.0 triggered membrane depolarizations too, but no sustained [Ca²⁺]_cyt increases (Extended Data Fig. 4a). For physiological investigations we finally developed a global light-application protocol (520 nm, 9 µW mm⁻²) to induce membrane depolarizations or Ca²⁺ influx in plant leaf cells stably expressing Ret-ACR1 2.0 or Ret-XXM 2.0 (hereafter referred to as ACR1 and XXM; Fig. 2a,b and Extended Data Fig. 4b).

Extended Data Fig. 3 — 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 — 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.

XXM and ACR1 elicit distinct phenotypes

When exposed to continuous global low-intensity green light illumination, XXM plants developed necrotic spots after 24 h (Fig. 2c and Extended Data Fig. 5a,b). In support of a pathogen-associated PCD²⁷ response governed by Ca²⁺ signalling, the necrotic phenotype was suppressed by chelating extracellular Ca²⁺ (Extended Data Fig. 5c,d). A nonlinear increase in apoplastic conductivity further indicated that PCD develops about 4–8 h after XXM activation (Fig. 2d). By contrast, ACR1 triggered steady anion release from mesophyll cells, resulting in a linear increase in ion leakage towards the apoplast and plant wilting after 4 h continuous low-intensity green light illumination (Fig. 2c,d and Extended Data Figs. 5e–j and 6a). The rapid recovery of leaf turgor within 20 min after ACR1 shut-off (Supplementary Video 2) supports the idea that the wilting phenotype results from reversible leaf cell water loss initiated by ACR1-driven anion efflux and concomitant potassium release through depolarization-activated K⁺ channels¹⁴.

Watering plants with 35% polyethylene glycol 6000 (PEG) mimicked the wilting time course observed in ACR1 plants (Extended Data Fig. 5e–j) and provided suitable experimental conditions for analysing ACR1 responses in a physiological context. Similarly, infection with Pseudomonas syringae pv. tomato strain DC3000 (Pst) was utilized as a suitable physiological control, reproducing the phenotypic characteristics observed with XXM (Extended Data Fig. 5a,b).

Nicotiana tabacum leaves inoculated with Pst triggered robust [Ca²⁺]_cyt increases (Extended Data Fig. 6b,c). As Ca²⁺-dependent ROS production is key for plant immunity and abiotic stress signalling^10,28,29, we monitored ROS production. We observed strong and progressive ROS generation in leaves 4 h after XXM stimulation as well as Pst inoculation. ROS production, however, was significantly lower in leaves of ACR1 or PEG-treated plants compared to XXM leaves and was barely detectable in control leaves (Fig. 2e and Extended Data Fig. 6d–g). In vivo quantitative real-time amperometric H₂O₂ measurements revealed a rapid H₂O₂ transient, reaching peak values of 15 µM within 4 min following light stimulation in XXM leaves, that levelled down to sustained steady-state values of 4–5 µM and returned to control levels upon light off (Fig. 2f and Extended Data Fig. 6f). The H₂O₂ signal lagging about 1 min behind the XXM-induced [Ca²⁺]_cyt increase and depolarization (Extended Data Fig. 6h–j) supports voltage- or Ca²⁺-dependent ROS production. Despite comparable amplitudes of electrical signals triggered by both optotools, ACR1 provoked only a small ROS rise of about 1 µM after 1 h stimulation (Extended Data Fig. 6f). Thus, our data support Ca²⁺-dependent profound H₂O₂ production in plants³⁰.

Metabolic rearrangement by XXM and ACR1

The observed phenotypes indicated that XXM and ACR1 address signalling pathways related to immune responses and osmotic stress, respectively. The phytohormone ABA is central to drought adaptation whereas the interplay of JA isoleucine (JA-Ile) and SA orchestrates defence signalling pathways^29,31. In general, JA is rapidly synthesized through its intermediate cis-(+)-12-oxo phytodienoic acid (OPDA) and is subsequently conjugated with l-isoleucine to its active variant JA-Ile^30,32. In this context, OPDA is discussed as a signalling molecule that regulates diverse biological processes in a JA-independent manner^33,34.

We used a targeted metabolomic approach to quantify the aforementioned marker metabolites. After 1 h, 4 h and 24 h green light treatment, ABA and proline levels increased strongly in ACR1 and PEG-treated control plants, whereas no significant changes were detected in XXM stimulated plants and Pst-treated plants or red light control plants (Fig. 3a–d and Extended Data Fig. 7a–c). OPDA, JA, JA-Ile and SA remained at low levels in PEG-treated plants or ACR1 stimulated plants and remained at basal levels under control conditions (Fig. 3e–l and Extended Data Fig. 7d–o). By contrast, in XXM plants JA-Ile, JA and OPDA strongly accumulated already after 1 h, returning to control levels within 4 h. In comparison, the onset of the SA transient was delayed and returned to basal levels after about 24 h (Fig. 3f–h,j–l and Extended Data Fig. 7i–k,m–o). Following Pst leaf inoculation, levels of JA-Ile, JA and OPDA were comparable to those of control plants, whereas SA levels increased significantly but less than with XXM stimulation (Fig. 3e,i–l and Extended Data Fig. 7h,l). These data corroborate the phenotypes as well as the kinetics of ROS generation and ion leakage (Extended Data Figs. 5a,b and 6a,d,e). In conclusion, spray inoculation and optotool activation seem to act on different timescales. The slow, successive Pst infection process^35,36 contrasts with the immediate impact of XXM stimulation, which affects all leaf cells simultaneously. This difference is evident in the unchanged SA levels observed 2 h after Pst infection, compared to the tenfold increase in SA levels observed at the same time point following XXM stimulation (Fig. 3i versus Fig. 3j inset). The minor transient increase of JA-Ile and JA observed after control spray inoculation probably resulted from mechanical cues³⁷. However, the possibility of a rapid, transient increase in JA-Ile and its precursors being missed during Pst treatment cannot be excluded. Overall, our results probably indicate that the two different optogenetic tools addressed distinct metabolic pathways in leaves.

Fig. 3 — 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

Extended Data Fig. 7 — 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

XXM and ACR1 control transcript profiles

To substantiate the notion that the optogenetic tools specifically address pathogen and wounding responses with XXM and drought signalling with ACR1, we complemented our metabolite profiling with transcriptomics. For this purpose, we subjected ACR1 and XXM plants to 1 h or 4 h low-intensity green light episodes and followed transcriptome profiles along a time course of 1, 4 and 8 h (Extended Data Fig. 8a).

Extended Data Fig. 8 — 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 (*Pst*8h) 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 *Pst*8h 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 *Pst*8h. 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 *Pst*8h. 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.

Activation of either ACR1 or XXM evoked significant transcriptional reprogramming: more than 1,200 (1 h) and 2,300 (4 h) differentially expressed genes (DEGs) in ACR1 plants, and about 11,000 (1 h) and 13,000 (4 h) DEGs in XXM plants, respectively (Fig. 4a,b and Supplementary Table 2, tabs 1–5). The number of DEGs rapidly declined after the light stimulus ceased, resembling optogenetic on–off switching (Fig. 4a,b). In contrast to the optotools, and in line with a steady stress response manifestation during water stress³⁸ or pathogen attack³⁵, DEGs exhibited a gradual increase over time following physiological stress (Fig. 4a–d and Supplementary Table 2, tabs 1–7). About 20% (1 h) and 50% (4 h) of the 1,333 DEGs addressed by drought stress (8 h PEG treated) were shared with ACR1, respectively. Similarly, 73% and 77% of 1,638 DEGs addressed by biotic stress (8 h Pst inoculation) were covered by the XXM dataset (Extended Data Fig. 8b,c).

Fig. 4 — 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 *Pst*8h 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

Gene ontology (GO)-based annotation of DEGs (plant GOSlim) revealed that abiotic (PEG8h) and biotic (Pst8h) stress responses matched best to those obtained with the 1 h stimulated ACR1 and XXM plants, respectively (Extended Data Fig. 8d–g and Supplementary Table 2, tab 8). Accordingly, at the level of gene function, the upregulated GO terms related to ABA signalling and water transport were enriched following ACR1 activation and 8 h PEG treatment. Conversely, biological processes related to transport and signalling of auxin were downregulated in ACR1 plants (Extended Data Fig. 8f,h,i and Supplementary Table 2, tabs 9–14). These results indicate that after activation, ACR1 plants face water deprivation and respond correspondingly by activating ABA signalling and biosynthesis and shutting down auxin-controlled plant growth (Extended Data Fig. 8h and Supplementary Table 2, tabs 9–12).

Biological processes associated with hormonal responses, cell recognition, immune responses and wounding were upregulated in XXM and Pst-inoculated plants, whereas GO terms related to photosynthesis were downregulated (Extended Data Fig. 8g,j,k and Supplementary Table 2, tabs 15–20). Chlorophyll fluorescence measurements corroborated a rapid shutdown of photosynthesis in light-stimulated XXM plants and to a lesser extend in Pst-inoculated leaves (Extended Data Fig. 9a). Compared to that for Pst inoculation, the six to eight times higher number of XXM-addressed DEGs highlights the numerous genes specifically targeted by XXM. Functional classification (plant GOSlim) of these genes again identified signal transduction, stress responses and cell death as enriched upregulated biological processes whereas photosynthesis as well as growth and development were downregulated (Supplementary Table 3). The observation that many upregulated XXM DEGs are involved in Ca²⁺ signalling (Supplementary Table 4), suggests that—compared to receptor-based Pst recognition—the Ca²⁺-permeable optotool activates a ‘global’ Ca²⁺ response, addressing several Ca²⁺-dependent pathways.

ACR1 and XXM shared common DEGs and GO terms (Extended Data Fig. 9b–e). However, expression profiles of top regulated genes addressed by either ACR1 or XXM activation showed that genes for proline synthesis, water transport, ABA synthesis and signalling were regulated by ACR1 (Fig. 4e,f and Supplementary Table 5). Genes associated with photosynthesis and auxin-dependent growth regulation were repressed with both optotools (Fig. 4g,h). Many genes related to JA, SA, ethylene, PCD and ROS pathways showed specific regulation in XXM plants but remained largely unchanged in ACR1 (Fig. 4i–m). These involved transcriptional master regulators of JA signalling and biosynthesis (Fig. 4i) that fine-tune plant immunity³¹. XXM-induced DEGs also included SA receptors and hub regulators (Fig. 4j) essential for pathogen resistance³⁹. Furthermore, XXM activation strongly triggered ethylene-associated plant immune responses⁴⁰ (Fig. 4k). The overall picture that XXM, but not ACR1, activates immune responses is also reflected by specific upregulation of genes associated with ROS production and redox regulation or PCD (Fig. 4l,m).

Tuning plant responses optogenetically

Green light has minor effects on photosynthesis and photomorphogenesis compared to red and blue light^41–43. It addressed few DEGs in mature N. tabacum leaves (Supplementary Table 6), making it a minimally invasive condition for rhodopsin-based optogenetic plant manipulation.

We introduced XXM as a new tool to orchestrate Ca²⁺ signals and downstream signalling pathways in planta by applying tailored light protocols (Figs. 1 and 2 and Extended Data Fig. 3). Of note, XXM additionally conducts Na⁺ in animal cell systems²⁰. In contrast to that in animal cells, the extracellular Na⁺ concentration in non-salt-stressed plants is about 1 mM (refs. ^44,45), not exceeding 40 mM in the cytosol⁴⁶. Given a resting potential in plant leaf cells of about −120 to −160 mV, a small inward-directed Na⁺ movement through XXM is outcompeted by Ca²⁺, driven by its steep inward gradient (>10,000-fold). Experimental evidence showed that an XXM-mediated necrotic phenotype occurred in Ca²⁺- but not Na⁺-rich media (Extended Data Fig. 5d). Further, typical sodium stress markers were barely induced by XXM, whereas many more genes related to Ca²⁺ transport and signalling were upregulated (Supplementary Table 4). Thus, in the plant context, the Na⁺ conductance of XXM can be disregarded, making XXM ideal to study the role of Ca²⁺ signals.

Using XXM and ACR1 side by side, we were able to tackle a long-standing question of how membrane potential changes and/or [Ca²⁺]_cyt variations shape plant stress responses. XXM and ACR1 both trigger comparable membrane depolarizations but led to clearly separable plant stress responses (Figs. 1–4). Our study allows the following interpretation: a long-term voltage change may not represent a signalling cue per se to convey specificity for the drought or immune response observed here. Instead, an ACR1-mediated light-induced hydraulic signal, resulting from anion efflux, accompanied by potassium and water efflux, initiated the drought stress response (Figs. 2–4). By contrast, the XXM-mediated Ca²⁺ influx triggered an immune response and suggests [Ca²⁺]_cyt to represent the key signal. Further studies should identify turgor sensors or Ca²⁺-dependent immune responses, possibly with the help of optotools. Of note, our studies are based on physiological responses observed in leaf cells, still leaving open the possibility that in distinct cell types—such as guard cells—signalling may be different⁴⁷. Defined oscillations in [Ca²⁺]_cyt or membrane voltage are considered to address specific signalling networks and physiological plant responses^48–50. Precise spatial activation of XXM or ACR1 may help to answer how [Ca²⁺]_cyt together with voltage signatures encodes information to communicate local and long-range stress-related cues in plants (Extended Data Fig. 10). Moreover, considering the observed dynamic kinetics of optogenetically induced physiological responses (Figs. 3 and 4 and Extended Data Fig. 9f–m), switching off optotools could allow for temporally resolved investigation of the underlying restoring mechanisms. Given the wilting phenotype induced by ACR1, there is potential to screen for mutants that show enhanced osmolyte uptake, making them more resistant to salt stress or drought. Overall, experimental approaches in this area would enhance our understanding of plant plasticity dynamics and resilience to changing environmental conditions.

Extended Data Fig. 10 — 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

Methods

Ethics

The laparotomy to obtain oocytes from X. laevis was carried out in accordance with the principles of the Basel Declaration and recommendations of Landratsamt Wuerzburg Veterinaeramt. The protocol under License number 70/14 from Landratsamt Wuerzburg, Veterinaeramt, was approved by the responsible veterinarian.

Molecular cloning

The pGEMHE vector⁵¹ was used for in vitro RNA synthesis and the following expression in X. aevis oocytes. The binary pCAMBIA3300 vector with the UBQ10 promoter or the pCAMBIA1300 vector with the CaMV 35S promoter was used for Agrobacterium infiltration and stable transformation of plants.

Substitutions were introduced by QuikChange site-directed mutagenesis PCR. The pCambia1300 NES-2×R-GECO1 was cloned using the USER cloning technique⁵². For other constructs, all of the fragments with suitable restriction sites were introduced into the pGEMHE vector or the binary vectors pCAMBIA3300 with T4 DNA ligase (Thermo Fisher Scientific).

Plasmid extractions from Escherichia coli cultures were carried out using the MiniPrep 250 Kit (QIAGEN) according to the manufacturer’s instructions. All constructs were verified by sequencing (Eurofins Genomics). The sequences of XXM 1.1 and XXM 2.0 are available in the Supplementary Information.

Molecular engineering and development of XXM 2.0

ChR2-XXM (D156H) was selected as a template to start molecular engineering owing to the high expression level and already enhanced Ca²⁺ permeability¹⁹. As no specific ion-selective filter is identified in ChR2, we anticipated that the three (extracellular, centre and intracellular) molecular gates (ECG, CG and ICG) might contribute to modulating ion-selective properties of ChR2, in addition to its gating function. Considering known substitution sites of ChR2 variants with modified ion selectivity in relation to the crystal structure indeed suggests a possibility to modulate ion selectivity using the ChR2 structure generated by PyMOL⁵³ (Extended Data Fig. 1b). Within the three-dimensional structure, the internal cavities were calculated using HOLLOW, which is a published python script⁵⁴. Some gating residues (H134 and E83 in ICG, and Q117 and R120 in ECG) and residues located in close proximity to the gate (E101 near ECG) were therefore substituted (Extended Data Fig. 1b). S63 (when combined with D156H) and E90 substitutions in CG have been reported to decrease Ca²⁺ conductance^55,56, and therefore were not tested here. The resulting variants were first tested by two-electrode voltage-clamp analysis with X. laevis oocytes for comparison of photocurrent amplitude (Extended Data Fig. 1c). Variants exhibiting high photocurrent amplitude were next studied to determine the ion selectivity (reversal potential shift comparison) by changing extracellular ion concentrations (Extended Data Fig. 1d, see details in the following section) for selecting the superior candidate with high Ca²⁺ conductance (Fig. 1). To further enhance its membrane trafficking, N-terminal truncation of ChR2 and addition of signal peptides as recently reported⁴ were explored and examined by fluorescence imaging along with a comparison of photocurrents in X. laevis oocytes.

Two-electrode voltage-clamp analysis with X.laevis oocytes

The AmpliCap-MaxT7 High Yield Message Maker Kit (Epicentre Biotechnologies) was used to synthesize XXM, XXM 1.1, XXM 1.2, XXM 1.3 and XXM 2.0 complementary RNA (cRNA). All of the cRNAs were stored in nuclease-free water at −20 °C. Oocytes were injected with 30 ng cRNA and incubated in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4) at 16 °C for 2 days. Two-electrode voltage-clamp recordings were carried out at room temperature with a two-electrode voltage-clamp amplifier (TURBO TEC-03X, NPI Electronic). Electrode capillaries (diameter 1.5 mm, wall thickness 0.178 mm; Hilgenberg) were pulled by a vertical puller (PC-10, Narishige) and filled with 3 M KCl, with tip resistance of 0.4–1 MΩ. A USB-6221 DAQ interface (National Instruments) and WinWCP V5.3.4 software (University of Strathclyde, UK) were used for data acquisition. Blue light illumination was supplied by a 473-nm laser (Changchun New Industries Optoelectronics Tech).

Photocurrents of different XXM versions in response to blue light illumination (473 nm, 3 mW mm⁻²) were compared at a holding potential of −90 mV in a standard recording solution (96 mM NaCl, 2 mM KCl, 1 mM BaCl₂, 1 mM MgCl₂, 10 mM HEPES, pH 7.6). For the reversal potential shift (mV) comparison, photocurrents of XXM and XXM 1.1 were recorded in both buffer A (119 mM N-methyl-d-glucamine, 0.8 mM BaCl₂, 5 mM HEPES, pH 7.6) and buffer B (80 mM BaCl₂, 5 mM HEPES, pH 7.6). Blue light (473 nm, 3 mW mm⁻²) was applied to activate different XXM variants. In the experiment for the comparison of calcium currents between XXM and XXM 1.1, 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid (BAPTA) was injected to block the endogenous Ca²⁺-activated chloride channels of oocytes. The Ca²⁺ currents triggered by 3 mW mm⁻² blue light were measured in the bath solution containing 80 mM CaCl₂ and 10 mM 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) at pH 9 and a holding potential of −90 mV.

The action spectrum for XXM 2.0 was detected with light of different wavelengths. Light of different wavelengths (399.3 nm, 422 nm, 440.7 nm, 456 nm, 479.5 nm, 496 nm, 516 nm, 541 nm, 562 nm and 595 nm) was obtained by narrow bandwidth interference filters (Edmund Optics) together with a PhotoFluor II light source (89 North). Equal photon flux was set for each wavelength. Photocurrents were detected when treated with light of distinct wavelength and normalized to the maximal stationary currents triggered by blue light (456 nm). The light intensities were measured with a Plus 2 Power & Energy Meter (Laserpoint).

Agrobacterium transformation

The Agrobacterium tumefaciens strain GV3101 was collected by centrifugation after 1 day of culture and washed twice with sterile distilled water. All of the plasmids described for plant expression were transformed into A. tumefaciens by an electroporation protocol⁵⁷. Monoclonal colonies were selected from lysogeny broth (LB)–agar plates with 100 μg ml⁻¹ kanamycin, 25 μg ml⁻¹ gentamycin and 10 μg ml⁻¹ rifampicin at 28 °C and cultured in LB medium with 100 μg ml⁻¹ kanamycin, 25 μg ml⁻¹ gentamycin and 10 μg ml⁻¹ rifampicin at 28 °C. The colonies were confirmed by PCR.

Agrobacterium infiltration of N.benthamiana leaves

For Agrobacterium infiltration, 30–37-day-old N. benthamiana plants grown in the greenhouse (40–60 kilolux light irradiation from 08:00–20:00, 24–26 °C) were used. Transient transformation of N. benthamiana plants was carried out according to the protocol of ref. ⁵⁸. Briefly, agrobacteria were cultured in LB medium with 150 µM acetosyringone at 28 °C for about 16–18 h, collected by centrifugation and washed twice with infiltration buffer (10 mM MgCl₂, 10 mM 2-morpholinoethanesulfonic acid (MES) (pH was adjusted to 5.6 by KOH), 150 μM acetosyringone). The final concentration was adjusted to 0.4 at an optical density of 600 nm (OD_600nm) in infiltration buffer. A 1-ml syringe was used to infiltrate the resuspended agrobacteria into the leaves through the abaxial epidermis. The infiltrated plants were grown in 650 nm red light (light intensity of about 30 µW mm⁻²; cycles of 14 h light at 26 °C/10 h dark at 16 °C).

Stable transformation of N.tabacum plants

N. tabacum (N. tabacum cultivar Petit Havana SR1) seeds sterilized by 6% NaOCl were germinated and grown in 500-ml sterile plastic boxes on agar plates (Murashige and Skoog medium including vitamins and MES (Duchefa Biochemie), 3% sucrose, 0.8% Gelzan (Sigma-Aldrich), pH 5.8 with KOH) in stable culture conditions of cycles of 14 h light at 26 °C/10 h dark at 16 °C. The A. tumefaciens strain GV3101 harbouring the pCAMBIA3300 vector with BASTA resistance or the pCAMBIA1300 vector with hygromycin resistance was used for N. tabacum transformation as described previously⁵⁹ with some minor modifications. Agrobacteria were collected and washed twice with sterilized MS solution (Murashige and Skoog medium including vitamins and MES (Duchefa Biochemie), 3% sucrose, pH 5.8 with KOH). The final concentration was adjusted to OD_600nm = 0.1. Sterilized leaves were cut into pieces of about 2 cm² and soaked in the resuspended agrobacteria solution for 20 min. The wet leaf pieces were dried, placed on plant growth medium and transferred after 3 days to a callus-inducing medium (Murashige and Skoog medium including vitamins and MES (Duchefa Biochemie), 3% sucrose, 0.8% Gelzan (Sigma-Aldrich), pH 5.8 with KOH, 20 µg ml⁻¹ dl-phosphinothricin (Duchefa Biochemie) or 30 mg l⁻¹ hygromycin B (Thermo Fisher Scientific), 500 µg ml⁻¹ ticarcillin disodium (Duchefa Biochemie), 100 mg l⁻¹ myo-inositol, 1 mg l⁻¹ thiamine hydrochloride, 1 mg l⁻¹ 6-benzylaminopurine and 100 µg l⁻¹ 1-naphthaleneacetic acid (Sigma-Aldrich)) and cultured under 650-nm light-emitting diode (LED) red light with light intensity of about 30 µW mm⁻². The pieces of leaves, explants and calli were transferred to new callus-inducing medium every 2 weeks. Generated shoots were decapitated and moved onto rooting medium (Murashige and Skoog medium including vitamins and MES (Duchefa Biochemie), 3% sucrose, 0.8% Gelzan (Sigma-Aldrich), pH 5.8 with KOH, 20 µg ml⁻¹ dl-phosphinothricin (Duchefa Biochemie) or 30 mg l⁻¹ hygromycin B (Thermo Fisher Scientific), 500 µg ml⁻¹ ticarcillin disodium (Duchefa Biochemie), 100 mg l⁻¹ myo-inositol, 1 mg l⁻¹ thiamine hydrochloride)) and, after root formation, were grown on soil in 650 nm red light (about 30 µW mm⁻², cycles of 14 h light at 26 °C/10 h dark at 16 °C).

The transgenic plants were verified by eYFP fluorescence or R-GECO1 fluorescence in leaves. Seeds of individual plants were collected and selected for BASTA or hygromycin resistance using selection medium (Murashige and Skoog medium including vitamins and MES (Duchefa Biochemie), 3% sucrose, 0.8% Gelzan, pH 5.8 with KOH, 20 µg ml⁻¹ dl-phosphinothricin (Duchefa Biochemie) or 30 mg l⁻¹ hygromycin B (Thermo Fisher Scientific)). Homozygous lines were used for experimental studies.

Confocal microscopy and image processing

A confocal laser scanning microscope (Leica SP5, Leica Microsystems CMS) controlled by Leica LAS AF (Version 2.7.3.9723, Leica Microsystems) was used to subcellularly localize the rhodopsin–eYFP fusions in plant cells or X. laevis oocytes. The yellow fluorescence was observed with a dipping 25× HCX IRAPO 925/0.95 objective in N. benthamiana leaves 3 days post Agrobacterium infiltration, in N. tabacum leaves after 45 days grown in red light (light intensity of about 30 µW mm⁻²; cycles of 14 h light at 26 °C/10 h dark at 16 °C) and in X. laevis oocytes 2 days post injection. eYFP was excited at 496 nm and fluorescence was captured between 520 and 580 nm. N. benthamiana and N. tabacum leaf discs were placed upside down for yellow fluorescence detection. FIJI IMAGEJ-win64 software⁶⁰ was used for image processing.

Aequorin-based cytoplasmic free Ca²⁺ measurements

Co-infiltration of agrobacteria with 10 μM of coelenterazine (PJK Biotech) was carried out as described in the ‘Agrobacterium infiltration of N. benthamiana leaves’ section under red light conditions. After 2 days in the red light growth room, aequorin luminescence from the infiltrated leaves was measured by a homemade luminometer. Luminescence was detected by a photomultiplier (Photo Counting Module MP 1983 RS CPM, Perkin Elmer) controlled by IGI-MPRS232 (IGIsystems). Labview 14.0.0 (National Instruments) was used to control the shutter and LEDs. A 520-nm green light LED (from WINGER, WEPGN3-S1) with a light intensity of 50 μW mm⁻² was used to activate rhodopsins. To prevent the LED light from being detected by the photomultiplier, an additional shutter (Uniblitz, VCM-D1) was installed.

Live-cell imaging and all-optical physiology measurements

Live-cell imaging experiments were carried out using transiently transformed N. benthamiana leaves, transgenic N. tabacum leaves (Ret-XXM 2.0 with R-GECO1, Ret-ACR1 2.0 with R-GECO1, R-GECO1, Ret-XXM 2.0 with pHuji and pHuji) or transiently transformed N. benthamiana mesophyll protoplasts (R-GECO1 and Ret-XXM 2.0 with R-GECO1). The A. tumefaciens strain GV3101 harbouring the corresponding pCAMBIA vectors and A. tumefaciens strain K19 were cultured at 28 °C overnight. The infiltration solution for co-expression of Ret-XXM 2.0 with R-GECO1 contained A. tumefaciens strain K19 (OD_600nm = 0.3) and A. tumefaciens strain GV3101 harbouring the pCAMBIA plasmids (pCAMBIA3300 vector carrying Ret-XXM 2.0, pCAMBIA1300 vector carrying NES-2× R-GECO1; OD_600nm = 0.4 for both). Control plants were infiltrated with infiltration solution containing A. tumefaciens strain K19 (OD_600nm = 0.3) and A. tumefaciens strain GV3101 harbouring the pCAMBIA1300 vector carrying NES-2× R-GECO1 (OD_600nm = 0.4). [Ca²⁺]_cyt measurements were carried out 3 days post infiltration. Mesophyll protoplasts were prepared from the transiently transformed N. benthamiana leaves from which the abaxial epidermis was peeled off. The leaf pieces without the main vein were incubated in enzyme solution (1% BSA, 0.05% pectolyase Y23, 0.5% cellulase R-10, 0.5% macerozym R-10, 1 mM CaCl₂, 10 mM MES, 500 mM d-sorbitol, pH 5.6 with Tris) for 2 h. Following enzymatic digestion, cells were filtered through a 100-µm mesh. Protoplasts were collected by low-speed centrifugation (80g) without acceleration at 4 °C. Protoplasts were washed twice using precooled wash solution (1 mM CaCl₂, 500 mM d-sorbitol, pH 5.6 with Tris) and finally resuspended in precooled wash solution and stored on ice until use. Leaf disc samples with a diameter of 5 mm were prepared by peeling the abaxial epidermis off and gluing leaf discs upside down with medical adhesive (ULRICH Swiss) on custom-made recording chambers. The samples were allowed to recover in the dark in bath solution (1 mM KCl, 1 mM CaCl₂, 10 mM MES, and 1,3-bis(tris(hydroxymethyl)methylamino)propane (BTP), pH 6.0) at room temperature (about 25 °C) overnight before R-GECO1 or pHuji fluorescence measurement and all-optical experiments were carried out.

The microscope setup to carry out live-cell imaging is described in detail elsewhere⁶¹. R-GECO1²⁵ and pHuji²⁶ were excited with 570 nm excitation light. VisiView software (Version 2.1.1) was used to simultaneously control R-GECO1 imaging and triggering of local green light (532 nm, 180 μW mm⁻²) illumination by a solid-state laser (Changchun New Industries Optoelectronics Tech) or global green light (520 nm, 9 μW mm⁻²) illumination by a homemade LED device (LED from WINGER, WEPGN3-S1). XXM 2.0 activation by green light was carried out in the 5 s interval time during R-GECO1 or pHuji imaging. Green light illumination was terminated more than 1 s before R-GECO1 was excited to avoid photoswitching effects of R-GECO1 during optogenetic stimulation as reported previously⁶². A dichroic mirror (HC593 (F38-593), AHF Analysetechnik) combined with a high-speed filter wheel equipped with bandpass filters for R-GECO1 or pHuji (ET 624/20 nm) was used to detect the red fluorescence. During the detection of [Ca²⁺]_cyt signal triggered by XXM 2.0 stimulation when a different light condition was used, a 2 s interval time was set during R-GECO1 illuminations. It should be noted that the light pulse protocols used to set defined [Ca²⁺]_cyt signatures must be customized for each particular cell system or plant line and cannot act as a blueprint as features such as the expression level and cell type used or the Ca²⁺ homeostasis will probably influence the signature.

To avoid undesirable XXM 2.0 or ACR1 2.0 activations during bright-field imaging, the microscope white light source was covered by a primary red filter (Lee filter 106). Simultaneous plasma membrane potential and R-GECO1 fluorescence recordings were carried out in R-GECO1, Ret-XXM 2.0 with R-GECO1, Ret-ACR1 2.0 with R-GECO1 samples. Current-clamp-based voltage recordings were carried out by microelectrode impalement as described elsewhere⁶¹. Glass microelectrodes filled with 300 mM KCl were connected to the microelectrode amplifier (TEC-05X; NPI Electronic) equipped with head stages of more than 10¹³ Ω input impedance. The reference electrodes were filled with 300 mM KCl. A piezo-driven micromanipulator (Sensapex) was used to direct the glass electrode. The current-clamp protocols were applied by WinWCP V5.3.4 software (University of Strathclyde, UK). R-GECO1 fluorescence intensities were recorded using FIJI IMAGEJ-win64 software⁶⁰.

Ca²⁺ signals were detected in N. tabacum leaves utilizing the R-GECO1 reporter following Pst treatment using a perfusion system (780 μl min⁻¹), which prevents motion and touch-induced imaging artefacts^47,63–65. Pst inoculated from LB–agar plates containing 10 µg ml⁻¹ rifampicin was cultured in LB medium with 10 µg ml⁻¹ rifampicin for 1.5 days (28 °C, 200 r.p.m.) and subsequently subcultured in 500 ml LB medium containing 10 µg ml⁻¹ rifampicin for 16 h. Pst cells were washed twice using sterile deionized water and suspended in bath solution (1 mM KCl, 1 mM CaCl₂, 10 mM MES and BTP, pH 6.0), resulting in a final perfusion solution with an OD_600nm of 0.5. Leaf disc samples from R-GECO1 transgenic N. tabacum plants without abaxial epidermis were glued with the adaxial side down to the coverslip in custom-made chambers using Medical Adhesive B (Ulrich Swiss) and allowed to recover in bath solution overnight before Ca²⁺ imaging.

Membrane voltage recordings in mesophyll cells

Microelectrodes for mesophyll cell impalement were pulled from borosilicate glass capillaries (inner diameter 0.58 mm, outer diameter 1.0 mm, Hilgenberg) using a horizontal laser puller (P2000, Sutter). Microelectrodes filled with 300 mM KCl having an electrode resistance of 60–110 MΩ were connected by Ag/AgCl wires to the microelectrode amplifier (Axon geneclamp 500 or VF-102; BioLogic). Reference electrodes were filled with 300 mM KCl, and plugged with 2% agar in 300 mM KCl. The NA USB-6221 interface (National Instruments) was used to digitalize data. Cells were impaled by an electronic micromanipulator (NC-30, Kleindiek Nanotechnik) and current-clamp protocols were applied with the WinWCP V5.3.4 software (University of Strathclyde, UK).

Plant growth conditions and sample collection

All of the WT and transgenic N. tabacum plants were grown under constant red light (650 nm, 30 μW mm⁻², 26 °C) for 45 days. For ACR1 2.0 and XXM 2.0 stimulation, 0 h, 1 h, 4 h and 24 h additional global green light (520 nm, 9 μW mm⁻²) were applied for the experimental groups. For osmotic stress treatment, 35% PEG was used to water Ret-eYFP #1 transgenic N. tabacum plants. This PEG concentration was selected after initial experiments with different PEG concentrations that would cause wilting phenotypes in a similar time frame (after 4–5 h) to that for the green-light-treated Ret-ACR1 2.0 plants. For Pst treatment, the Pst was washed twice with sterile deionized water and suspended in 10 mM MgCl₂ containing 0.04% Silwet L-77 with a final OD_600nm of 0.5 and sprayed on the entire Ret-eYFP #1 transgenic N. tabacum plants. Pst treatment was applied by spray inoculation to prevent wounding effects taking place that would unequivocally occur during infiltration by a syringe. The same amount (about 25 ml for each plant) of 10 mM MgCl₂ containing 0.04% Silwet L-77 was sprayed on Ret-eYFP #1 transgenic N. tabacum as the negative control. At t = 0 h before the additional green light illumination, PEG, Pst or 10 mM MgCl₂ was applied. For plants treated with 0 h, 1 h and 4 h green light illumination, the fifth leaves of N. tabacum were collected at different time points (t = 0 h, 1 h, 4 h and 8 h) and frozen in liquid nitrogen quickly for metabolite measurement and transcriptomics analysis. For plants treated with 24 h global green light, PEG, Pst or 10 mM MgCl₂, the fifth leaves from N. tabacum at different time points (t = 0 min, 3 min, 10 min, 0.5 h, 1 h, 2 h, 4 h, 8h and 24 h) were used for detection and quantification of ROS, electrolyte leakage estimations, chlorophyll fluorescence detection, metabolite measurement and transcriptomics analysis.

Detection of necrosis in leaf discs

A tissue puncher (Stiefel Disposable biopsy punch, diameter of 6 mm) was used to prepare leaf discs from the fifth leaf of 45-day-old N. tabacum plants. Leaf discs were washed twice with deionized water and transferred into 24-well plates containing 0.4 ml ultrapure H₂O that contained 1 mM CaCl₂, 10 mM CaCl₂, 5 mM EGTA (pH 7.0, KOH), 5 mM K₄BAPTA or 5 mM K₄BAPTA plus 10 mM NaCl as indicated in Extended Data Fig. 5c,d. Samples were placed in the dark for 1 h before exposing them to the light condition (growth chamber with constant red light (650 nm, 30 μW mm⁻²) plus constant green light (520 nm, 9 µW mm⁻², 26 °C)). Images were captured after 24 h treatment.

Chlorophyll fluorescence measurements

N. tabacum plants treated as described in the ‘Plant growth conditions and sample collection’ section were used to quantify photosynthesis performance with a pulse-amplitude modulation fluorometer. The fifth leaf was fixed and monitored with a Maxi pulse-amplitude modulation fluorometer (AVT 033); chlorophyll fluorescence measurements were recorded with IMAGING WIN v.2.41a FW MULTI RGB (Walz). The dark-adapted N. tabacum leaf was exposed to actinic light with intensity 7 (photosynthetically active radiation as 146 μmol m⁻² s⁻¹). The maximal fluorescence yield of a dark-adapted sample (Fm) and dark-level fluorescence yield (Fo) were detected. The quantum yield of the dark-adapted leaf samples is a measure of the potential quantum yield of the samples, which was calculated according to the equation: Yield = (Fm − Fo)/Fm.

Electrolyte leakage estimations

The membrane integrity of N. tabacum leaf cells was estimated by electrolyte leakage of leaf samples. Ion conductivity was measured as described previously⁶⁶. Seven leaf discs (5 mm diameter) were detached from the fifth leaves of 45-day-old plants and equilibrated together in 0.3 ml of ultrapure H₂O after washing twice with ultrapure H₂O. Ion conductivity was quantified 20 min after leaf disc equilibration in ultrapure H₂O (EC1) using a LAQUAtwin EC-11 conductivity meter (Horiba). The samples were then heated at 99 °C for 1 h to measure the final electrical conductivity (EC2) when the samples reached room temperature again. The relative electrolyte leakage was calculated, as a percentage, by the formula: EL = EC1/EC2 × 100.

Detection of ROS

Chemical detection of ROS in green light (520 nm, 9 μW mm⁻²)-treated N. tabacum leaves was carried out by 3,3-diaminobenzidine (DAB, Sigma-Aldrich) staining as described previously⁶⁷. The fifth leaves of N. tabacum plants were stained with fresh DAB staining solution (10 mM Na₂HPO₄, 1 mg ml⁻¹ DAB, 0.05% Tween 20, pH 3.0) by application of negative pressure for 5 min in dark. After 5 h incubation (shaking speed of 80–100 r.p.m.), the stained leaves were moved into fresh chlorophyll destaining solution (ethanol/acetic acid/glycerol, 3:1:1) and bathed in hot water (about 90–95 °C) for 15 min. Finally, the stained leaves were put into cold fresh chlorophyll destaining solution for 30 min. Images were taken with a plain white background under uniform lighting.

The chemiluminescent ‘superoxide probe’ luminol can be applied to indicate the ROS production⁶⁸. Superoxide released from leaf tissues was detected by the luminescence of luminol with Skanlt software (Version 6.1) according to the method described previously⁶⁹ with minor modifications. Leaf discs were prepared from the fifth leaves of 45-day-old N. tabacum plants using a tissue puncher (Stiefel Disposable biopsy punch, diameter of 6 mm). Leaf discs were washed with deionized water twice and transferred into the 96-well assay plate (black plate, clear bottom with lid, Corning) and incubated in the dark overnight to recover. Water was replaced with 200 µl of luminol–peroxidase working solution (30 mg l⁻¹ luminol (Sigma) and 20 mg l⁻¹ horseradish peroxidase (Sigma)) in each well containing leaf discs. Samples were kept in the dark for 1 h before measurement. Luminescence was measured in a microplate reader (Luminoskan Ascent, Thermo Labsystems) and 5 min global constant green light (9 µW mm⁻²) illumination was applied during the rest periods.

The method for in vivo measuring the production of H₂O₂ amperometrically from mesophyll cells in parallel with intracellular membrane potential recordings was described previously⁷⁰. Measurements were carried out in standard bath solution (1 mM KCl, 1 mM CaCl₂, 10 mM MES, and BTP, pH 6.0) with a platinum–iridium electrode (MicroProbes) cut back to an active (uninsulated) area of about 1 mm length. ROS detection was carried out by Patch-Master software V2x90 (HEKA). The platinum–iridium disc was gently placed in close proximity to mesophyll cells and held at a constant voltage of 600 mV with an amperometry amplifier (VA 10X, NPI Electronic). Oxidation of H₂O₂ at the active microelectrode surface resulted in a positive current signal, which was low-pass-filtered at 1 Hz and recorded with Patch-Master software V2x90 (HEKA). The electrode was calibrated in freshly prepared bath solutions with defined H₂O₂ concentrations. Green light (520 nm, about 9 µW mm⁻²) illumination on the N. tabacum leaves was carried out by green LEDs (WINGER, WEPGN3-S1).

All-trans retinal and carotenoid measurements

All-trans retinal and carotenoids were measured according to a protocol published elsewhere⁴. The fifth leaf of transgenic N. tabacum plants grown for 45 days in red light was triturated in liquid nitrogen and 200 mg leaf material was extracted with 500 µl of chloroform. The extract was centrifuged for 5 min at 18,400g and 50 µl of the organic phase was evaporated in a SpeedVac at 40 °C and dissolved in 50 µl of a 1:1 ethanol and chloroform mixture. A 5 µl volume of dissolved solution was analysed by ultrahigh-performance liquid chromatography (UPLC) combined with ultraviolet and tandem mass spectrometry detection using a Waters Acquity UPLC system coupled to a Waters Quattro Premier triple-quadrupole mass spectrometer equipped with an electrospray interface. Ten plants were used for retinal and 12 plants were used for carotenoid quantification.

Phytohormone measurement

All of the samples were prepared as described in the ‘Plant growth conditions and sample collection’ section. Ground samples (150 mg) were lyophilized in a laboratory freeze dryer (CHRIST, Laboratory freeze dryer Alpha 1-2) and subsequently used for phytohormone extraction. The extraction and chromatographic separation was carried out as described previously⁷¹, using 5 ng of dihydro-JA, JA–norvaline, [¹⁸O₂]OPDA, [D₄]SA and [D₆]ABA as phytohormone internal standard. The extraction solution contained ethylacetate (p.a.) and formic acid (p.a.) (99:1 in volume) to which 5 ng phytohormone internal standard was added. All samples were fixed on a TissueLyser with shaking for 3 min at a speed of 23 Hz. After that, samples were centrifuged and the supernatant was dried in a SpeedVac at 45 °C and finally dissolved in 40 μl liquid (acetonitrile (for high-performance liquid chromatography)/water (MilliQ), 1:1 (v/v)). Phytohormones were analysed by UPLC–electrospray interface–tandem mass spectrometry using a Waters Acquity I-Class UPLC system coupled to an AB Sciex 6500+ QTRAP tandem mass spectrometer (AB Sciex), operated in negative ionization mode as described elsewhere^72,73. Analyst (Version 1.6.3) software and MultiQuant (Version 3.0.2) software from Sciex were used for mass spectrometry detection of hormones and metabolites.

Proline quantification

The proline content of N. tabacum leaves was measured according to the spectroscopic method of ref. ⁷⁴. Samples were prepared as described in the section ‘Plant growth conditions and sample collection’. Ground samples (150 mg) were lyophilized in a laboratory freeze dryer (CHRIST, Laboratory freeze dryer Alpha 1-2). The dry samples were mixed in 40% ethanol and incubated at 4 °C overnight. The supernatant was collected after centrifugation at 13,500g for 5 min. A 500 µl volume of the ethanol extraction or 100 µl standard solution was mixed with 1,000 µl reaction mix (1% ninhydrin (w/v) in 60% acetic acid (v/v) and 20% ethanol (v/v)). After incubation at 95 °C for 20 min and subsequent centrifugation for 1 min at 9,000g, the samples (supernatant) were subjected to absorption measurement at 520 nm with a spectrophotometer (Hitachi U-1500). Proline concentration was determined according to the standard curve, and concentrations were calculated on the basis of dry weight.

Transcriptomics analysis

Three replicates of leaf samples from two batches of N. tabacum plants were collected for RNA sequencing. The experimental design for transcriptomics is shown in Extended Data Fig. 8a. Samples at 0 h, 1 h, 4 h and 8 h from plants growing in red light conditions were used as the biological controls to compare the expression levels of Ret-XXM 2.0 and Ret-ACR1 2.0 transgenic plants during or after the global green light illumination. In these experiments PEG-treated plants and Pst inoculation served as possible physiological controls to ACR1 2.0 and XXM 2.0 activation, and spraying leaves with buffer (MgCl₂) served as an additional control to Pst-sprayed plants (Extended Data Fig. 8a). RNA extraction was carried out using ground samples (150 mg) with the Macherey-Nagel NucleoSpin RNA Plant Kit (https://www.takarabio.com/documents/User%20Manual/UM/UM_TotalRNAPlant_Rev_07.pdf). DNase1 (Thermo Fisher) was used to digest DNA. RNA sequencing was carried out by Novogene (UK) with an Illumina NovaSeq 6000 Sequencing System. Paired-end 150 bp was the read length. Data processing (fastp) and mapping to the N. tabacum genome (kallisto)⁷⁵ was carried out using Amalgkit (https://github.com/kfuku52/amalgkit). Functional annotations of N. tabacum genes for subsequent bioinformatic analyses were retrieved from the Dicots PLAZA 5.0 repository^76,77.

Normalization and DEG analysis were carried out employing the DIANE package using DESeq2 and default parameters⁷⁷. DESeq2 uses a Wald test, in which the shrunken estimate of log fold change is divided by the standard error to produce a z-statistic. This z-statistic is then compared against a standard normal distribution⁷⁸. A prefiltering step eliminated genes exhibiting rowMeans over all conditions ≤ 5 counts, reducing the number of input genes from 69,500 to 42,196. Unless stated otherwise, |log₂[fold change]| ≥ 2 with a false discovery rate of 0.01 was taken as the cutoff for DEG identification. Only a small number of DEGs were identified when comparing Ret-ACR1 2.0 or Ret-XXM 2.0 transgenes with Ret-eYFP control plants grown under non-stimulating red light conditions (Supplementary Table 2, tab 1), demonstrating that ACR1 and XXM expression have virtually no impact on the transcript profiles of plants and plants growing in red light conditions are proper biological controls. Likewise, GO enrichment analysis on DEGs was carried out using the DIANE suite and corresponding enrichment plots were created using the srplot web interface (https://www.bioinformatics.com.cn/en). Venn diagrams were generated with the GOVenn script of the GOPlot package⁷⁹ and subsequent GO analysis on Venn subsets was carried out using gprofiler2⁸⁰. g:Profiler functional enrichment analysis is conducted using the g:GOSt tool that carries out over-representation analysis via the hypergeometric test⁸⁰. Finally, heat maps were generated with the pheatmap R package (version 1.0.12; https://CRAN.R-project.org/package=pheatmap). N. tabacum genes were further annotated manually according to their A. thaliana orthologues⁸¹ and corresponding gene symbols from the Aramemnon database⁸².

Quantitative real-time PCR

Quantification of gene transcripts was carried out by real-time PCR as described elsewhere⁸³. The samples were prepared as described in the section ‘Plant growth conditions and sample collection’. Ground samples (100 mg) were used for RNA extraction by the NucleoSpin RNA Plant Kit (Macherey-Nagel). cDNA was synthesized from 2.5 g of total RNA using oligo(dT) primer (Thermo Fisher Scientific) and M-MLV Reverse Transcriptase (Promega). All quantitative real-time PCR reactions were carried out with the Eppendorf Mastercycler ep realplex 2 system and Eppendorf Mastercycler ep realplex (Version 2.2) software, in a 20 µl reaction volume containing 2 µl diluted cDNA, 0.8 µM primer pairs and 10 µl ABsolute qPCR SYBR Green Capillary Mix (Thermo Scientific). Information on the genes and primers used is provided in Supplementary Table 7. Transcripts were normalized to that of 10,000 molecules of actin.

Surface potential recording on N.tabacum leaves

The design for long-range electrical signal measurements in N. tabacum leaves is shown in a diagram in Extended Data Fig. 10a. The surface potential recordings were carried out on 6–7-week-old N. tabacum leaves according to a previously described protocol⁸⁴ with minor modifications. A USB-6221 interface (National Instruments) was used to digitalize the electrical signals, which were recorded with WinWCP V5.3.4 software (University of Strathclyde, UK). The electrode silver wires (Ag/AgCl) connected to a microelectrode amplifier (Axon geneclamp 500) were wrapped around the petiole of the fifth leaves gently. Electrode gel (Auxynhairol) was used to cover the surface of the wrapped wires to aid connectivity between the electrodes and the petiole. The reference electrode consisting of an Ag/AgCl electrode was placed in a 200-ml pipette tip filled with electrode gel (Auxynhairol), which was inserted in the soil of the pots the N. tabacum plants grew in. Nine hours after mounting the electrodes, the surface potential was recorded when applying a 600-ms green light (532 nm, 5.3 mW mm⁻²) pulse at the main vein. A popular Technology Enhanced Clad Silica multimode optical fibre (diameter of 1,500 µm, 0.39 NA, Thorlabs) was placed directly on the top of main vein for light application. To prevent scattering of light and to guarantee local green light application, the optical fibre was covered by a non-transparent black plastic pipe up to the tip. The electrical signals were monitored at exactly 5 cm away from the illumination spot.

Significance analysis

Student’s t-test or ANOVA was used to analyse significant differences between groups. Significance analysis among more than three groups was carried out with one-way ANOVA using IBM SPSS statistics (version 26.0). For the post hoc multiple comparisons, the homogeneity of variances was tested first. If the variances were homogeneous (P > 0.05), the Tukey test was used for significance analysis. If the variances were not homogeneous (P < 0.05), either the Dunnett T3 or Games-Howell test was chosen, depending on whether the sample sizes were equal or not. Different letters indicate significant differences among the samples (lowercase letters indicate P values at the 0.05 level and capital letters indicate P values at the 0.01 level). Significance analysis among two groups was carried out with a two-sided Student’s t-test. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001. The significance analysis is performed with a 95% confidence of interval. All of the P values are listed in Supplementary Table 8.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Online content

Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-024-07884-1.

Supplementary information

Supplementary Information^{(15.4KB, docx)}

Coding sequences of XXM variants and legends for Supplementary Tables 1–8 and Videos 1 and 2.

Reporting Summary^{(3.2MB, pdf)}

Peer Review File^{(2.2MB, pdf)}

Supplementary Table 1^{(16.7KB, xlsx)}

Exact numbers of samples in Fig. 3 and Extended Data Figs. 7 and 9.

Supplementary Table 2^{(11MB, xlsx)}

Summary of DEGs and GO terms.

Supplementary Table 3^{(1.1MB, xlsx)}

Functional classification of genes specifically addressed by XXM .

Supplementary Table 4^{(1.1MB, xlsx)}

Calcium-related genes and sodium-related genes triggered by XXM 2.0 stimulation.

Supplementary Table 5^{(32.8KB, xlsx)}

Gene information for Fig. 4.

Supplementary Table 6^{(287.2KB, xlsx)}

Green-light-addressed DEGs.

Supplementary Table 7^{(12.1KB, xlsx)}

Gene information and primers for qPCR.

Supplementary Table 8^{(47.1KB, xlsx)}

P values for significance analysis.

Supplementary Video 1^{(20MB, mp4)}

Cytosolic free Ca²⁺ recording in a N. benthamiana mesophyll cell in response to XXM 2.0 activation. Live-cell [Ca²⁺]_cyt imaging in the mesophyll of transiently transformed N. benthamiana leaves expressing Ret-XXM 2.0 upon green light (532 nm, 180 µW mm⁻²) illumination. Scale bar, 50 μm.

Supplementary Video 2^{(31.7MB, mp4)}

Phenotypes of ACR1 2.0 or XXM 2.0 transgenic N. tabacum plants in red light when additional green light is switched on. Transgenic N. tabacum plants (Ret-eYFP, Ret-ACR1 2.0 and Ret-XXM 2.0) were grown in red light (650 nm, 30 μW mm⁻², 26 °C) for 45 days and additional green light (520 nm, 9 μW mm⁻²) was added at t = 1 h. Only Ret-ACR1 2.0 N. tabacum leaves wilt at the edges after 4 h of green light illumination, but when green light was switched off after 8 h illumination, turgor recovered within 20 min. Scale bar, 10 cm.

Source data

Source Data Fig. 1^{(141.1KB, xlsx)}

Source Data Fig. 2^{(1.1MB, xlsx)}

Source Data Fig. 3^{(46KB, xlsx)}

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Source Data Extended Data Fig. 1^{(10.8KB, xlsx)}

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Acknowledgements

This study is financed by the Deutsche Forschungsgemeinschaft (DFG) Projektnummer 374031971 TRR 240 A04 and 417451587 granted to G.N. Financial support provided by the Prix Louis-Jeantet to G.N. and by the DFG to K.R.K. (KO3657/2-3) and R.H. (Reinhart Koselleck Grant 415282803) is acknowledged. Y.Z. and M.D. were supported by the Chinese Scholarship Council and the German Academic Exchange Service (DAAD). In addition M.D. was supported by DFG Projekt 415282803 to R.H. We thank J. P. Rathjen and C. Zipfel for sharing seeds of aequorin-expressing N. benthamiana plants. Pst isolated by B. Staskawicz was provided by S. Berger. X. laevis oocytes were either bought from EcoCyte Bioscience or obtained from frogs in the Julius-von-Sachs-Institute, Wuerzburg University. We thank F. Waller for sharing expertise and equipment for ion conductivity measurements and K. Fukushima for providing the AMALGKIT pipeline and transcript mapping (https://github.com/kfuku52/amalgkit). Open access funding was enabled and organized by the DEAL-Agreement.

Extended data figures and tables

Author contributions

G.N., S.G., M.K., M.J.M., R.H., D.B. and K.R.K. conceived the project. S.G., K.R.K., M.D. and G.N. designed and S.G., K.R.K. and G.N. supervised the experiments. M.D., Y.Z., J.Y.-S. and S.G. carried out the in planta voltage recordings, confocal microscopy and aequorin experiments and analysed the data. Y.Z., S.Y. and M.D. generated all constructs used in this study. M.D. generated the transgenic plants and carried out metabolite analysis, ion leakage measurement, quantitative real-time PCR, Ca²⁺ and pH imaging experiments, and phenotype characterization. S.Y. and S.G. designed the development of XXM 2.0 and S.Y. carried out the functional characterization in X. laevis oocyte experiments. M.K. and M.D. carried out the hormone and metabolite analysis. M.D. and S.S. carried out ROS detection and analysed the data. D.B. and M.D. identified the orthologues of the N. tabacum genes and designed primers for quantitative real-time PCR. D.B. carried out all bioinformatic analyses. K.R.K., M.D., S.G., D.B., R.H. and G.N. wrote the paper and the initial draft was written by K.R.K. All authors edited and approved the final version of the manuscript to be published.

Peer review

Peer review information

Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Funding

Open access funding provided by Julius-Maximilians-Universität Würzburg.

Data availability

All the data generated in this study are available in the paper and the Supplementary Information. The RNA-sequencing data have been deposited in the National Center for Biotechnology Information database (Bioproject ID: PRJNA1108451). Source data are provided with this paper.

Code availability

No customized code was generated in this study.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Meiqi Ding, Yang Zhou, Dirk Becker, Shang Yang

Contributor Information

Rainer Hedrich, Email: rainer.hedrich@uni-wuerzburg.de.

Georg Nagel, Email: nagel@uni-wuerzburg.de.

Shiqiang Gao, Email: gao.shiqiang@uni-wuerzburg.de.

Kai R. Konrad, Email: kai.konrad@uni-wuerzburg.de

Extended data

is available for this paper at 10.1038/s41586-024-07884-1.

Supplementary information

The online version contains supplementary material available at 10.1038/s41586-024-07884-1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information^{(15.4KB, docx)}

Coding sequences of XXM variants and legends for Supplementary Tables 1–8 and Videos 1 and 2.

Reporting Summary^{(3.2MB, pdf)}

Peer Review File^{(2.2MB, pdf)}

Supplementary Table 1^{(16.7KB, xlsx)}

Exact numbers of samples in Fig. 3 and Extended Data Figs. 7 and 9.

Supplementary Table 2^{(11MB, xlsx)}

Summary of DEGs and GO terms.

Supplementary Table 3^{(1.1MB, xlsx)}

Functional classification of genes specifically addressed by XXM .

Supplementary Table 4^{(1.1MB, xlsx)}

Calcium-related genes and sodium-related genes triggered by XXM 2.0 stimulation.

Supplementary Table 5^{(32.8KB, xlsx)}

Gene information for Fig. 4.

Supplementary Table 6^{(287.2KB, xlsx)}

Green-light-addressed DEGs.

Supplementary Table 7^{(12.1KB, xlsx)}

Gene information and primers for qPCR.

Supplementary Table 8^{(47.1KB, xlsx)}

P values for significance analysis.

Supplementary Video 1^{(20MB, mp4)}

Supplementary Video 2^{(31.7MB, mp4)}

Source Data Fig. 1^{(141.1KB, xlsx)}

Source Data Fig. 2^{(1.1MB, xlsx)}

Source Data Fig. 3^{(46KB, xlsx)}

Source Data Fig. 4^{(24.5KB, xlsx)}

Source Data Extended Data Fig. 1^{(10.8KB, xlsx)}

Source Data Extended Data Fig. 2^{(12.4KB, xlsx)}

Source Data Extended Data Fig. 3^{(291.4KB, xlsx)}

Source Data Extended Data Fig. 6^{(3.4MB, xlsx)}

Source Data Extended Data Fig. 7^{(32KB, xlsx)}

Source Data Extended Data Fig. 9^{(21.3KB, xlsx)}

Source Data Extended Data Fig. 10^{(10KB, xlsx)}

Data Availability Statement

No customized code was generated in this study.

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PERMALINK

Probing plant signal processing optogenetically by two channelrhodopsins

Meiqi Ding

Yang Zhou

Dirk Becker

Shang Yang

Markus Krischke

Sönke Scherzer

Jing Yu-Strzelczyk

Martin J Mueller

Rainer Hedrich

Georg Nagel

Shiqiang Gao

Kai R Konrad

Abstract

Main

A light-gated Ca2+-permeable channel

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

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

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

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

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

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

XXM and ACR1 elicit distinct phenotypes

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.

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

Metabolic rearrangement by XXM and ACR1

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

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

XXM and ACR1 control transcript profiles

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

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

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

Tuning plant responses optogenetically

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

Methods

Ethics

Molecular cloning

Molecular engineering and development of XXM 2.0

Two-electrode voltage-clamp analysis with X.laevis oocytes

Agrobacterium transformation

Agrobacterium infiltration of N.benthamiana leaves

Stable transformation of N.tabacum plants

Confocal microscopy and image processing

Aequorin-based cytoplasmic free Ca2+ measurements

Live-cell imaging and all-optical physiology measurements

Membrane voltage recordings in mesophyll cells

Plant growth conditions and sample collection

Detection of necrosis in leaf discs

Chlorophyll fluorescence measurements

Electrolyte leakage estimations

Detection of ROS

All-trans retinal and carotenoid measurements

Phytohormone measurement

Proline quantification

Transcriptomics analysis

Quantitative real-time PCR

Surface potential recording on N.tabacum leaves

Significance analysis

Reporting summary

Online content

Supplementary information

Source data

Acknowledgements

Extended data figures and tables

Author contributions

Peer review

Peer review information

Funding

Data availability

Code availability

Competing interests

Footnotes

Contributor Information

Extended data

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

A light-gated Ca²⁺-permeable channel

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

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

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

Aequorin-based cytoplasmic free Ca²⁺ measurements