Structural insights into the coordinated regulation of the SLAH family in Arabidopsis thaliana

Domain: biology
Published: 2025-12-17
Editorial outcome: minor_revision

Abstract
S-type anion channel homologs (SLAH) are widely expressed in various plant tissues and play a key role in anion transport, which is crucial for plant adaptation to both biotic and abiotic stresses. In this study, we employ cryo-electron microscopy (cryo-EM) to analyze four SLAH channel complexes from Arabidopsis thaliana: the homotrimeric SLAH3 channel, the 2SLAH1 + SLAH3 + tRNA complex, the 1SLAH1 + 2SLAH3 complex, and the 3SLAH1 + tRNA complex. Critically, our studies reveal that tRNA directly binds to and occupies the intracellular entrance of the SLAH1 homotrimer and the 2SLAH1 + SLAH3 heterocomplex. Electrophysiological experiments confirm tRNA’s role as a potent inhibitory regulatory subunit: RNase-mediated tRNA degradation robustly activates SLAH1 currents, while targeted mutagenesis of SLAH1 tRNA-interacting residues phenocopy this activation and enhanced ABA-induced stomatal closure. Combining with structural biology, electrophysiology, and biochemistry, we comprehensively examine the key residues in SLAH1 and SLAH3 that are responsible for the anion permeation. This mechanistic advancement provides a deeper understanding of the molecular basis for plant stress tolerance and identifies specific molecular targets for future engineering crops.

Introduction
Plants face diverse biotic and abiotic stresses. Biotic stress includes biological factors like infections, diseases, or pests that impair growth1–3, whereas abiotic stress involves environmental conditions such as salinity, drought, high temperatures, UV radiation, or nutrient deficiencies4. Investigating plant responses and regulatory pathways under stress is crucial for improving resilience and crop yield5. Guard cells and stomata, essential for gas exchange, photosynthesis, transpiration, and immunity6,7, also impact global carbon and water cycles, playing a critical role in enhancing crop productivity and water-use efficiency8,9. Electrophysiological studies have identified two types of membrane anion channels in guard cells10–13: R-type (rapid-type) and S-type (slow-type). R-type14,15, also known as quick-activating anion channel, mediates rapid currents16 and is activated by depolarization, inactivating with hyperpolarization. S-type channels, including the SLAC/SLAH family, activate more slowly11,12 and regulate stomatal opening via anionic efflux11,12.

The Arabidopsis genome encodes five SLAC/SLAH members: SLAC1, SLAH1, SLAH2, SLAH3, and SLAH417,18. Among these, SLAC1 has been the most extensively researched19–24, focusing on its structure, function, and regulatory mechanisms. SLAC1 primarily mediates the efflux of NO3− and Cl−, with a stronger preference for NO3−. Since the discovery of anion currents in plants and the functional characterization of SLAC1 from the SLAC/SLAH family, additional SLAH channel proteins have been identified17,18. SLAH3 is broadly expressed in roots, guard cells, mesophyll cells, pollen tubes, stems, hypocotyls, and nectar glands25–30. SLAH3 shows permeability to both NO3− and Cl−, though Cl− permeability is relatively lower, with a NO3−:Cl− permeability ratio of 20:128,29,31,32. SLAH3 activation requires kinase activity and increased extracellular NO3− concentrations. Functional studies reveal SLAH3’s involvement in regulating stomatal movements29, balancing nitrogen and potassium33, sensing acid toxicity in root cells34, facilitating the transport of Cl− from roots to shoots35, influencing pollen tube growth36, and reducing ammonium toxicity in roots26.

While research on SLAC1 and SLAH3 is thorough, fewer studies address SLAH1 and SLAH217,18, and SLAH4 remains largely unexplored17,18. Currently, only the three-dimensional structure of SLAC1 has been resolved23,37, while the structural and functional details of other members are still unknown. In this work, we employ cryo-EM to analyze the structures of the SLAH3 homotrimer, SLAH1-tRNA complex, and the heteromeric complexes of 2SLAH1 + 1SLAH3 + tRNA and 1SLAH1 + 2SLAH3. We identify tRNA molecules that inhibit SLAH1 channel activity, validate kinase-mediated regulation of SLAH proteins, and elucidate how different SLAH protein combinations control anion transport through electrophysiological assays. These findings enhance our understanding of plant stress resistance mechanisms and contribute valuable knowledge for advancing agricultural development.

Results
Structural determination of the AtSLAH3 channel
To explore the molecular mechanism of SLAH family-mediated anion transport, we transfected Chinese Hamster Ovary (CHO) cells with a GFP-tagged AtSLAH3 plasmid and a plant protein kinase CPK21 plasmid. With the increase in the clamped voltage, AtSLAH3 generated substantial currents (Fig. 1a), findings consistent with the previous study29 that SLAH3 mediates S-type anion currents when co-expressed with CPK21 in Xenopus oocytes. To delve deeper into the underlying anion transport mechanism of AtSLAH3, we overexpressed and purified the AtSLAH3 protein in HEK293F cells and resolved its structure at 2.63Å using cryo-EM (Fig. 1b and Extended Data Figs. 1 and 2). The map reconstructed using single-particle cryo-EM revealed clear structural details for the transmembrane region, extracellular loops, and most intracellular loops, except for the loop between TM8 and TM9, which might be due to its flexibility (Fig. 1c, d and Extended Data Fig. 2). Like AtSLAC1, AtSLAH3 forms a trimer, with each monomer consisting of 10 transmembrane helices. TM1, TM3, TM5, TM7, and TM9 are arranged in a pentagonal structure, critical for substrate transport. TM2, TM4, TM6, TM8, and TM10 create the outer layer, with longer helices at greater inclinations. Each monomer also contains two additional cytosol helix (CH) domains (CH1 and CH2), which may contribute to channel gating.

The channel narrows on the extracellular side and widens intracellularly. A “plug”-like structure composed of the N- and C-termini, along with CH1 and CH2, blocks the channel in its inactive state (Fig. 1c, d). We further analyzed the ion conduction pathway and identified four key residues involved in transport: F346, V403, F517, and F336 (Fig. 1e). Sequence alignment between AtSLAH3 and AtSLAC1 revealed high similarity, particularly in elongated N- and C-termini (Extended Data Fig. 3). The structural comparison showed that these key residues correspond to F276, V336, F450, and F266 in AtSLAC1 (Extended Data Fig. 4). Their arrangement is consistent in both channels. Notably, F517 (F450 in AtSLAC1) is the only fully conserved residue across the SLAC/SLAH family, located centrally in the pore and playing a pivotal role in gating (Extended Data Figs. 3 and 4). This suggests that AtSLAH3 and AtSLAC1 might share similar ion transport mechanisms, driven by these key residues. To gain deeper insights into the functional roles of these amino acids in the SLAH3 channel, we systematically introduced alanine substitutions at a total of 10 residues located within the N-termini, C-termini, CH-domains, and loop regions connecting the transmembrane domains (Fig. 1f). Current measurements of the mutants revealed that the N-terminal mutations Y227A, E234A, E236A, and R257A significantly enhanced the anion currents. Additionally, the R333A mutation on the lateral side of the TM3 helix and the Y515A mutation on the lateral side of the TM9 helix also increased the currents (Fig. 1g). These results further confirm that disrupting both the intracellular plug of SLAH3 and the interactions within its transmembrane region can partially activate SLAH3 channel activity.

Biochemical characterization and structural determination of the AtSLAH3-SLAH1 heterocomplex
A previous study35 showed that SLAH1 and SLAH3 are co-expressed in xylem parenchyma cells, and their interaction was further suggested when co-expressed in oocytes. To verify whether SLAH1 and SLAH3 co-localize in planta, we transiently co-expressed SLAH1-GFP and SLAH3-mCherry in Arabidopsis protoplasts and performed fluorescence co-localization analysis. The analysis revealed that SLAH1 and SLAH3 exhibited co-localization at the membrane (Fig. 2a), findings consistent with the previous study35. To verify the interaction between SLAH1 and SLAH3, we performed pull-down assays with tagged versions of SLAH1 and SLAH3 (Strep tag and Flag tag) (Fig. 2b). We confirmed the interaction between SLAH1 and SLAH3 by co-expressing and purifying them with different tags, demonstrating that SLAH1 and SLAH3 can form heterodimeric channel complexes in vitro. Expression tests confirmed that SLAH1 is more highly expressed than SLAH3 in vitro (Fig. 2b). To further investigate, we overexpressed SLAH3 (Strep-tagged) and SLAH1 (without tag), followed by affinity purification and cryo-EM analysis. Two distinct conformations of the complex emerged from subsequent 3D classification. Finally, we obtained a 3.03Å heterocomplex for conformation 1 and a 3.18Å heterocomplex for conformations 2 (Extended Data Fig. 5). These high-resolution maps enabled us to confirm that the conformation 1 heterocomplex contains 2 SLAH1 and 1 SLAH3, whereas the conformation 2 heterocomplex contains 1 SLAH1 and 2 SLAH3.

tRNA binding on the SLAH3-SLAH1 heterocomplexes
Interestingly, in the cryo-EM map of the 2SLAH1 + 1SLAH3 complex, we identified an additional density shaped like an inverted “L” (Extended Data Fig. 5). This region did not correspond to unmodeled domains of SLAH1 or SLAH3 and was absent in both the 1SLAH1 + 2SLAH3 and SLAH3 trimer complexes. This density, containing a double-helical structure, suggested the presence of nucleic acids. To identify the type of nucleic acid, we performed phenol-chloroform extraction on the proteins purified via affinity chromatography, followed by nucleic acid electrophoresis (Fig. 2c). The results showed nucleic acid fragments between 50 bp and 100 bp. DNase treatment did not affect the bands, while RNase treatment eliminated them, confirming that the nucleic acids isolated from the SLAH1/SLAH3 complex are RNA (Fig. 2c). Further RNA sequencing revealed that 87.45% of the nucleic acids were tRNA (Fig. 2d). These tRNAs were approximately 75 nucleotides long (Fig. 2e), consistent with our gel results. Sequencing data identified that tRNA-His-GTG-1-1 is the predominant type of tRNA, responsible for histidine transport within the cell (Fig. 2f). Hence, we incorporate this representative tRNA model into our heterocomplex (Fig. 2g).

In the 2SLAH1 + 1SLAH3 + tRNA complex, tRNA binds asymmetrically, forming two contact interfaces between SLAH1 and SLAH3 (Fig. 3a). Hydrophobic interactions between their transmembrane regions primarily sustain these interfaces. At interface 1, near the anticodon loop of tRNA, R287 from TM1 of SLAH1 interacts with the carbonyl oxygen of SLAH3. TM1 of SLAH1 also forms extensive hydrophobic contacts with TM3 and TM5 of SLAH3, stabilizing this interface (Fig. 3b). At interface 2, closer to the T loop of tRNA, interactions mainly involve hydrophobic contacts between TM3 of SLAH3 and TM4 and TM5 of SLAH1. Additionally, Y440 and Q441 from the intracellular loop of SLAH3 interact hydrophobically with Y167 and T174 of SLAH1. In the 1SLAH1 + 2SLAH3 complex without tRNA (Fig. 3c), similar polar and hydrophobic interactions stabilize the heterotrimer. Due to the heterotrimeric nature of the complex, two distinct interfaces (designated Interface 3 and Interface 4) are formed between SLAH1 and SLAH3. Interface 3 is supported by polar interactions involving R287 and Y319 from SLAH1 with K377 and E419. At interface 4, R499, Y480, K483, and Y529 form polar interactions with I164 and E207 (Fig. 3d).

tRNA binds on the SLAH1 to inhibit channel function
Our aforementioned studies suggested that tRNA primarily binds between two SLAH1 monomers but is absent in SLAH3 homomeric complexes and the 2SLAH3 + 1SLAH1 heteromeric complex. We speculate that tRNA preferentially interacts with SLAH1. To validate this, we overexpressed and purified SLAH1 and determined its 3D structure via cryo-EM. We observed tRNA binding in the SLAH1 homotrimer (Fig. 4a, b and Extended Data Fig. 6). Pore radius analysis of AtSLAH1 reveals the existence of three-layer constriction sites that maintain the AtSLAH1 in a closed state. The first layer, formed by L133, S54, and F311, functions as the gate-controlled hydrophobic layer. F307, located in the middle section of the channel and conserved among the SLAH family, constitutes the second layer. Of additional importance at the third layer, Y123 interacts with R47, constituting the narrowest region of SLAH1 together with I244 (Fig. 4c, d). Notably, R47 and Y123 are conserved only in the SLAH1/4 branch, whereas a glycine residue and a phenylalanine occupy the corresponding positions in SLAH2, SLAH3, and SLAC1, respectively (Fig. 4d).

Unlike SLAH3, SLAH1 lacks sufficient termini to form a “plug” below the transmembrane region, allowing tRNA to occupy the channel aperture, maintaining the channel in an inactive state (Extended Data Fig. 7). Electrophysiological recordings of SLAH1 on CHO cells revealed no significant currents, even with increased depolarized voltage and/or co-expressed with OST1 kinase (Fig. 4e). We assumed that the binding of tRNA might exert a regulatory role for the function of SLAH1. As expected, with the adding of 10 mM RNase A in the pipette solution, presumed to degrade tRNA to further decrease the binding extent of tRNA on SLAH1, we observed robust currents of SLAH1 (Fig. 4e). These results suggested that tRNA blocks the channel aperture, and its removal by RNase A activates the activity of channel, underscoring the inhibitory role of tRNA.

Electrostatic analysis of the SLAH1 homotrimer indicates a strong positive charge in the intracellular region, facilitating interactions with negatively charged tRNA (Fig. 5a, b). Since tRNA spans two SLAH1 monomers, only one tRNA binds in both homomeric (3SLAH1) and heteromeric (2SLAH1 + SLAH3) complexes. Hydrogen bonds between tRNA’s phosphate diester groups and lysine/arginine residues in the SLAH1 channel—K175, R176, K240, R242, K293, K296, R297, R356—ensure stable binding and block ion transport (Fig. 5c). Among these, only R176 exhibits high homology within the SLAH/SLAC family, whereas the other positively charged residues are exclusive to the SLAH1/SLAH4 branch (Extended Data Fig. 4). To validate the inhibitory effect of tRNA on SLAH channels, we generated alanine substitutions at eight positively charged lysine (K) and arginine (R) residues (K/R mutant) within the SLAH1-tRNA interaction interface, as identified in the structural model. First, electrophysiological measurements in CHO cells revealed that this SLAH1(K/R mutant) significantly enhanced SLAH1 anion currents without requiring exogenous RNase treatment (Fig. 5d). Subsequently, validation in Arabidopsis protoplasts demonstrated that, phenocopying the CHO cell results, the SLAH1(K/R mutant) attenuated the tRNA-mediated suppression of SLAH1 channel activity (Fig. 5e).

To further investigate the role of tRNA binding in SLAH1 during stomatal closure, we transiently expressed AtSLAH1, AtSLAH1(K/R mutant), AtSLAH3, and an empty vector control (EV) in leaves of Nicotiana benthamiana (Nb) and assessed their responses to ABA-induced stomatal closure. AtSLAH1-expressing leaves exhibited a degree of ABA-induced stomatal closure similar to the EV control (Fig. 5f). In contrast, leaves expressing the AtSLAH1(K/R mutant) showed enhanced ABA-induced stomatal closure, comparable to leaves expressing AtSLAH3 (Fig. 5f). These findings indicate that, unlike wild-type AtSLAH1, the AtSLAH1(K/R mutant) activates SLAH1 channel function, likely due to the disruption of tRNA-mediated inhibition.

Structural comparison between the four SLAH homo and hetero complex
In total, we resolved four complexes: SLAH1-tRNA, 2SLAH1 + SLAH3 + tRNA, SLAH1 + 2SLAH3, and 3SLAH3 (Extended Data Fig. 8). Structural comparison of the 3SLAH1 + tRNA and 2SLAH1 + SLAH3 + tRNA complexes revealed that tRNA resides between two SLAH1 monomers in both, indicating that the binding of tRNA requires at least two SLAH1 monomers in the complex. However, spatial displacement of the bound tRNAs occurs due to complex composition differences. Specifically, in transitioning from the 3SLAH1 to the 2SLAH1 + SLAH3 complex, the SLAH1 monomer near the 5′ and 3′ end of tRNA shifts clockwise, particularly in the outer helix (Extended Data Fig. 8a). No major conformational changes occurred in the other SLAH1 monomer present in both complexes. In addition, the 3SLAH3 and 1SLAH1 + 2SLAH3 complexes showed minimal conformational changes in their SLAH3 proteins (Extended Data Fig. 8b).

In the four SLAH transporter complex structures we resolved, the asymmetric binding of tRNA and the structural differences between the SLAH1 and SLAH3 monomers result in the present structures theoretically containing six distinct SLAH1 monomer (designated as SLAH1-1′ to SLAH1-6′) and four distinct SLAH3 monomer (designated as SLAH3-1′ to SLAH3-4′) (Extended Data Fig. 9a). Pore size analysis of the individual SLAH1 and SLAH3 monomers reveals that all monomers adopt a closed conformation with the narrowest constriction site below 1Å (Extended Data Fig. 9b, d, e, and g), which likely represents the lowest-energy state of SLAH family proteins in the absence of external stimuli. While minor conformational variations are observed among the key pore-lining residues across different SLAH1 and SLAH3 monomers (Extended Data Fig. 9c and f), all structures consistently correspond to the closed state.

Ion permeation of the SLAH family
Previous studies have shown that SLAH1 cooperates with SLAH3 to regulate the anion transportation from root to shoot35, of which the molecular mechanisms remain unclear. Akin to previous study, SLAH3 exhibits a marked anion (Cl−) preference, which was demonstrated by a positively shifted reversal potentials (Erev) when the decrease of Cl− rather than Na+ concentration in bath solution (Fig. 6a). For SLAH1, even with the adding of 10 mM intracellular RNase A, we could only obtain very small currents in the NaCl solution that was set to assess the ion selectivity (Fig. 6b). The small currents made us impossible to assess the anion preference directly, as the influence of background currents of blank cell became non-ignorable at this condition. However, the comparison of Erev at 0 and 10 min showed that along with the activation of SLAH1, the Erev was significantly right shifted, indicating an increase of anion preference that might be mediated by the activation of SLAH1 (Fig. 6c). These findings suggest that both SLAH1 and SLAH3 facilitate anion transport.

We next sought to explore the molecular basis of how SLAH1 and SLAH3 control the transport ratio of NO3−/Cl−. An almost equimolar nitrate-to-chloride solutions (pipette/bath: 140 mM Cl−/135 mM NO3−) were set to assess the relative nitrate-to-chloride of SLAHs. When SLAH3 channels were expressed alone, a reversal voltage of approximately −32 mV was observed, suggesting that SLAH3 channels have notably greater permeability to NO3− than to Cl− (Extended Data Fig. 10a). Mutation of key hydrophobic phenylalanine residues in the permeation pathway (F336A, F346A, F517A) didn’t significantly alter SLAH3 nitrate ion selectivity (Extended Data Fig. 10a). Similarly, when we expressed the SLAH1 channel protein alone, the Erev was only very slightly shifted (−7 ± 2 mV), demonstrating a comparable permeability to NO3− and Cl− (Extended Data Fig. 10b, c). Mutagenesis at the key hydrophobic phenylalanine residues (F307A and F311A) or the lower constriction site (Y123A) also didn’t significantly alter nitrate-to-chloride permeation of SLAH1 (Extended Data Fig. 10b, c).

However, the R47A and R198A mutations dramatically reduced whole-cell SLAH1 currents to near-background levels, indicating that residues R47 and R198 are critical for anion permeation through the channel (Extended Data Fig. 11). Notably, R47 and R198 are positively charged amino acids whose side chains project into the permeation pathway. These residues are highly conserved within the SLAH1/SLAH4 clade but are absent in other SLAH family members (Extended Data Fig. 3). R47 is positioned at the lower end and R198 at the upper part of the transmembrane region. The presence of these positive charges on opposite sides of the membrane influences anion recruitment and transport function, underlying the differences in anion permeability among SLAH family proteins.

Dynamic interactions within SLAH homo and heterocomplex in planta
To further validate and investigate its potential dynamics interactions between SLAH1 and SLAH3 in planta, we employed Bimolecular Fluorescence Complementation (BiFC) to assess their interactions under ABA treatment (Fig. 7). In Nicotiana benthamiana (Nb) leaves transiently co-expressing AtSLAH1-cYFP and AtSLAH3-nYFP, weak BiFC signal was detected under mock conditions. However, the interaction intensity increased after 3h of ABA treatment, and subsequently diminished after 6h of ABA exposure. This dynamic pattern confirmed their intrinsic interactions within SLAH1 and SLAH3 and suggests ABA-induced changes in the heterocomplex formation.

To model the condition where both SLAH1 and SLAH3 are present, we consistently co-expressed both channels in subsequent experiments (Fig. 7 and Extended Data Fig. 12). When SLAH1 was co-expressed with AtSLAH3-cYFP and AtSLAH3-nYFP in Nb, the homomeric interaction of SLAH3 showed a tendency to strengthen with ABA treatment. Conversely, when SLAH3 was co-expressed with AtSLAH1-cYFP and AtSHAH1-nYFP, the homomeric interaction of SLAH1 exhibited a weakening trend upon ABA application. Following the wash-out of the ABA stress signal, the signal intensity of the SLAH1 homomeric complex recovered. These findings suggest that the assembly of SLAH channels in an overexpression system in planta is a dynamic process. Over the course of ABA induction, the SLAH channel complex appears to transition from SLAH1 homomers, to SLAH1-SLAH3 heterocomplex, and finally to SLAH3 homomers. This dynamic shift in heteromeric complex assembly likely represents an adaptive response mechanism enabling plants to adjust to changing environmental conditions.

Methods
Materials
The following reagents were purchased from Sigma-Aldrich: NaCl (S9888); HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; H3375). In addition, protease Inhibitor Cocktail Tablets (04693116001) were obtained from Roche. Detergent LMNG (NG310) was obtained from Anatrace. Strep-Tactin resin (2-1208-500) and D-desthiobiotin (2-1000-005) were ordered from IBA. For cell culture, the human embryonic kidney (HEK) 293F cell line (R79007) was obtained from Thermo Fisher Scientific. The HEK293F cell culture medium (SMM293-TII) was obtained from Sino Biological; the Penicillin-Streptomycin Solution (SV30010) was ordered from Hyclone. We obtained the transfect reagents Polyethylenimine (PEI, Linear, MW 25000; 23966) from Polysciences.

Cell culture and transfection
For protein expression and purification, the optimized coding cDNA for Arabidopsis thaliana SLAH1 (Uniprot: Q5E930) and SLAH3 (Uniprot: Q9FLV9), including a C-terminus tandem twin Strep-tag, was cloned into the pcDNA3.1(−) vector. HEK293F cells were cultured in medium supplemented with 1× penicillin/streptomycin in a Multitron-Pro shaker (Infors, 120 r.p.m.) at 37 °C with 5% CO2. In brief, for 1 L culture of HEK293F cells, 1 mg SLAH1 or SLAH3 plasmid was pre-incubated with 3 mg 25-kD a linear PEIs (Polysciences) in 50 ml fresh medium for 30 min prior to adding the mixture to cells. The transfected cells were cultured for 48 h before harvest. For the expression of the SLAH1/SLAH3 heterocomplex, 0.75 mg of SLAH1 plasmid and 0.75 mg of SLAH3 plasmid were mixed with 3 mg of PEI per 1 L of HEK293F cells.

Protein expression and purification for cryo-EM analysis
For one batch of protein purification, about six liters of transfected cells were harvested by centrifugation at 3000×g. All procedures below were carried out at 4 °C or on ice.

For SLAH3 complex purification, harvested cells were resuspended in lysis buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM DTT, and protease inhibitor, and then lysed by high-pressure homogenizer. After removal of cell debris by centrifugation at 10,000×g for 45 min, the cell membrane fraction was pelleted by a 150,000×g ultracentrifugation for 1 h. The membrane fraction was resuspended and solubilized in lysis buffer plus 1% (w/v) LMNG for 2 h with gentle rotation. After ultracentrifugation at 150,000×g for 30 min, the supernatant was passed through a column filled with Strep-Tactin Sepharose resin. The resin was washed 50 CV (column volume) with wash buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM DTT, 0.005% (w/v) LMNG. The target SLAH3 protein was eluted with wash buffer plus 10 mM desthiobiotin. The eluted SLAH3 protein was concentrated to a final volume of approximately 100 μl by a 100 kDa cut-off centrifugal filter (Millipore) and further purified by size-exclusion chromatography (SEC) (Superose 6 5/150, GE Healthcare) in SEC buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 0.002% (w/v) LMNG, and 0.02% (w/v) DDM. The SEC fractions corresponding to the SLAH3 complex were collected for cryo-EM sample preparation. The peak fractions were concentrated to 20 mg/ml for grid preparation. A similar protocol was employed for the purification of the SLAH1 complex and the SLAH1/SLAH3 heterocomplex.

Electron microscopy sample preparation and imaging
The cryo-EM grids of SLAH family proteins were prepared using Vitrobot Mark IV (FEI) at 8 °C and 100% humidity. 3–4 μL aliquots of samples at a concentration of 20 mg/ml were applied onto glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3 300 mesh). After awaiting time of 5 s, the grids were blotted for 3 s and plunged into liquid ethane for quick freezing. The grids were screened on a Tecnai Arctica microscope (FEI) operated at 200 kV using a Falcon II direct electron detector (FEI). The qualified grids were transferred into a Titan Krios microscope (FEI) operated at 300 kV equipped with an energy filter (slit width 20 eV; GIF Quantum LS, Gatan) for data acquisition.

For the SLAH3 complex, images were recorded using a K3 submit direct electron detector (Gatan) and a GIF Quantum energy filter (slit width of 20 eV) in a super mode at a nominal magnification of 105,000×g, corresponding to a calibrated pixel size of 0.4187 Å. Date acquisition was performed automatically using AutoEMation 2.058 in a movie mode, with a frame exposure time of 0.08 s and a total exposure time of 2.56 s, resulting in a total of 32 frames per stack, and the total dose rate for each stack was approximately 50 e− Å−2. All 32 frames in each stack were aligned and summed using the whole-image motion correction program MotionCor259 and binned to a pixel size of 0.8374 Å.

For the SLAH1 complex, images were recorded similar to those of SLAH3 samples, except that the detector was equipped with a spherical aberration (CS) corrector and the final binned pixel size was 0.8697 Å.

For the SLAH1/SLAH3 heterocomplex, cryo-EM data were collected on a 300 kV Titan Krios G3i equipped with a Gatan K3 detector, a GIF Quantum energy filter (slit width of 20 eV), and a spherical aberration (CS) corrector. Micrographs were recorded with a defocus range of −1.3 to –1.8 μm. The total dose was about 50 e− Å−2 for each stack. Data collection was done using EPU data acquisition software in super resolution mode with a calibrated pixel size of 0.54 Å. All 32 frames in each stack were aligned and summed using the MotionCor2 and binned to a pixel size of 1.08 Å.

Image processing and 3D reconstruction
To investigate the transport mechanism of the SLAH family, we collected three data batches: 1329 images of SLAH3, 2862 images of the SLAH1 + SLAH3 heteromeric complex, and 1013 images of SLAH1. Image processing was performed using cryoSPARC software60.

For the data set of the SLAH3 complex, micrographs were imported, and the CTF corrections were performed using Patch CTF estimation. After the deletion of bad micrographs, 200 micrographs were used for automatic picking by blob picker, and these particles were subjected to 2D classification. The class averages representing projections of the SLAH3 complex in different orientations were chosen as templates for template picking from the whole dataset. A total of 1418 K particles were picked from 1329 micrographs. These particles were extracted and binned 4 times and subjected to 2D classification. After three rounds of 2D classification, 586 K particles in good 2D averages were chosen. These particles were subjected to the ab initio reconstruction. The maps of each class were measured in Chimera61, and the particles from the best class were selected and re-extracted to the original pixel size. After another round of ab initio reconstruction, three different states of the SLAH1 complex were identified. The best particles for SLAH3 complex were used for 3D reconstruction by non-uniform (NU) refinement62 to further improved data quality. Finally, a 2.63 Å resolution map with C3 symmetry imposed was obtained. The local resolution maps of the SLAH3 complex were calculated using local resolution estimation in cryoSPARC and displayed in ChimeraX63.

Similar data processing procedures were applied for the dataset of the SLAH1 complex. Finally, due to the binding of tRNA in the cytosol side of SLAH1, we imposed C1 on the final best particles and obtained a 3.18 Å resolution map of the SLAH1-tRNA complex.

For the dataset of SLAH1 + SLAH3 heterocomplex, ab-intial classification clearly distinguished two different states of the complex. From further calculations using NU-refinement and CTF refinement, we obtained a 3.03 Å resolution map for 2SLAH1 + 1SLAH3 + tRNA complex and a 3.18 Å resolution map for 1SLAH1 + 2SLAH3 complex.

RNA sequencing and data analysis
Overexpressed SLAH1 proteins were obtained following affinity chromatography and gel-filtration chromatography. RNA was extracted using a phenol-chloroform extraction followed by ethanol precipitation.

tRNA was isolated from total RNA using the Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE) method. Over 2 μg total RNA was resolved on 7.5% Urea PAGE gels (7M Urea) and recovered within a size window of 60–100 nt for tRNA.

The demethylation section in rtStarTM tRF & tiRNA Pretreatment Kit was used for tRNA m1A & m3C demethylation treatment. A 50 μL demethylation reaction mixture was prepared according to the manufacturer’s protocol and incubated at 37 °C for 2 h. Then, 40 μL nuclease-free water and 10 μL 5× Stop Buffer were added to terminate the reaction. Demethylated tRNA was purified by phenol-chloroform extraction and ethanol precipitation. Demethylated tRNA was partially hydrolyzed according to Hydro-tRNA seq method. Partially hydrolyzed tRNA fragments were converted to barcoded small RNA sequencing libraries using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit according to the manufacturer’s instructions.

The completed libraries were qualified and quantified by the Agilent 2100 Bioanalyzer. An equal amount of Hydro-tRNA seq libraries was mixed for the following sequencing on an Illumina sequencer. The DNA fragments in well-mixed libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, and loaded onto the reagent cartridge at a loading concentration. The sequencing run was performed on Illumina sequencer.

Sequencing quality was examined by FastQC software, and trimmed reads (trimmed 3′-adaptor bases by cutadapt) were aligned to the cytoplasmic mature-tRNA sequence obtained from GtRNAdb and the mitochondrial mature-tRNA sequences obtained from mitotRNAdb using BWA software. For tRNA alignment, the maximum mismatch was 2. The tRNAs expression profiling was calculated based on uniquely mapped reads and including mapped reads, respectively. The differentially expressed tRNAs were screened based on the count value with the R package edgeR. Principal Component Analysis, correlation analysis, Hierarchical Clustering, scatterplots, venn plots, and volcanoplots were performed with the differentially expressed tRNAs in R or Python environment for statistical computing and graphics.

Model building
Models of full-length SLAH1 and SLAH3 were predicted on AlphaFold64,65. Sequence alignment and secondary structure prediction of SLAH1 and SLAH3 were used to aid the model building. The predicted model of SLAH3 was docked into the cryo-EM map with a resolution of 2.63 Å in Chimera and manually adjusted in Coot to acquire the atomic model of SLAH361,66. Model refinement was performed on the main chain of the atomic models using the real_space_refine module of PHENIX67 with secondary structure and geometry restraints to avoid over-fitting.

According to small RNA sequencing, the tRNA (His-GTG-1-1) is the most dominant class among the 75 nt tRNA candidates. The tRNA (His-GTG-1-1) sequence is: GCCGUGAUCGUAUAGUGGUUAGUACUCUGCGUUGUGGCCGCAGCAACCUCGGUUCGAAUCCGAGUCACGGCACCA.

The 3D structure model for tRNA was predicted using trRosettaRNA68 and manually adjusted to fit in the map using COOT.

Then, the model of SLAH1 and tRNA was used initial model for the model building of the 3SLAH1 + tRNA complex. Similar procedures were performed for the 2SLAH1 + 1SLAH1 + tRNA complex and the 1SLAH1 + 2SLAH3 complex. Cryo-EM data collection and refinement statistics are shown in Table S1.

Whole-cell patch-clamp recording and data analysis
The DNA encoding SLAH1 or SLAH3 was subcloned into the pcDNA3.1(−) with or without an N-terminal GFP-tag. Chinese hamster ovary cells (CHO) were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (Gibco) and 100 μg/mL penicillin-streptomycin. Cells were transiently transfected with SLAH1 and/or SLAH3 or both using Lipofectamine 3000 reagent (Thermo Fisher Scientific). Electrophysiology experiments were performed 24−48 h after transfection. Patch pipettes were pulled with P-97 (SUTTER INSTRUMENT) and heat polished to a resistance of 3–8 MΩ. Patch-clamp recordings were done with an Axopatch 200B amplifier and Axon Digidata 1550A driven by Clampex 10 software (Molecular Devices).

For the recording of anion (Cl−) mediated currents, pipettes were filled with the solution containing 150 mM HCl and 20 mM HEPES, pH 7.4 adjusted with NMDG, and cells were bathed with bath solution containing 150 mM HCl, 1 mM CaCl2 and 20 mM HEPES, pH 7.4 with NMDG. For the assessment of anion (Cl−) selectivity of SLAH3, pipettes were filled with the solution containing 140 mM NaCl, 5 mM EGTA, 3 mM Mg-ATP and 10 mM HEPES, pH 7.2 with NMDG, bath solution including three types that were high NaCl bath solution (135 mM NaCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose and 10 mM HEPES, pH 7.4 with NMDG), low NaCl bath solution (9 mM NaCl, 1 mM MgCl2, 1.5 mM CaCl2, 260 mM glucose and 10 mM HEPES, pH 7.4 with NMDG) and low Na+ bath solution (9 mM NaCl, 126 mM NMDG-Cl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose and 10 mM HEPES, pH 7.4 with NMDG). For the assessment of anion (Cl−) selectivity of SLAH1, the testing paradigm was the same as that of SLAH3, except that the pipette solution was supplemented with 10 mM RNase A, and the bath solution applied was only the low NaCl solution. For the assessment of relative nitrate-to-chloride permeabilities, the pipette solution containing 140 mM NaCl, 5 mM EGTA, 3 mM MgATP, and 10 mM HEPES, pH 7.2 with NMDG, and the bath solution containing 135 mM NaNO3, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4 with NMDG. Whole-cell currents were evoked by 800 ms voltage steps from −100 mV to +100 mV in 20 mV increments. All recordings were performed at room temperature (approximately 25 °C). All values were measured using Clampfit 10.6 Software.

Plant materials, vector construction, and growth conditions
For protoplast transformation, A. thaliana Col-0 were grown under an 8 h-light/16 h-dark photoperiod (22 °C–18 °C). Nicotiana benthamiana (Nb) plants were grown under a 16 h-light/8 h-dark photoperiod (24 °C–26 °C). For protoplast transformation, the coding sequences of AtSLAH1, AtSLAH17M, AtSLAH3, and AtOST1 were amplified and inserted into pUC19 vector.

For the BIFC assay, the coding sequences of AtSLAH1, AtSLAH1(R/K mutant), or AtSLAH3 were amplified and inserted into pEG100-cYFP/nYFP vector. For Stomatal aperture quantification, the coding sequences of AtSLAH1, AtSLAH1(R/K mutant), or AtSLAH3 were amplified and inserted into pE1766 vector.

Transient expression in A. thaliana protoplasts and electrophysiological recording
Procedures for protoplast transformation were described previously69. A. thaliana mesophyll protoplasts were isolated from 3 to 4-week-old Col-0 plants. Leaf slices from the fifth, sixth, and seventh true leaves of these plants were digested in enzyme solution for 3 h. The suspension was centrifuged and washed twice with W5 solution. Plasmids were transformed into protoplasts using DNA-PEG–calcium transfection. The transfected protoplasts were incubated at 22 °C for 16 h in WI solution and then were used for subsequent assays.

For the electrophysiological recording in protoplast, pipettes with a resistance of 10–20 MΩ were filled with the solution containing 140 mM NaCl, 5 mM EGTA, 3 mM Mg-ATP, 10 mM HEPES, 1 mM mannitol, pH 7.2 with NMDG. Three concentrations of mannitol (150 mM, 100 mM, and 50 mM) were tested in the bath solution, and the final bath solution composition was determined to be 135 mM NaCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, 10 mM HEPES, and 100 mM mannitol, pH 7.4 with NMDG. Whole-cell currents were evoked by 800 ms voltage steps from −100 mV to +100 mV in 20 mV increments. All recordings were performed at room temperature (approximately 25 °C). All values were measured using Clampfit 10.6 Software.

Agrobacterium-mediated transient expression
The Agrobacterium tumefaciens (GV3101) strains containing the indicated constructs were cultivated at 28 °C overnight, pelleted by centrifugation, and resuspended in infiltration buffer (10 mM MES, 200 μM acetosyringone, 10 mM MgCl2, pH 5.6), kept under dark for 2 h (OD600 = 0.1–0.6), and subsequently syringe-infiltrated into Nb leaves as Agro-infiltration.

Bimolecular fluorescence complementation
For BIFC assay, the coding sequences of AtSLAH1 and AtSLAH3 were cloned into pEG100 vector to construct fusions expressing cYFP and nYFP. All of the constructs were introduced into the A. tumefaciens strain GV3101 and infiltrated into N. benthamiana leaves. Discs were removed from these leaves using a hole puncher at 42 h, 45 h, 48 h, and treated with ABA (final concentration: 100 μM) for time gradients. Discs were removed from these leaves using a hole puncher at 36 h and treated with water for 6 h after ABA (final concentration: 100 μM) for 6 h. Leaf discs subjected to different ABA treatments were harvested from the same leaf following the groupings scheme of the BiFC assay. Confocal imaging was performed using the Zeiss LSM880 at 48 h after Agro-infiltration.

Stomatal aperture quantification
For AtSLAH1, AtSLAH1(R/K mutant) or AtSLAH3 overexpression regulated stomatal aperture, 3–4 leaves from 4-week-old Nb plants were syringe infiltrated and subsequently flooded with Agrobacterium strains containing the indicated constructs in a buffer with 0.01% (v/v) Tween-20. At 36 h after Agro-infiltration, Stomatal aperture quantification leaves of the Nb were harvested and floated on stomatal opening buffer (50 mM KCl, 10 mM MES-KOH, pH 6.15) with the abaxial surfaces exposed to light for 3 h, and subsequently treated with mock or ABA (final concentration: 10 μM) for 1 h.

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

Data availability
The 3D cryo-electron microscopy density map has been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) with the following accession codes: 3SLAH3 EMDB: 63691; PDB: 9M7R, 1SLAH1 + 2SLAH3 EMDB: 63692; PDB: 9M7S, 3SLAH1 + tRNA EMDB: 63693; PDB: 9M7U, 2SLAH1 + 1SLAH3 + tRNA EMDB: 63695, PDB: 9M7W. The source data underlying Figs. 1a, g, 2b, c, 4e, 5d–f, 6a–d and Supplementary Figs. 10, 12, and 14 are provided as a Source Data file. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Zhang et al. report the cryo-EM structures of A. thaliana S-type anion channel homologs (SLAH) in\ndistinct states. Notably, they identify tRNA as a binding partner of SLAH1 that inhibits channel activity. Overall, the\nmanuscript is intriguing, and the cryo-EM data appear solid and promising. This reviewer has the following suggestions and\nquestions for the authors’ consideration during revision:\n1. What is the physiological role of tRNA in regulating channel activity? While the authors provide biochemical evidence, it\nremains unclear why tRNA, rather than another protein or small molecule, specifically binds to SLAH1 and inhibits its\nfunction.\n2. The authors claim that tRNA inhibits channel activity. Could they provide a pore-size curve comparing channels with and\nwithout tRNA binding?\n3. In Fig. 5c, the authors highlight several basic residues essential for tRNA engagement. Have they tested mutations of\nthese residues and assessed their functional impact?\n4. In Fig. 2a, why are there two bands in each pull-down lane? Are they input and elution fractions? This should be clarified.\n5. Fig. 3c should be labeled as Fig. 3b, and Fig. 3b should be 3c to align with their order of citation in the manuscript.\n6. The abstract should be refined to better emphasize the role of tRNA in channel regulation.", "reviewer_2": "Evaluation des Manuskripts #NCOMMS-25-16898-T „Structural insights into the coordinated regulation of the SLAH family\nin Arabidopsis thaliana“ by Zhang et al.\nWhile the structure and function of the SLAC/SLAH family founder, SLAC1, is well documented, the remaining members of\nthe SLAH family and heteromers thereof are only poorly understood. Here, the authors provide CryoEM structures of\nhomomeric SLAH1 and 3 channels as well as of heterotrimers between SLAH1 and 3 subunits with different stoichiometries.\nBy chance, they discovered that a tRNA molecule blocks the cytosolic entrance of the pore of SLAH1 subunits. In principle,\nboth, the new structures and the putative role of regulatory tRNAs are of high interest. However, the MS is of insuffficant\nquality and conclusions drawn are often not supported by corresponding experiments. Since, the quality of the paper in ist\npresent form does not meet the standards of Nature Communication the referee lists only the major shortcomings of the MS:\n• Neither the description of the new SLAH structures nor the proposed regulatory role of the tRNA are sufficiently\nunderpinned by mutant analysis (e.g. binding sites for the tRNA, mutants changing the permeability/selectivity, the role of the\ntermini, CH-domains), and comprehensive electrophysiological measurements.\n• The reader does not find sufficient description/explanation of the results in the text, the figure legends, and in the figures\nitself.\n• The description of results is too sparse (especially the 3D structures) and the authors do not follow a logical story board.\nE.g. inhibition of anion currents by tRNA is the prime new finding but the authors do not provide any mutant analysis to\nsupport their structural findings and do not provide any hint whether there are tRNAs interacting with SLAH1 subunits in\nplanta.\n• Often only minor Erev shifts (e.g. 10 to 20 mV upon a 10-fold shift in external chloride concentration) are shown to provide\nevidence for permeability changes between different subunit stoichiometries but the current amplitudes and kinetics are not\nmentioned/analysed. In stark contrast to the S-type anion current electrophysiology in guard cells analysis of SLAH3 in\noocytes, SLAH3 does not show any deactivation kinetics in the hands oft he authors. The reader wonders about this strong\ndiscrepancy.\n• In addition the reader wonders about another strong discrepancy oocyte data published and CHO cells data shown by the\nauthors. In oocytes coexpression of SLAH1 and 3 is sufficient to generate a chloride and nitrate permeable anion channel\nwithout the need of a kinase (Lehmann et al.,2021), while in CHO cells it is not?\n• The reader is not informed by the authors, how a putative regulation via tRNA should works in planta?", "reviewer_3": "In this manuscript, Sensen and colleagues investigate the molecular mechanisms of SLAH proteins, S-type anion channel\nhomologs from Arabidopsis thaliana, using structural, biochemical, and electrophysiological approaches. SLAHs are widely\nexpressed in various plant tissues and are responsible for anion transport, playing a crucial role in plant adaptation to both\nbiotic and abiotic stresses. The authors determined several cryo-EM structures of AtSLAH at different functional states,\nincluding the homotrimeric SLAH3, a heterotrimeric complex comprising two copies of SLAH1 and one copy of SLAH3 with\ntRNA, another heterotrimeric complex with one copy of SLAH1 and two copies of SLAH3, and the\nhomotrimeric SLAH1 with tRNA bound. Structural comparisons of these complexes revealed the binding mode of tRNA on\nSLAH1, where two subunits coordinate a single tRNA molecule. Additionally, the authors identified key residues essential for the\nnitrate-to-chloride permeability ratio. Together, these findings provide valuable insights into how SLAH proteins respond to\nextreme environmental conditions.\nThe complex, particularly the heterotrimer containing SLAH1 and SLAH3, generated using overexpressed proteins, is\nintriguing. However, it remains unclear whether these complexes exist under physiological conditions. The authors should\nprovide evidence to demonstrate their physiological relevance. In Figure 7, they propose a model linking observations from\ntheir structural and functional studies. It is uncertain whether the different complexes of SLAH1 and SLAH3 co-exist or if the\ncomplex transitions from an SLAH1 trimer to an SLAH3 trimer under stress. Additionally, will other SLAH members also form\nheterotrimers, given their high sequence similarity?\nAnother related question arises: in the heterotrimeric complex, if tRNA binding inhibits the activity of SLAH1, how will the\nactivity of SLAH3 be affected?\nThe authors refer to two heterocomplexes of SLAH1 and SLAH3 as state 1 and state 2, suggesting they are two subsequent\ntransient states in complex formation. Given the uncertainty regarding their physiological relevance, it would be better to\nrename these complexes.\nHere are my comments:\n1. Abstract: It is unclear how this study \"establishes a new paradigm for coordinated regulation\" and \"opens new avenues for\nengineering crops under extreme environmental conditions.\"\n2. Line 88: The map reconstructed using single-particle cryo-EM is not an electron density map; please correct this.\n3. Line 89: The authors need to provide evidence to support the claim of “cleared structural details for extracellular loops and\nmost intracellular loops.”\n4. Line 65: “Cl-from” should be corrected to “Cl- from.”\n5. Line 95: Define \"CH\" for a broader audience.\n6. Figure 3a: Part of the protein density is colored as tRNA.\n7. Both interface 1 and 2 are used to describe the interactions between SLAH1 and SLAH3 in the two complexes. Consider\nusing different terms to avoid potential confusion.\n8. Line 161: The statement \"The SLAH1 channel narrows extracellularly but expands intracellularly\" is difficult to understand\nbased on the current version of Figure 4c-d.\n9. The pore profile should be presented for SLAH1 and SLAH3 in the heterotrimeric complexes, particularly to support the\nclaim that “the tRNA maintains the channel in an inactive state.”\n10. Scale bars are missing in the raw micrographs in the supplemental materials.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Response: We sincerely appreciate the reviewer's insightful question regarding the specific\nphysiological role of tRNA in regulating SLAH1 channel activity. Our findings that tRNA, rather\nthan another protein or small molecule, acts as a direct inhibitory regulator of SLAH1 are\nsupported by multiple lines of evidence and structural rationale:\n(1) Biochemical Identification: We isolated the SLAH1 protein complex in vitro and identified\nco-purifying nucleic acids via gel electrophoresis. Subsequent DNase/RNase sensitivity\ntests confirmed these were RNA molecules. RNA sequencing definitively revealed that\ntRNA constituted the vast majority (87.45%) of this bound RNA, with tRNA-His-GTG-1-1\nbeing predominant (Figure 2).\n(2) Functional Validation via Mutagenesis: The cryo-EM structure of the SLAH1-tRNA\ncomplex revealed key positively charged residues (Lysine/Arginine, K/R) within the\nintracellular entrance of SLAH1 that form hydrogen bonds with the phosphate backbone\nof tRNA. We generated a SLAH1(K/R mutant) targeting these specific interaction sites.\nElectrophysiological recordings in both CHO cells and Arabidopsis protoplasts\ndemonstrated that this mutant exhibited significantly enhanced anion currents without\nrequiring exogenous RNase treatment, phenocopying the activation seen when wild-type\nSLAH1 is treated with RNase. This directly links disruption of the tRNA-binding interface\nto channel activation, confirming tRNA's inhibitory role.\n(3) Physiological Relevance in Stomatal Closure: Transient expression in Nicotiana\nbenthamiana leaves showed that while wild-type AtSLAH1 expression resulted in ABA-\ninduced stomatal closure similar to the empty vector control, expression of the\nAtSLAH1(K/R mutant) significantly enhanced ABA-induced closure, comparable to the\neffect of expressing the SLAH3 channel. This demonstrates that attenuating tRNA-\nmediated inhibition of SLAH1 potentiates its physiological function in stress response\n(stomatal closure).\nWhy tRNA specifically? The strong electrostatic complementarity provides a compelling\nexplanation. The intracellular region of the SLAH1 homotrimer exhibits a pronounced positive\nelectrostatic potential, creating an ideal binding surface for the highly negatively charged\ntRNA molecule. This electrostatic attraction is a primary driver for the specific interaction.\nWhile tRNA is classically known for its role in protein synthesis, its ability to act as a regulatory\nligand through charge-based interactions with proteins is increasingly recognized, paralleling\nrecent findings where tRNA binds cytochrome c to inhibit apoptosis or bridges subunits of\nproton-activated chloride channels. Therefore, tRNA's inherent polyanionic nature allows it\nto function as a specific, potent inhibitory subunit for the SLAH1 channel, modulating anion\nflux and consequently, plant stress responses like stomatal closure.\nIn summary, our combined structural, biochemical, electrophysiological, and physiological\ndata establish tRNA as a specific endogenous ligand that binds the positively charged\nintracellular aperture of SLAH1 via defined residues, maintaining the channel in a closed state.\nIts degradation or displacement releases this inhibition, activating anion efflux critical for\nstress adaptation.\n2. The authors claim that tRNA inhibits channel activity. Could they provide a pore-size curve\ncomparing channels with and without tRNA binding?\nResponse: We sincerely thank the reviewer for their constructive comment regarding the\npore-size analysis. Our structural analysis using the HOLE program indeed provides insights\ninto the closed state induced by tRNA binding:\n(1): Structural Evidence for Closure: Pore radius analysis of all resolved SLAH complexes –\nincluding the SLAH1 homotrimer with bound tRNA, the 2SLAH1+SLAH3+tRNA\nheterocomplex, the 1SLAH1+2SLAH3 heterocomplex, and the SLAH3 homotrimer –\nconsistently revealed a closed conformation. Each individual monomer within these\ncomplexes exhibited a narrowest constriction site below 1 Å (Extended Data Fig. 9).\nThis represents the lowest-energy state of these channels in the absence of activating stimuli.\n(2): Conformational Impact of tRNA: Within the SLAH1 homotrimer + tRNA complex, we\nanalyzed the pore profile of all three monomers. While subtle conformational variations exist\namong the monomers (Extended Data Fig. 9c), tRNA binding resulted in all adopting a closed\npore conformation with sub-1 Å constrictions. Critically, this tRNA-bound state corresponds\nto the functional observation of no significant currents in SLAH1-expressing cells, even when\nco-expressed with the activating kinase OST1 (Fig. 4e).\n(3): Functional Evidence for tRNA-Mediated Inhibition: The key functional evidence for tRNA's\ninhibitory role comes from electrophysiology: Robust SLAH1 currents were only observed\nupon exogenous addition of RNase A (presumed to degrade bound tRNA) to the pipette\nsolution (Fig. 4e). Similarly, targeted mutagenesis of key tRNA-interacting residues\n(SLAH1(K/R mutant)) in SLAH1 resulted in significantly enhanced anion currents without\nRNase treatment (Fig. 5d, e), phenocopying the RNase effect.\nThese combined structural and functional data lead us to propose that tRNA binding,\nmediated by hydrogen bonds to key residues lining the intracellular vestibule of SLAH1 (Fig.\n5c), stabilizes the channel in a closed conformation. This likely impedes conformational\nchanges necessary for activation, even in the presence of regulatory kinases like OST1.\nThe pore constriction observed in the tRNA-bound structures is consistent with this mechanism\nof inhibition. While we have not resolved an activated open-state structure of SLAH1 without\ntRNA for direct pore-size comparison, the clear activation of currents upon tRNA removal (via\nRNase or mutagenesis) provides compelling functional evidence that tRNA binding is the\ncritical factor maintaining the channel in its closed, non-conducting state observed\nstructurally.\n3. In Fig. 5c, the authors highlight several basic residues essential for tRNA engagement. Have\nthey tested mutations of these residues and assessed their functional impact?\nResponse: We sincerely thank the reviewer for this valuable suggestion. Based on the\nstructural analysis identifying key positively charged residues within the SLAH1 intracellular\nvestibule that form hydrogen bonds with tRNA (Fig. 5c), we generated a SLAH1(K/R mutant)\ntargeting all eight highlighted basic residues (Lysine/Arginine) crucial for tRNA engagement.\nWe then rigorously assessed the functional impact of these mutations using three\ncomplementary experimental approaches:\n(1): Electrophysiology in CHO Cells: Whole-cell patch-clamp recordings demonstrated that\nthe SLAH1(K/R mutant) exhibited significantly enhanced anion currents compared to wild-\ntype (WT) SLAH1, even in the absence of exogenous RNase treatment (Fig. 5d). This\nphenocopies the activation seen with RNase degradation of tRNA.\n(2): Electrophysiology in Arabidopsis Protoplasts: Consistent with the CHO cell results,\nexpression of the SLAH1(K/R mutant) in native plant protoplasts also resulted in robustly\nenhanced anion currents relative to WT SLAH1 (Fig. 5e).\n(3): Stomatal Response Assay in Nicotiana benthamiana: Transient expression of the\nAtSLAH1(K/R mutant) in Nb cells significantly enhanced ABA-induced stomatal closure\ncompared to leaves expressing WT AtSLAH1 (Fig. 5f).\nThese three independent lines of evidence—enhanced currents in heterologous (CHO) and\nnative (protoplast) systems, coupled with potentiated physiological function (stomatal\nclosure)—conclusively establish that the specific basic residues identified structurally (Fig. 5c)\nare functionally essential for mediating tRNA binding and its inhibitory regulation of SLAH1\nchannel activity. Disruption of this interaction site releases the tRNA-mediated inhibition,\nactivating the channel.\n4. In Fig. 2a, why are there two bands in each pull-down lane? Are they input and elution\nfractions? This should be clarified.\nResponse: We sincerely apologize for any confusion caused by this figure. For the\nheterocomplexes formed by SLAH1 and SLAH3 with different tags, we performed separate in\nvitro overexpression followed by affinity purification and gel filtration chromatography. The\ntwo bands shown in each pull-down lane in Fig. 2a represent protein samples from two\nadjacent fractions near the peak apex of the gel filtration elution profile. To eliminate this\npotential confusion, we have removed one of the bands in the revised version of the\nmanuscript and have provided a clarifying explanation in the corresponding figure legend.\n5. Fig. 3c should be labeled as Fig. 3b, and Fig. 3b should be 3c to align with their order of\ncitation in the manuscript.\nResponse: We sincerely appreciate the reviewer for point-outing this mistake; we have\ncarefully corrected the figure panel labeling order (swapping what was originally labeled as\nFig. 3c to Fig. 3b, and Fig. 3b to Fig. 3c) in the revised manuscript to ensure proper alignment\nwith their citation sequence in the text.\n6. The abstract should be refined to better emphasize the role of tRNA in channel regulation.\nResponse: Thanks for this suggestion. We have refined the abstract to better summarize the\nrole of tRNA in channel regulation.\nReviewer #2 (Remarks to the Author):\nEvaluation des Manuskripts #NCOMMS-25-16898-T „ Structural insights into the\ncoordinated regulation of the SLAH family in Arabidopsis thaliana“ by Zhang et al.\nWhile the structure and function of the SLAC/SLAH family founder, SLAC1, is well documented,\nthe remaining members of the SLAH family and heteromers thereof are only poorly\nunderstood. Here, the authors provide CryoEM structures of homomeric SLAH1 and 3\nchannels as well as of heterotrimers between SLAH1 and 3 subunits with different\nstoichiometries. By chance, they discovered that a tRNA molecule blocks the cytosolic\nentrance of the pore of SLAH1 subunits. In principle, both, the new structures and the putative\nrole of regulatory tRNAs are of high interest. However, the MS is of insuffficant quality and\nconclusions drawn are often not supported by corresponding experiments. Since, the quality\nof the paper in ist present form does not meet the standards of Nature Communication the\nreferee lists only the major shortcomings of the MS:\n• Neither the description of the new SLAH structures nor the proposed regulatory role of the\ntRNA are sufficiently underpinned by mutant analysis (e.g. binding sites for the tRNA, mutants\nchanging the permeability/selectivity, the role of the termini, CH-domains), and\ncomprehensive electrophysiological measurements.\nResponse: We sincerely appreciate the reviewer's suggestions regarding the need for deeper\nmutant analysis to underpin our structural findings and tRNA regulatory mechanism. To\ncomprehensively address this, we performed extensive mutagenesis coupled with\nelectrophysiological characterization across three critical domains:\n(1): tRNA-Binding Site Mutants: Based on the SLAH1-tRNA complex structure (Fig. 5c), we\ngenerated a SLAH1(K/R mutant) targeting key positively charged residues essential for tRNA\nengagement within the intracellular vestibule. Electrophysiological recordings in both\nheterologous (CHO cells, Fig. 5d) and native (Arabidopsis protoplasts, Fig. 5e) systems\ndemonstrated that this mutant exhibited significantly enhanced anion currents compared to\nwild-type (WT) SLAH1, phenocopying the activation seen with RNase degradation of tRNA.\nThis provides direct functional validation that disrupting the identified tRNA-binding interface\nreleases inhibition, confirming tRNA's regulatory role.\n(2): Permeation Pathway Mutants in SLAH1: We systematically mutated charged and polar\nresidues lining the SLAH1 permeation pathway (R47A, Y123A, F307A, F311A, I244A, Q136A,\nR198A, Q318A). Crucially, R47A and R198A mutations dramatically reduced whole-cell SLAH1\ncurrents to near-background levels (Extended Data Fig. 11), establishing these residues as\nessential for anion conduction. Mutations at other sites (F307A, F311A, Y123A) did not\nsignificantly alter the NO₃⁻/Cl⁻ permeability ratio compared to WT SLAH1 (Extended Data Fig.\n10b-c), indicating these residues primarily influence conductance magnitude rather than\nselectivity under the conditions tested.\n(3): N/C-Termini, CH-Domains, and Loop Mutants in SLAH3: To probe the role of the\nintracellular \"plug\" and gating domains identified in the SLAH3 structure (Fig. 1f), we mutated\n10 residues in the N-terminus, C-terminus, CH-domains, and loops connecting\ntransmembrane helices. Electrophysiological analysis revealed that mutations disrupting the\nplug/interactions (Y227A, E234A, E236A, R257A in N-terminus; R333A in CH2 domain; Y515A\nlateral to TM9) significantly enhanced SLAH3 anion currents (Fig. 1g). This functionally\nconfirms that the intracellular domains act as a regulatory module whose disruption partially\nactivates the channel.\nThese comprehensive mutant analyses—spanning tRNA interaction sites, permeation\npathway residues critical for conduction, and regulatory termini/CH-domains—coupled with\ndetailed electrophysiological characterization across multiple systems (Figs. 1g, 4e, 5d-e,\nExtended Data Figs. 10, 11) provide robust functional underpinning for our newly resolved\nSLAH structures and the proposed inhibitory mechanism mediated by tRNA binding to SLAH1.\nThe data collectively demonstrate how specific structural elements govern channel gating,\nanion permeation, and tRNA-dependent regulation.\n• The reader does not find sufficient description/explanation of the results in the text, the\nfigure legends, and in the figures itself.\nResponse: Thank you very much for this valuable feedback regarding the clarity and depth\nof our results presentation. We have carefully revised the manuscript to significantly enhance\nthe description and explanation of our findings throughout the main text, figure legends, and\nthe figures themselves.\nSpecifically, we have added substantial new data and corresponding detailed descriptions\nfocused on key findings:\n(1) Electrophysiological Validation of tRNA Inhibition: We have added detailed\nelectrophysiological data demonstrating the inhibitory effect of tRNA on the SLAH1 channel.\nThis includes: Current recordings from both CHO cells and Arabidopsis protoplasts expressing\nthe SLAH1(K/R mutant) (targeting tRNA-binding residues). Results consistently show\nsignificantly enhanced currents compared to wild-type SLAH1 (Figs. 5d, 5e), directly\nconfirming the functional consequence of disrupting tRNA binding.\n(2) Validation of Complex Dynamics in planta: To solidify our findings on complex assembly\nand transitions, we have added: Fluorescence co-localization analysis demonstrating the\ninteraction between SLAH1 and SLAH3 at the membrane in Arabidopsis protoplasts (Fig. 2a).\nBimolecular Fluorescence Complementation (BiFC) assays in Nicotiana benthamiana leaves.\nThese assays visually confirm the interaction between SLAH1 and SLAH3 and, critically, reveal\nthe dynamic state transitions occurring in response to ABA treatment, reflecting adaptation\nto abiotic stress (Fig. 7).\nPhysiological Relevance in Stomatal Regulation: We have incorporated stomatal aperture\nassays in Nicotiana benthamiana leaves. These assays clearly demonstrate the distinct\nfunctional roles.\n(3) Enhanced Presentation: Furthermore, we have thoroughly revised the narrative flow of the\nResults section to ensure logical progression and improved integration of these new findings.\nCrucially, we have significantly expanded the descriptions within the figure legends to provide\nstandalone clarity, explicitly stating the biological question addressed, the experimental\nsystem used, key observations, and the interpretation/conclusion drawn from each panel. We\nhave also ensured that all critical features discussed in the text are clearly labeled and\nannotated within the figures themselves.\nWe believe these comprehensive revisions substantially improve the clarity, completeness,\nand rigor of our presentation, providing the reader with a much more robust and well-\nexplained account of our structural, functional, and physiological findings.\n• The description of results is too sparse (especially the 3D structures) and the authors do not\nfollow a logical story board. E.g. inhibition of anion currents by tRNA is the prime new finding\nbut the authors do not provide any mutant analysis to support their structural findings and do not\nprovide any hint whether there are tRNAs interacting with SLAH1 subunits in planta.\nResponse: We sincerely appreciate the reviewer's helpful suggestions regarding the need for\ndeeper functional validation of tRNA inhibition and its physiological relevance. We have\nconducted critical experiments to directly address these points:\n(1) Mutant Analysis Validating tRNA Inhibition Mechanism:\nBased on the SLAH1-tRNA complex structure identifying specific interaction sites (Fig. 5c),\nwe generated a mutant targeting key tRNA-binding residues, designated AtSLAH1(K/R mutant).\nElectrophysiological characterization in both heterologous (CHO cells, Fig. 5d) and native\nplant systems (Arabidopsis protoplasts, Fig. 5e) demonstrated that this mutant exhibited\nsignificantly enhanced anion currents compared to wild-type (WT) AtSLAH1. Crucially, this\nactivation occurred without exogenous RNase treatment, phenocopying the effect of RNase\non WT SLAH1 (Fig. 4e). This provides direct functional evidence that disrupting the structurally\nidentified tRNA-binding interface releases the channel from inhibition, confirming tRNA's role\nas the inhibitory ligand.\n(2) Physiological Relevance in planta: Enhanced Stomatal Response in Mutant:\nTo unequivocally demonstrate that disrupting tRNA binding activates SLAH1 function in a\nphysiological context, we transiently expressed AtSLAH1(K/R mutant) in Nicotiana\nbenthamiana leaves and assessed its impact on ABA-induced stomatal closure. Leaves\nexpressing WT AtSLAH1 exhibited stomatal closure levels similar to the empty vector (EV)\ncontrol under ABA treatment. In stark contrast, leaves expressing the AtSLAH1(K/R mutant)\nshowed significantly enhanced ABA-induced stomatal closure (Fig. 5f). The magnitude of\nclosure enhancement by the mutant was comparable to leaves expressing the constitutively\nmore active AtSLAH3 channel (Fig. 5f).\nThis mutant analysis provides strong mechanistic support for our prime finding of tRNA-\nmediated inhibition and directly links the structural observations to a key physiological\nresponse (stomatal closure) in planta.\n• Often only minor Erev shifts (e.g. 10 to 20 mV upon a 10-fold shift in external chloride\nconcentration) are shown to provide evidence for permeability changes between different\nsubunit stoichiometries but the current amplitudes and kinetics are not mentioned/analysed.\nIn stark contrast to the S-type anion current electrophysiology in guard cells analysis of SLAH3\nin oocytes, SLAH3 does not show any deactivation kinetics in the hands oft he authors. The\nreader wonders about this strong discrepancy.\nResponse: We sincerely appreciate the reviewer’s insightful observation regarding the\ndiscrepancies in deactivation kinetics between SLAH3 expressed in oocytes and S-type anion\ncurrents in guard cells. We acknowledge that this difference warrants further investigation. In\nour study, we followed methodologies similar to those employed in recent SLAC1 channel\nresearch (Li Y, et al. Nat Commun. 2022. PMID: 35523967), which yielded consistent results\nunder our experimental conditions. However, we agree that the absence of deactivation\nkinetics in our SLAH3 recordings contrasts with guard cell observations. This divergence may\narise from intrinsic differences in the expression systems or variations in ionic compositions\nof the intracellular/extracellular solutions, though the exact mechanism remains unclear at\nthis stage. Regarding anion selectivity, we adopted an equimolar nitrate-chloride gradient\n(pipette/bath: 140 mM Cl⁻/135 mM NO₃⁻) to evaluate the relative NO₃⁻/Cl⁻ permeability of\nSLAHs, complemented by mutagenesis to identify key residues governing selectivity. To\nensure transparency, we have provided full current traces and I-V curves in Supplementary\nFigure S10 which comprehensively depict the electrophysiological profiles.\n• In addition the reader wonders about another strong discrepancy oocyte data published\nand CHO cells data shown by the authors. In oocytes coexpression of SLAH1 and 3 is sufficient\nto generate a chloride and nitrate permeable anion channel without the need of a kinase\n(Lehmann et al.,2021), while in CHO cells it is not?\nResponse: We sincerely appreciate the reviewer’s insightful observation regarding the\ndifferences in SLAH1/SLAH3 activity between oocytes and CHO cells. We agree that this\ndiscrepancy is intriguing and warrants further discussion. In our experiments using CHO cells,\nco-expression of SLAH1 and SLAH3 alone yielded currents close to background levels, unlike\nthe robust anion currents reported in oocytes (Lehmann et al., 2021). However, our cryo-EM\nstructure of the SLAH1/SLAH3 complex reveals extensive intermolecular interactions at their\ninterface, strongly supporting a functional interplay between these subunits. To facilitate\ndetailed functional analyses—particularly regarding the stoichiometric regulation of NO₃⁻/Cl⁻\nselectivity—we co-expressed SLAH1 or SLAH3 with kinases in CHO cells. This approach\nprovided stable and reproducible currents, enabling rigorous structure-guided investigations.\nImportantly, we emphasize that our findings do not preclude the possibility of kinase-\nindependent SLAH1/SLAH3 activity in other systems (e.g., oocytes or plant cells). Rather, they\nhighlight cell-type-specific regulatory mechanisms that may influence channel behavior. We\nfully agree with the reviewer that understanding the physiological interplay between SLAH1\nand SLAH3 is critical. This represents a key focus of our ongoing work, where we aim to dissect\nthe precise rules governing their regulation across different cellular contexts.\nIn addition, we have also discussed the potential role of histidine residues in SLAH3 for acid-\ninduced activation, as described by Lehmann et al. (2021), within the Discussion section and\nFigure S13.\"\n.\n• The reader is not informed by the authors, how a putative regulation via tRNA should works\nin planta?\nResponse: We appreciate the reviewer’s query regarding the physiological relevance of\ntRNA-mediated regulation in planta. Our integrated structural, biochemical, and functional\ndata support a coherent model for tRNA-dependent regulation of SLAH1 in vivo. During the\nprotein purification process, we observed the binding of tRNA and confirmed its presence\nthrough biochemical assays.\n(1). Functional Validation in Plant Systems: Critically, targeted mutagenesis of SLAH1 tRNA-\nbinding residues (AtSLAH1(K/R mutant)) disrupted this interaction in planta. Expression of the\nmutant in Arabidopsis protoplasts yielded significantly enhanced anion currents compared to\nwild-type SLAH1 (Fig. 5e), directly mirroring the activation seen with RNase treatment in CHO\ncells (Fig. 4e). Transient expression in Nicotiana benthamiana leaves revealed a key\nphysiological consequence: while wild-type AtSLAH1 expression resulted in ABA-induced\nstomatal closure similar to the empty vector (EV) control, expression of the AtSLAH1(K/R\nmutant) caused significantly enhanced stomatal closure (Fig. 5f).\n(2). Proposed Mechanism of tRNA Regulation in Planta:\nBased on our integrated structural, electrophysiological, and biochemical data, we propose\nthe following model for tRNA-dependent regulation of the SLAH family in planta\n(summarized in Extended Data Fig. 12):\nUnder normal physiological conditions, SLAH1 is highly expressed and predominantly forms\nSLAH1-SLAH3 heterocomplexes in root xylem parenchyma cells. This complex configuration\nfacilitates Cl⁻ efflux, enabling efficient root-to-shoot Cl⁻ translocation. This process supports\noptimal growth, increasing fresh weight and biomass. Upon abiotic stress (e.g., drought,\nsalinity), the ABA signaling pathway is activated. Recent studies indicate stress triggers\naccumulation of free amino acids via enhanced synthesis. This metabolic shift leads to a\ncorresponding increase in tRNA abundance within the cell. The elevated tRNA pool provides\nligands for specific binding to SLAH1. As revealed by our structural and mutagenesis data,\ntRNA binds via hydrogen bonds to key positively charged residues (Lys/Arg) within the\nintracellular vestibule of SLAH1. This binding stabilizes SLAH1 in a closed conformation,\nphysically occluding the ion conduction pathway and preventing anion efflux. Crucially, this\ninhibition likely overrides activation by kinases like OST1 (Fig. 4e), effectively locking the\nchannel closed. Concomitantly, ABA signaling downregulates SLAH1 expression. The\ncombination of SLAH1 inhibition by tRNA and reduced SLAH1 protein levels shifts the\nequilibrium towards the formation of SLAH3 homotrimers. SLAH3, activated by stress-\ninduced signals (e.g., cytosolic acidosis, kinases), mediates robust NO₃⁻ efflux. This anion flux\nis critical for driving stomatal closure, reducing transpirational water loss, and enhancing plant\nsurvival under stress.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Sensen and colleagues investigate the molecular mechanisms of SLAH\nproteins, S-type anion channel homologs from Arabidopsis thaliana, using structural,\nbiochemical, and electrophysiological approaches. SLAHs are widely expressed in various\nplant tissues and are responsible for anion transport, playing a crucial role in plant adaptation\nto both biotic and abiotic stresses. The authors determined several cryo-EM structures of\nAtSLAH at different functional states, including the homotrimeric SLAH3, a heterotrimeric\ncomplex comprising two copies of SLAH1 and one copy of SLAH3 with tRNA, another\nheterotrimeric complex with one copy of SLAH1 and two copies of SLAH3, and the\nhomotrimeric SLAH1 with tRNA bound. Structural comparisons of these complexes revealed\nthe binding mode of tRNA on SLAH1, where two subunits coordinate a single tRNA molecule.\nAdditionally, the authors identified key residues essential for the nitrate-to-chloride\npermeability ratio. Together, these findings provide valuable insights into how SLAH proteins\nrespond to extreme environmental conditions.\nThe complex, particularly the heterotrimer containing SLAH1 and SLAH3, generated using\noverexpressed proteins, is intriguing. However, it remains unclear whether these complexes\nexist under physiological conditions.\nThe authors should provide evidence to demonstrate their physiological relevance. In Figure\n7, they propose a model linking observations from their structural and functional studies. It is\nuncertain whether the different complexes of SLAH1 and SLAH3 co-exist or if the complex\ntransitions from an SLAH1 trimer to an SLAH3 trimer under stress. Additionally, will other\nSLAH members also form heterotrimers, given their high sequence similarity?\nResponse: We sincerely appreciate these constructive comments regarding the physiological\nrelevance of the SLAH1-SLAH3 complexes and the potential for heteromerization with other\nfamily members. We provide the following evidence addressing these points:\n(1) Dynamic Complex Assembly in planta under ABA Stress:\nTo directly probe the interaction and assembly dynamics of SLAH1 and SLAH3 in planta, we\nemployed Bimolecular Fluorescence Complementation (BiFC) in Nicotiana benthamiana\nleaves under ABA treatment (Fig. 7). Under mock conditions, co-expression of AtSLAH1-cYFP\nand AtSLAH3-nYFP yielded a weak BiFC signal, indicating basal interaction.Crucially, after 3\nhours of ABA treatment, the BiFC signal significantly intensified, demonstrating enhanced\ninteraction and likely increased formation of SLAH1-SLAH3 heterocomplexes. Following 6\nhours of ABA exposure, the BiFC signal diminished, suggesting a reduction in heterocomplex\nformation. To model the scenario where both proteins coexist, we monitored homomeric\ninteractions in the presence of the partner: Co-expression of SLAH1 with AtSLAH3-cYFP +\nAtSLAH3-nYFP showed that SLAH3 homomeric interaction strengthened upon ABA\ntreatment. Conversely, co-expression of SLAH3 with AtSLAH1-cYFP + AtSLAH1-nYFP\nrevealed that SLAH1 homomeric interaction weakened under ABA treatment.\nThese results demonstrate that the assembly of SLAH channels in planta is a dynamic\nprocess. In response to ABA-induced stress, the predominant complex transitions sequentially\nfrom SLAH1 homomers, to SLAH1-SLAH3 heterocomplexes, and finally to SLAH3 homomers.\nThis dynamic shift represents an adaptive mechanism allowing plants to modulate anion\ntransport in response to stress.\n(2) Heteromerization Potential of Other SLAH Members:\nGiven the high sequence homology within the SLAH family, we investigated whether other\nmembers form heteromers. We differentially tagged all four SLAH proteins (SLAH1,\nSLAH2,\nSLAH3, SLAH4) with either Strep or Flag tags. We performed in vitro pull-down assays on\npairwise combinations: SLAH1+SLAH2, SLAH1+SLAH4, SLAH2+SLAH3, SLAH2+SLAH4,\nSLAH3+SLAH4, alongside SLAH1+SLAH3. Western blot analysis confirmed that all tested\npairwise combinations of SLAH family proteins can form heteromeric complexes in vitro\n(Extended Data Fig. 14). While promiscuous physical interactions occur in vitro, functional\nstudies by Cubero-Font et al. (2016) demonstrated that SLAH1 specifically activates only\nSLAH3 when co-expressed, showing no functional activation of SLAC1, SLAH2, or SLAH4.\nOur in vitro data confirms the structural potential for promiscuous heteromer formation\nwithin the SLAH family. However, the established in vivo functional specificity, where SLAH1\nexclusively partners with SLAH3 to form an active transport complex, highlights the critical\nrole of cellular context, regulatory factors, or specific protein interfaces in determining\nphysiologically relevant and functional heteromeric assemblies.\nAnother related question arises: in the heterotrimeric complex, if tRNA binding inhibits the\nactivity of SLAH1, how will the activity of SLAH3 be affected?\nResponse: We thank the reviewer for raising this critical point regarding SLAH3 functionality\nwithin the heterotrimeric complex. Our structural and functional analyses reveal that SLAH3\nactivity remains inhibited in the 2SLAH1+1SLAH3+tRNA complex.\nHOLE pore radius analysis of individual protomers across all resolved complexes (including\nthe heterotrimer) consistently shows sub-1 Å constriction sites in every monomer (Extended\nData Fig. 9b, d, e, g). This confirms all protomers—including the SLAH3 subunit in the\nheterotrimer—adopt a closed conformation characteristic of the inhibited state.\nThe SLAH3 monomer within the 2SLAH1+1SLAH3+tRNA complex exhibits near-identical\ntransmembrane helix arrangement and intracellular domain positioning to SLAH3 protomers\nin the inhibited SLAH3 homotrimer (Extended Data Fig. 9f). While minor side-chain variations\nexist (Extended Data Fig. 9c), they do not alter the fundamental closed-pore architecture.\nThe authors refer to two heterocomplexes of SLAH1 and SLAH3 as state 1 and state 2,\nsuggesting they are two subsequent transient states in complex formation. Given the\nuncertainty regarding their physiological relevance, it would be better to rename these\ncomplexes.\nResponse: We appreciate your valuable suggestion. To prevent potential ambiguity, we have\nrenamed these complexes (conformation 1 and 2) in the revised manuscript accordingly.\nHere are my comments:\n1. Abstract: It is unclear how this study \"establishes a new paradigm for coordinated\nregulation\" and \"opens new avenues for engineering crops under extreme environmental\nconditions.\"\nResponse: We have refined the abstract to better reflect the key findings of our study and\navoid using “establishes a new paradigm and opens new avenues”\n2. Line 88: The map reconstructed using single-particle cryo-EM is not an electron density\nmap; please correct this.\nResponse: we have corrected this issue in the revised manuscript.\n3. Line 89: The authors need to provide evidence to support the claim of “cleared structural\ndetails for extracellular loops and most intracellular loops.”\nResponse: we have provided evidence as Figure S2 in the revised manuscript.\n4. Line 65: “Cl-from” should be corrected to “Cl- from.”\nResponse: we have corrected this issue in the revised manuscript.\n5. Line 95: Define \"CH\" for a broader audience.\nResponse: we have corrected this issue in the revised manuscript.\n6. Figure 3a: Part of the protein density is colored as tRNA.\nResponse: we have corrected this issue in the revised manuscript.\n7. Both interface 1 and 2 are used to describe the interactions between SLAH1 and SLAH3 in\nthe two complexes. Consider using different terms to avoid potential confusion.\nResponse: We sincerely apologize for any misunderstanding and potential confusion caused\nby this. In the revised version, we have defined the contact surfaces of the initially mentioned\n2SLAH1+1SLAH3+tRNA complex as Interface 1 and 2, while the contact surfaces of the\nsubsequently mentioned 1SLAH1+2SLAH3 complex are defined as Interface 3 and 4.\nAdditionally, we have clearly labeled these in the figure and Figure legends.\n8. Line 161: The statement \"The SLAH1 channel narrows extracellularly but expands\nintracellularly\" is difficult to understand based on the current version of Figure 4c-d.\nResponse: Sorry for this confusing issue and we have deleted this statement in the revised\nmanuscript, see line 177-185.\n9. The pore profile should be presented for SLAH1 and SLAH3 in the heterotrimeric complexes,\nparticularly to support the claim that “the tRNA maintains the channel in an inactive state.”\nResponse: In the revised manuscript, we performed HOLE pore radius analysis for each\nprotomer of SLAH1 and SLAH3 separately to more clearly illustrate the structural basis of our\nfindings and conclusions.\n10. Scale bars are missing in the raw micrographs in the supplemental materials.\nResponse: we have added the Scale bars in the raw micrographs in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns.", "reviewer_3": "In the revised manuscript, the author has addressed most of the questions I raised in the previous review. I have an additional question related to the revised manuscript. The author proposed a working model in response to ABA treatment, suggesting that the SLAH1 homotrimer dynamically shifts to an SLAH1-SLAH3 heterotrimer, and ultimately to a SLAH3 homotrimer. However, it is unclear whether this transition requires the upregulation of SLAH3 expression. Additionally, for the dissociated SLAH1 from the complex, will it reform a homotrimer or be degraded? Finally, how is this cycle completed to return to the starting point, i.e., the homotrimeric SLAH1?\nMinor points:\n1. Line 145: It is improper to refer to the cryo-EM map as electron density map, as the nucleus also contributes to the density map. The author should carefully verify and consistently use either “cryo-EM map” or “Coulomb potential map” throughout the manuscript.\n2. Line 622: The citation for HOLE is missing.\n3. Line 650: Consider using ribbon model rather than stick model to maintain consistency.\n4. Line 653: Should be interface 1 and 2.\n5. Line 665: transmembrane and intracellular view according to the presented figure.\n6. Line 669: channel rather than Channel.\n7. Supplementary materials: There are multiple typos, including missing spaces between digits and their units, which should be corrected.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We sincerely thank the reviewer for their valuable suggestions during the review\nprocess. In response to the concerns and questions raised, we have performed an additional\nset of BiFC experiments. Specifically, leaf discs initially induced in ABA solution for 6 hours\nwere transferred to water for a further 6-hour treatment to remove the ABA stress signal. We\nobserved that, following this 6-hour washout period, the protein complexes reverted\npredominantly to the SLAH1 homomeric state.\nTo monitor potential changes in protein expression levels, we constructed SLAH1-Flag\nand SLAH3-HA vectors and co-expressed them in tobacco leaves. These samples underwent\nthe same treatment protocol as the BiFC experiments and were subsequently harvested for\nWestern blot analysis. The results indicated that the overall protein expression levels of both\nSLAH1 and SLAH3 remained largely unchanged, supporting the feasibility of this dynamic\nequilibrium.\nIt is important to note that our current methodology relies on Agrobacterium-\nmediated transient overexpression in Nicotiana benthamiana, a system widely used for\nstudying plant protein-protein interactions and stress responses. However, compared to\nstable transgenic Arabidopsis lines, transient overexpression can lead to exceptionally high\nprotein levels that might mask expression changes resulting from endogenous regulatory\nmechanisms. Furthermore, N. benthamiana may lack some relevant transcription factors\npresent in Arabidopsis. Consequently, in our transient overexpression system, we did not\nobserve changes in SLAH1 expression levels after 3-6 hours of ABA induction, which differs\nfrom findings in previous foundational studies (2016, Current Biology, PubMed ID:\n27397895). Additionally, during this dynamic cycle, dissociated SLAH1 or SLAH3 may exist in\nmonomeric forms. This is supported by previous research, such as a 2021 Current Biology\nstudy (PubMed ID: 34233161) demonstrating that acidosis can influence the conversion\nbetween SLAH3 monomers and multimers, and similar dynamic transitions between\nmonomeric and multimeric states regulated by specific cues have been reported for plant\nNLR proteins (PubMed ID: 37523563).\nFurthermore, regarding the elaboration of our proposed model, we have tempered\nthe interpretation in the Discussion section. We also explicitly acknowledged that the\nproposed model requires further validation at the physiological level in Arabidopsis.\nMinor points:\n1. Line 145: It is improper to refer to the cryo-EM map as electron density map, as the nucleus\nalso contributes to the density map. The author should carefully verify and consistently use\neither “cryo-EM map” or “Coulomb potential map” throughout the manuscript.\nResponse: We sincerely appreciate the reviewer for point-outing this mistake and we have\ncorrected this in the revised manuscript.\n2. Line 622: The citation for HOLE is missing.\nResponse: Thanks for this suggestion. We have added this citation for HOLE.\n3. Line 650: Consider using ribbon model rather than stick model to maintain consistency.\nResponse: We sincerely thank the reviewer for their comments and we have replaced this\nwith ribbon model.\n4. Line 653: Should be interface 1 and 2.\nResponse: We sincerely thank the reviewer for their comments and we have corrected this.\n5. Line 665: transmembrane and intracellular view according to the presented figure.\nResponse: We have corrected this description.\n6. Line 669: channel rather than Channel.\nResponse: We have revised this as suggested.\n7. Supplementary materials: There are multiple typos, including missing spaces between digits\nand their units, which should be corrected.\nResponse: We sincerely thank the reviewer for their suggestions. We have thoroughly\nchecked the supplementary material and corrected the relevant errors."}]

Decision letter