The ROS-producing enzyme NADPH oxidase/RBOH is a Ca2+ channel regulated by protons in the eukaryotic kingdom

doi:10.21203/rs.3.rs-4840681/v1

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The ROS-producing enzyme NADPH oxidase/RBOH is a Ca2+ channel regulated by protons in the eukaryotic kingdom

https://doi.org/10.21203/rs.3.rs-4840681/v1

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NADPH oxidase (NOX) is crucial for the production of reactive oxygen species (ROS) during developmental processes and stress responses across plants, animals and fungi. The interdependence of ROS and Ca²⁺ signals in various cellular signaling pathways is well-established, but the specific calcium channel that collaborates with NADPH oxidase to generate Ca²⁺-ROS co-coupling signals remains unidentified. In this study, we demonstrate that eukaryotic NOXs, such as Arabidopsis RBOHD/C/F, human NOX2 and yeast YNO1, function as calcium channels that directly mediate Ca²⁺ influx. Additionally, we reveal that these eukaryotic NOX family members act as non-selective cation channels, similar to human TRP family channels, capable of transporting monovalent and bivalent cations. Furthermore, RBOHD's calcium channel activity is activated by protons, and mutation of RBOHD results in significantly reduced physiological responses to low pH in a Ca²⁺-dependent manner. Overall, we have identified for the first time the conserved calcium channel activity of NADPH oxidases, such as plant RBOHs, and human and yeast NOXs across eukaryotic species. This discovery introduces a new paradigm for NADPH oxidase research.

Biological sciences/Plant sciences/Plant signalling

Biological sciences/Plant sciences/Plant physiology

ROS

Ca2+

calcium channel

NADPH oxidase

NOX

RBOHD

eukaryote

Reactive oxygen species (ROS) play pivotal roles across animals, plants and fungi. A primary source of ROS is catalyzed by NADPH oxidase (NOX), which is prominent in developmental processes and stress responses ¹. Human phagocyte NOX2 (gp91^phox), the first cloned NOX, is crucial for pathogen resistance and mediates oxygen-depleting respiratory bursts, while plant NOX homologs are known as RBOHs (respiratory burst oxidase homologs). Human NOX functions are associated with hearing, kidney function and neurological development; and plant RBOHs also play critical roles during developmental growth and in responses to abiotic and biotic stresses ^2,3. Together with yeast and animal NOXs, it demonstrates that NOXs exhibit fundamental and conserved roles in ROS regulation.

Typically, the structures of NOX proteins contain 6-7 transmembrane (TM) domains. These proteins also feature a long C-terminal region bound by flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH). NOX mediates the transfer of electrons from NADPH, sequentially to FAD, heme (Fe²⁺), and oxygen to form superoxide anion (O₂^-), which is catalyzed by superoxide dismutase (SOD) to produce hydrogen peroxide (H₂O₂). O₂^-and H₂O₂ belong to ROS ³. ROS acts as a second messenger similar to signaling calcium ion (Ca²⁺). Both ROS and Ca²⁺ serve as universal languages/signals for sensing internal and external stimuli and facilitating cell-cell communication. However, the molecular mechanisms underlying the integration of ROS and Ca²⁺ remain elusive.

It is well-documented that ROS-Ca²⁺ interactions play a central role in early recognition and signaling of various physiological processes, such as growth, flowering, fertilization, pathogenesis, abiotic stress responses (e.g., heavy ions), long-distance wounding signal, systemic acquired resistance (SAR) signal, and recognition of volatile organic compounds (VOCs) ^2,4-6. AtRBOHs, HsNOX5/DUOXs and fungal NOXC all have calcium-bound EF-hands at the N-terminal ⁷. Furthermore, ROS production by AtRBOHs and HsNOX5 requires Ca²⁺ activation ^7,8. Despite extensive studies on the inseparable relationship between ROS and Ca²⁺, their co-coupling mechanism remains poorly understood.

Calcium channels are generally crucial for encoding Ca²⁺ signals in eukaryotic cells. In the context of long-distance transmission of wounding or heavy ionic signals, several models propose alternating activation and coupling of ROS-Ca²⁺ based on unidentified calcium channel together with RBOHs ^9,10. Additionally, it remains unclear which calcium channel activates AtRBOHD serving as a regulatory hub in plant immunity ^11,12. Moreover, Ca²⁺ signal is essential for sensing and transmitting wounding-induced long-distance signals ¹³, while potential SAR signals, which has puzzled plant pathologists for several decades, have begun to focus on RBOHD/ROS ¹⁴.

Why ROS and Ca²⁺ are so deeply interconnected in many biological processes, and the reasons for the lack of discovery of a specific Ca²⁺ encoder remain open questions. Taken together, based on the current knowledge of NOX/RBOH in eukaryotic species, we speculate that membrane-localized NOX/RBOH itself may serve as both a calcium ion channel and a ROS-producing enzyme, suggesting that the long-sought calcium channel may indeed be a functional feature of NOX/RBOH itself.

The NADPH oxidase RBOHD is a Ca²⁺channel

Hydrogen peroxide (H₂O₂)-induced the significant elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in plants carrying calcium indicator aequorin (Col-0^AEQ) was completely blocked by the calcium channel inhibitor LaCl₃, suggesting that calcium channels are essential for Ca²⁺ influx across the plasma membrane during ROS signaling (Fig 1a, Extended Data Fig. 1). RBOHD in Arabidopsis thaliana (RBOHD, or AtRBOHD) and other plants localized on the plasma membrane has been extensively studied and is predicted to couple with an unidentified calcium channel for encoding Ca²⁺-ROS waves in numerous proposed models ^2,10-12. We used rbohD mutant carrying a transposon insertion that results in no detectable full-length transcripts and lacks the specific ROS spike induced by pathogen-associated molecular pattern (PAMP) factor flg22 (Extended Data Fig. 2 and 3). Interestingly, the ROS-induced spike of [Ca²⁺]_cyt dramatically decreased in rbohD^AEQ in dependence of extra high Ca²⁺ (Extended Data Fig. 4), further supporting that RBOHD potentially integrates Ca²⁺-ROS signals.

To explore the physiological role of RBOHD in relation to calcium, we first examined the phenotypic responses of WT, rbohD and cngc2 plants under varying concentrations of additional calcium in the growth hydroponic media. Compared to WT, rbohD mutants exhibited significantly reduced plant size under low supplemental calcium conditions, whereas these phenotypes recovered under high calcium supplementation (Fig. 1b, c), contrasting with the phenotype observed in cngc2 controls ¹⁵. This highlights the critical role of RBOHD in sustaining plant growth in a Ca²⁺ concentration-dependent manner, thereby establishing a direct link between plant RBOHD function and calcium regulation.

To determine whether RBOHD exhibits calcium channel activity, we performed two-electrode voltage clamp (TEVC) recording assay in Xenopus oocytes, a widely adopted method for studying ion channels ^15,16. RBOHD-GFP fusion protein localizes at the plasma membrane of the oocytes (Extended Data Fig. 5). Surprisingly, upon injection of RBOHD capped RNA (cRNA), oocytes displayed significantly large currents at -140 mV in the presence of 30 mM CaCl₂ (Fig. 1d, e), which was significantly higher than that observed in the positive control CNGC14 ^17,18, whereas the negative control showed almost no detected current. Thus, we report for the first time that Arabidopsis NADPH oxidase RBOHD functions as a calcium channel.

To further validate the calcium channel activity of RBOHD, we conducted additional experiments in human embryonic kidney 293T (HEK293T) cells and the Ca²⁺-uptake deficient yeast mutant cch1 mid1 respectively. Stimulation of HEK293T cells expressing the calcium indicator GCaMP6s with exogenous 400 μM CaCl₂ elicited distinct calcium-induced specific spikes in both RBOHD- and CNGC14-expressing cells (Fig. 1f). Furthermore, we observed yeast mutant cch1 mid1 was rescued by RBOHD similar to the positive control CNGC14 and wild-type BY4741 (Fig. 1g). In summary, integrating data from TEVC recording, HEK293T and yeast systems reveal a novel function of Arabidopsis NADPH oxidase RBOHD as a calcium channel.

RBOHD behaves as a non-selective cation channel

Calcium channels, such as transient receptor potential (TRP) channels, are known for transporting various ions ¹⁹. To investigate whether RBOHD exhibits similar capabilities, we assessed the effects of multiple cations in Xenopus oocytes using the TEVC recording assay. The results demonstrated that RBOHD can transport not only monovalent ions like K⁺ and Na⁺, but also bivalent ions like Ca²⁺ and Mg²⁺. However, it exhibitedvery weak transport activity of Ba²⁺ (Fig. 2a, b). This indicates that RBOHD possesses characteristics of transporting multiple cationic substrates, similar to ion channels such as human TRPs and Arabidopsis CSCs/OSCAs ^19-21.

To further characterize the fundamental features of the calcium channel RBOHD, we analyzed its dependence on Ca²⁺ concentration and its pharmacological properties using the TEVC system. Increasing the CaCl₂ concentration from 5 mM to 30 mM resulted in a corresponding increase in current signals from 1.5 μA to 5 μA, demonstrating that RBOHD channel activity is Ca²⁺ concentration-dependent (Fig. 2c, d). However, when the calcium channel inhibitor LaCl₃ was added to the TEVC assay, the calcium channel activity of RBOHD was significantly reduced to levels comparable to negative controls (Fig. 2e, f). Furthermore, treatment with DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate), which blocks Cl^- channels known to amplify Ca²⁺ signals in TEVC ¹⁶, resulted in a significantly stronger primary Ca²⁺ signal compared to negative controls (Fig. 2e, f). In conclusion, RBOHD functions as a typical non-selective cation channel that is permeable to Ca²⁺ and other cations.

Human and yeast NOXs are universal Ca²⁺and cation channels

NOXs are predicted to have emerged in the early stages of eukaryotic evolution, with mammalian and fungal NOXs sharing conserved protein domains similar to those found in plant RBOHs (⁷ and Fig. 3a, b). We aimed to determine whether NOXs from human (Homo sapiens), yeast (Saccharomyces cerevisiae) and other species, exhibit general channel activity similar to RBOHD across eukaryotes. We selected Arabidopsis and rice RBOHs, Human NOX2/NOX5/DUOX1 and yeast YNO1 which is essential for responding to stresses like H₂O₂, apoptosis and actin function ^7,22. We also included SpNOX from the bacterium Streptococcus pneumoniae, given its potential role in ROS-related functions and its status as an exceptionally rare NOX homolog in prokaryotes ²³.

We evaluated the calcium channel activity of various NOXs in Xenopus oocytes by TEVC recording assay. Among the tested NOXs, AtRBOHC/F, closely related to AtRBOHD, exhibited calcium channel activity, whereas AtRBOHE/H, OsRBOHA/B, and HsNOX5/DUOX1 did not (Fig 3c, d, Extended Data Fig. 6). Using CNGC14 as a positive control, we observed significant currents at -140 mV for HsNOX2, ScYNO1 and AtRBOHC in the presence of 30 mM CaCl₂ (Fig. 3c, d). These results were further validated through independent yeast system to access calcium channel activity. HsNOX2, ScYNO1 and AtRBOHC were able to complement the growth phenotype of yeast mutant cch1 mid1, like CNGC14 and controls (Fig. 3e). However, SpNOX showed no calcium channel activity in either the TEVC or yeast assays (Fig. 3c, d, e). In summary, Arabidopsis, human and yeast NADPH oxidases universally exhibit calcium channel activity in eukaryotes.

To further investigate ion selectivity, we conducted assays similar to those used for RBOHD (Fig. 2a, b). The results showed that ScYNO1 and HsNOX2 can also transport the monovalent cation like Na⁺ and K⁺, as well as bivalent cations like Ca²⁺ and Mg²⁺, but exhibited very low activity for Ba²⁺ (Fig. 3f, g and Extended Data Fig. 7), consistent with the findings for RBOHD (Fig. 2a, b). Moreover, the signals observed with CaCl₂ and Ca-Glu in the HsNOX2 TEVC assays were similar (Fig. 3g), suggesting that the cation Ca²⁺, rather than anions, plays a critical role in NOX channel activity. Collectively, these findings demonstrate that eukaryotic NADPH oxidases function as Ca²⁺/cation channels.

The function of RBOHD is regulated by protons and depends on external Ca²⁺ concentrations

To assess the function of calcium channel NOXs in response to environmental stimuli and stress signals, we first tested the calcium permeability of RBOHD using the TEVC assay under different pH values. The results showed that when pH was reduced from 7.4 to 5.6, the currents of RBOHD increased from an undetectable background level like negative controls to a high level of 1.5 μA, whereas there was no change was observed in the control CNGC14 (Fig. 4a, b). This suggests that lower pH, or more protons H⁺, is required for switching on the calcium channel activity of RBOHD. When the pH value ranged from 10 to 4.5, wild-type roots became longer with the increase of H⁺ concentration and showed a gradient change. However, rbohD roots were relatively insensitive to pH changes (Fig. 4c, d), indicating that RBOHD function is dependent on ambient pH or proton concentration. Furthermore, to assess the calcium-dependent physiological function of RBOHD, we observed the increasement of additional Ca²⁺ enhanced a significant difference in root elongation between WT and rbohD, especially at low pH 5.8 versus high pH 8 (Fig. 4e, f). This further supports that the function of the RBOHD channel on root elongation is not only dependent on pH, but also requires sufficient exogenous Ca²⁺.

To further test the function of RBOHD on stress responses, we found that rbohD^AEQ seedlings treated with flg22 resulted in significantly lower [Ca²⁺]_cyt spikes compared to controls especially under low extra Ca²⁺ conditions (Extended Data Fig. 8). Compared to controls, rbohD plants were more susceptible to bacteria Pst DC3000, and also showed inhibited stomata closure in response to flg22, particularly under insufficient extra Ca²⁺ supply (Extended Data Fig. 8). When adding SA, rbohD roots were extremely insensitive to the changing low or high extra calcium (Extended Data Fig. 8). Collectively, it demonstrates that the calcium-dependent function of RBOHD integrates Ca²⁺-ROS signals in stress responses.

Taken together, NADPH oxidases mediated Ca²⁺ influx are widely important for growth and defense in eukaryotic organisms. The function of NOX channel activity is activated by protons and depends on calcium concentrations. Therefore, we speculate that Ca²⁺-ROS coupling signaling stimulated by multiple internal and external cues could require NOX as a key hub to recognize, integrate and transduce signals in cells(Fig. 4g).

In summary, we have identified NOX/RBOH as a novel class of calcium/cation channels regulated by protons, representing a channel-type enzyme responsible for integrating Ca²⁺-ROS signals. This discovery introduces a new paradigm and concept for ROS research within eukaryotes.

NOXs/RBOHs as new members of eukaryotic calcium channels

In this study, we discovered that NOX enzymes, known for catalyzing ROS, also function as conserved calcium channels in eukaryotes. As calcium channels are crucial for generating calcium spikes/waves in the cytoplasm through influx or efflux of calcium ions, and since Ca²⁺ serves as a key second messenger in eukaryotic signaling ^24,25, this newfound calcium channel function in NOX enzymes presents new opportunities for enhancing our understanding of Ca²⁺ signaling and re-evaluating the fundamental roles of NOXs in future study of ROS and calcium signaling.

NOX proteins belong to the family of 6- or 7-transmembrane proteins, and their permeability characteristics for calcium and cations are analogous to those of human temperature receptors known as TRPs, as shown in this study (Fig. 2). TRP channels mediate the transport of Ca²⁺ and other ions such as Mg²⁺ and K⁺ ^19,26. Moreover, TRP channel activity is regulated by calcium ion concentrations and can be modulated by specific inhibitors like LaCl₃. Extensive research on TRPs has significantly expanded our understanding of their roles in various fields related to human development and diseases ²⁶. Similarly, we anticipate that exploring the ion channel activity of NOX enzymes will deepen our understanding of their underlying mechanisms and broaden our knowledge of their diverse physiological roles across different organisms.

Redox hemostasis, which balances oxidizing and reducing reactions, is critical for cellular function and cell fate widely ²⁷. Excessive ROS is associated with diverse human diseases such as diabetes, cancers and neurodegenerative diseases, as well as plant and fungus anti-stress responses ^2,22,28. In this study, calcium channel function of RBOHD depends on pH (Fig. 4), which closely interacts with ROS and cellular redox states. Therefore, our discovery of the calcium channel function of NOXs opens new avenues for exploring calcium-redox biology.

Protons regulate channel activity of NOXs

The gradient of proton H⁺, forming the electrochemical potential, is a key driving force for substance transport and signal transduction across cell membranes. It has been found that protons can activate and regulate ion channels or proton-sensing G protein-coupled receptors (GPCRs), such as human TRPV1, GPR4, etc. which critical residues can bind to protons ^19,29-31. Recently, it has been reported that optogenetic manipulation of cellular H⁺ influx can activate an unknown calcium channel localized in the endoplasmic reticulum in Arabidopsis ³². So far, known cation channels directly activated by proton H⁺ have not been reported in plants. This study demonstrates that the channel activity of RBOHD can be directly activated by protons (Fig. 4). In addition, NOXs have calcium/cation channel characteristics similar to TRP family channels (Fig. 2 and 3). The questions, like whether critical amino acids of NOXs can directly bind to H⁺, and whether H⁺ is involved in the formation of NOX protein complex, need further studies in future.

Membrane potential, ROS and calcium signals are very early changing factors in long-distance transmission of systemic stimuli such as wounding, pathogens, drought, temperature, light and heavy ions ^2,10,13. RBOHD may couple together with calcium, ROS and proton potential as a key machinery to reveal the nature of systemic long-distance signal. In animals and fungi, NOXs also play integral roles in multiple signal transductions. The new concept of channel properties of NOXs identified in this study contributes to integrating and transmitting basic cellular signals or languages.

Plant materials and growth conditions

All Arabidopsis materials used in this study are Col-0 background. Line rbohD^AEQ was generated based on crossing rbohD mutant and calcium indicator plants. Arabidopsis seeds were surface-sterilized by soaking in 15% NaClO for 5 minutes, followed by washing three times with sterile ddH₂O. Subsequently, seeds were planted on 1/2 MS (Phyto Technology Laboratories, S806) medium supplemented with 0.8% agar, pH 5.8. Petri dishes were stratified at 4°C for 3 days, and then transferred to a long-day greenhouse (22℃, 16h light/8h dark). For experiments involving additional calcium treatments, 1/2 MS without Ca²⁺ is supplemented by 0.1-1.5 mM CaCl₂. For hydroponic experiments, transfer 7-day-old seedlings from 1/2 MS medium (1.5 mM Ca²⁺) to a liquid medium containing 1/6 MS (pH 5.8) and varying concentrations of CaCl₂. Plants were transferred to a short-day greenhouse (22℃, 8h light/16h dark) for additional 2-3 weeks.

Measurement of [Ca²⁺]_cyt burst

To measure [Ca²⁺]_cyt, seedlings of Col-0^AEQ and rbohD^AEQ were cultivated on 1/2 MS medium containing either 0.1 mM or 1.5 mM CaCl₂ in a long-day greenhouse (22℃, 16h light/8h dark) for 1 week. Seedlings were incubated overnight in 100 μl water containing 10 μM coelenterin (Nanolight Technology). Subsequently, they were stimulated with 500 nM flg22 (Genscript), or 4 mM H₂O₂ and the fluorescence change was measured using Varioskan Flash Spectral (ThermoFisher). The relative fluorescence value was represented by L/Lmax.

Aequorin bioluminescence-based Ca²⁺ imaging

[Ca²⁺]_cyt was measured in aequorin-expressing plants as previously described. Seedlings were grown in 150 mm × 15 mm Petri dishes for 7 days, and applied evenly with 2 ml water of 10 µM coelenterin (Nanolight Technology) per Petri dish 12 h before imaging. Bioluminescence imaging was conducted with the Tanon 5200 Multi chemiluminescent imaging system (Tanon), and images were analyzed using ImageJ. For LaCl₃ treatment, seedlings were incubated in 1 mM LaCl₃ for 20 minutes before imaging. The remaining aequorin luminescence (L_max) was measured using 1 M CaCl₂ and 10% (v/v) ethanol. All treatments were conducted in the dark, and experiments were carried out at room temperature (22-25°C).

Measurement of ROS burst

Leaf discs from 4-week old plants were placed into a 96-well plate and incubated with ddH₂O overnight. Then plants were treated with 30 μg/ml luminol (sigma, A8511) and 20 μg/ml horseradish peroxidase (HRP, sigma, P6782), followed by 100 μl of 500 nM flg22. Fluorescence was detected by Varioskan Flash Spectral (ThermoFisher).

RNA extraction and RT-qPCR

Total RNA was extracted by Trizol (LABLEAD, R1000), and 1 μg total RNA was used for reverse transcription (LABLEAD, F0202). The obtained cDNA was used as a template to detect the expression of the interest genes by qPCR assay (LABLEAD, R0202-02), with ACTIN8 serving as the internal control.

Two-electrode voltage clamp recording from Xenopus oocytes

The full-length coding sequences (CDS) of AtCNGC14, AtRBOHD, AtRBOHC, AtRBOHF, AtRBOHE, AtRBOHH, HsNOX2, HsNOX5, HsDUOX1, SpNOX, ScYNO1, OsRBOHA, or OsRBOHB were cloned into the expression vector pGEMHE via homologous recombination and expressed in Xenopus oocyte. Capped RNA (cRNA) was synthesized from 1 μg of linearized plasmid DNA template using the mMESSAGE mMACHINETM T7 kit (Themo Fisher Scientific, Ambion), following the manufacturer's instructions. The oocytes were isolated and incubated overnight at 18°C in calcium-free ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 10 mM HEPES/ NaOH, pH 7.4) supplemented with 50 mg/ml of gentamicin. After injection with 23 nl cRNA at a concentration of 1000 ng/μl per oocyte, cells were incubated for two days in ND96 solution containing calcium (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 10 mM HEPES/NaOH, pH 7.4). Oocytes were voltage-clamped using a TEV 200 A amplifier (Dagan) and monitored by computer using Digidata 1440 A converter and pCLAMP 10.4 software (Axon Instruments). The pipette solution contained 3 M KCl. For the identification of calcium channel activity, oocytes were immersed in perfusion fluid containing 30 mM CaCl₂, 2 mM KCl, 1 mM MgCl₂, and 10 mM MES/Tris (pH 5.6) during signal recording. For ion selectivity experiments, the perfusion solution contained 10 mM MES/Tris (pH 5.6) and 15 mM divalent ion solution or 30 mM univalent ion solution. For pharmacologic analysis, the perfusion solution contained 10 mM MES/Tris (pH 5.6) supplemented with 1 mM DIDS (ThermoFisher) or 1 mM LaCl₃. The osmolalities of all solutions were adjusted to 220 mOsmol kg⁻¹ using D-mannitol. Voltage steps were applied from +20 to -140 mV in -10 mV decrements during 2 s, with each step starting with 0.05 s and ending with 2.3 s at the resting potential of the oocyte membrane in the tested bath solution.

Calcium imaging in HEK293T cell

The full-length CDS of AtCNGC14 and AtRBOHD were cloned into pBudCE4.1-GCaMP6s vector by homologous recombination. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C with 5% CO_2. Plasmid was extracted with kit (TIANGEN) and transfected into HEK293T cells with PEI 25K (TIANGEN). After 2 days, samples were observed by Lecia SP8 microscope. Fluorescence signals from GCaMP6s were collected every 5 seconds and 180 photos were collected for each sample. The Ca²⁺ imaging solution contains 120 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 1.2 mM NaHCO₃, 10 mM Glucose and 10 mM HEPES. About 60 seconds after initiation of the imaging procedure, 2.5 mM CaCl₂ was added to elicit Ca²⁺ entry through any active channels.

Calcium uptake-deficient yeast assay

Full-length CDS of AtCNGC14, AtRBOHD, HsNOX2, HsNOX5, SpNOX, or ScYNO1 were cloned into yeast expression vector YEP124 by homologous recombination. Yeast strain BY4741 deficient in cch1 mid1 was cultured in YPD medium and aliquoted into 1.5 ml EP tubes. Add 100 μl 0.1 mol/L LiAC to each tube for re-suspension, and then incubate at 30°C for 10 min to prepare yeast component cells. The plasmid containing the interest gene was transformed into yeast cch1 mid1 by 50% PEG4000. After recovery at 28 °C for 3 h, yeast cells were collected and plated on SD/-His-Leu-Ura solid medium for selection. The correct colony was selected and the wild-type BY4741 colony was cultured with YPD medium to OD₆₀₀=3.0. 100 μl aliquot of yeast extract was mixed with 4 ml of sterilized top agar medium (20 g/L yeast extract, 20 g/L tryptone, 20 g/L glucose, 0.7% agar) and overlaid onto YPD solid medium. Place a round filter paper on the medium, then add 10 μl of 10 mg/ml alpha-factor (Macklin) to the center of the filter paper. Incubate at 28 °C for 12 h to observe the growth of yeast.

Root elongation assay

Seedlings were vertically cultured on 1/2 MS medium containing 1.5 mM calcium for 4 days. Subsequently, they were transferred to various treatment media, with the position of the root apices marked before continuing growth for an additional 4 days. Images were captured using a Canon EOS 550D camera, and root elongation was quantified using Image J software.

Bacterial growth assay

Seedlings were grown for 10 days in 1/2 MS medium（0.1 or 1.5 mM CaCl₂） on a 12-well tissue culture plate. Pseudomonas syringae pv. tomato (Pst) DC3000 was grown to OD₆₀₀=1.0, then resuspended twice in 10 mM MgCl₂ and adjusted to OD₆₀₀=0.0001. Seedlings were immersed in the bacterial suspension for 2 minutes, and then transferred to be incubated in a climate room for 3 days. After surface sterilization with 75% ethanol, equal weights of Arabidopsis seedlings were ground in 10 mM MgCl₂, diluted in a gradient and plated. Colonies on plates were counted after 2 days.

Stomatal aperture measurements

Leaves from 4-week-old plants were floated in a buffer solution (50 mM KCl, 10 mM MES, pH 6.15) for 2 hours. Subsequently, the buffer was supplemented with either 0.1 mM or 1.5 mM CaCl₂, and optionally treated with 1 μM flg22 solution, followed by an additional 2-hour incubation. Stomatal responses were then observed using a ZEISS microscope, and stomatal aperture measurements were conducted using Image J software.

Funding

This work was supported by the Program of Natural Science Foundation of China (No. 32222011 and 32170580 to L.Y.L, No. 32270265 and 31872170 to L.G.L, and No. 32170263 to D.K.), the Key Program of the National Natural Science Foundation of China (No. 31930010 to L.G. L.), the Capacity Building for Sci-Tech Innovation-Fundamental Scientiﬁc Research Funds (No. 19530050165 to L.G.L.), Beijing Natural Science Foundation (5222001 to L.Y.L and 5202003 to K.D.).

Author contributions

L.Y.L. and L.G.L. conceived the project and designed the research. Z.Y., Y.C., C.S., C.L., C.F., Z.L., Z.Y., W.G. and L.G. performed the experiments. L.Y.L., Z.Y., Y.C., C.S., S.L., H.T., E.Y., Y.S., D.K. and H.C. participated in the data analysis. L.Y.L. and L.G.L. wrote the manuscript with contributions from all authors. All authors contributed to the article discussed the results and have approved the submitted version.

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supplementaryfileplasmidsandprimerslist.xlsx
Dataset 1, Dataset 2
ExtendedDataFigures.docx
Extended Data 1-8

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The ROS-producing enzyme NADPH oxidase/RBOH is a Ca2+ channel regulated by protons in the eukaryotic kingdom

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