Potential abiotic stress targets for modern genetic manipulation.

Research into crop yield and resilience has underpinned global food security, evident in yields tripling in the past 5 decades. The challenges that global agriculture now faces are not just to feed 10+ billion people within a generation, but to do so under a harsher, more variable, and less predictable climate, and in many cases with less water, more expensive inputs, and declining soil quality. The challenges of climate change are not simply to breed for a "hotter drier climate," but to enable resilience to floods and droughts and frosts and heat waves, possibly even within a single growing season. How well we prepare for the coming decades of climate variability will depend on our ability to modify current practices, innovate with novel breeding methods, and communicate and work with farming communities to ensure viability and profitability. Here we define how future climates will impact farming systems and growing seasons, thereby identifying the traits and practices needed and including exemplars being implemented and developed. Critically, this review will also consider societal perspectives and public engagement about emerging technologies for climate resilience, with participatory approaches presented as the best approach.

The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants. Here, we found that the additional loss of GAD1, GAD4, and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2, which we link to increased cytosolic calcium (Ca2+ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild-type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard-cell ROS, restored the gad2-like greater stomatal apertures of gad1/2/4/5 beyond that of wild-type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca2+ -mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard-cell signal and the interactions it has with other established signals.

GABA does not regulate stomatal CO2 signalling in Arabidopsis.

Optimal stomatal regulation is important for plant adaptation to changing environmental conditions and for maintaining crop yield. The guard-cell signal GABA is produced from glutamate by Glutamate Decarboxylase (GAD) during a reaction that generates carbon dioxide (CO2) as a by-product. Here, we investigated a putative connection between GABA signalling and the more clearly defined CO2 signalling pathway in guard cells. The GABA-deficient mutant lines gad2-1, gad2-2 and gad1/2/4/5 were examined for stomatal sensitivity to various CO2 concentrations. Our findings show a phenotypical discrepancy between the allelic mutant lines gad2-1 and gad2-2 - a weakened CO2 response in gad2-1 (GABI_474_E05) in contrast to a wild-type response in gad2-2 (SALK_028819) and gad1/2/4/5. Through transcriptomic and genomic investigation, we traced the response of gad2-1 to a deletion of full-length Mitogen-activated protein kinase 12 (MPK12) in the GABI-KAT line, thereafter as renamed gad2-1*. Guard cell-specific complementation of MPK12 restored the gad2-1* CO2 phenotype, which confirms the proposed importance of MPK12 to CO2 sensitivity. Additionally, we found that stomatal opening under low atmospheric CO2 occurs independently of the GABA-modulated opening-channel ALMT9. Our results confirm that GABA has a role in modulating the rate of stomatal opening and closing - but not in response to CO2  per se.

StomaAI: an efficient and user-friendly tool for measurement of stomatal pores and density using deep computer vision.

Using microscopy to investigate stomatal behaviour is common in plant physiology research. Manual inspection and measurement of stomatal pore features is low throughput, relies upon expert knowledge to record stomatal features accurately, requires significant researcher time and investment, and can represent a significant bottleneck to research pipelines. To alleviate this, we introduce StomaAI (SAI): a reliable, user-friendly and adaptable tool for stomatal pore and density measurements via the application of deep computer vision, which has been initially calibrated and deployed for the model plant Arabidopsis (dicot) and the crop plant barley (monocot grass). SAI is capable of producing measurements consistent with human experts and successfully reproduced conclusions of published datasets. SAI boosts the number of images that can be evaluated in a fraction of the time, so can obtain a more accurate representation of stomatal traits than is routine through manual measurement. An online demonstration of SAI is hosted at https://sai.aiml.team, and the full local application is publicly available for free on GitHub through https://github.com/xdynames/sai-app.

Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

The function of the Medicago truncatula ZIP transporter MtZIP14 is linked to arbuscular mycorrhizal fungal colonization.

Soil micronutrient availability, including zinc (Zn), is a limiting factor for crop yield. Arbuscular mycorrhizal (AM) fungi can improve host plant growth and nutrition through the mycorrhizal pathway of nutrient uptake. Although the physiology of Zn uptake through the mycorrhizal pathway is well established, the identity of the related molecular components are unknown. Here, RNA-seq analysis was used to identify genes differentially-regulated by AM colonization and soil Zn concentration in roots of Medicago truncatula. The putative Zn transporter gene MtZIP14 was markedly up-regulated in M. truncatula roots when colonized by Rhizophagus irregularis. MtZIP14 restored yeast growth under low Zn availability. Loss-of-function mutant plants (mtzip14) had reduced shoot biomass compared to the wild-type when colonized by AM fungi and grown under low and sufficient soil Zn concentration; at high soil Zn concentration, there were no genotypic differences in shoot biomass. The vesicular and arbuscular colonization of roots was lower in the mtzip14 plants regardless of soil Zn concentration. We propose that MtZIP14 is linked to AM colonization in M. truncatula plants, with the possibility that MtZIP14 function with AM colonization is linked to plant Zn nutrition.

The emerging role of GABA as a transport regulator and physiological signal.

While the proposal that γ-aminobutyric acid (GABA) acts a signal in plants is decades old, a signaling mode of action for plant GABA has been unveiled only relatively recently. Here, we review the recent research that demonstrates how GABA regulates anion transport through aluminum-activated malate transporters (ALMTs) and speculation that GABA also targets other proteins. The ALMT family of anion channels modulates multiple physiological processes in plants, with many members still to be characterized, opening up the possibility that GABA has broad regulatory roles in plants. We focus on the role of GABA in regulating pollen tube growth and stomatal pore aperture, and we speculate on its role in long-distance signaling and how it might be involved in cross talk with hormonal signals. We show that in barley (Hordeum vulgare), guard cell opening is regulated by GABA, as it is in Arabidopsis (Arabidopsis thaliana), to regulate water use efficiency, which impacts drought tolerance. We also discuss the links between glutamate and GABA in generating signals in plants, particularly related to pollen tube growth, wounding, and long-distance electrical signaling, and explore potential interactions of GABA signals with hormones, such as abscisic acid, jasmonic acid, and ethylene. We conclude by postulating that GABA encodes a signal that links plant primary metabolism to physiological status to fine tune plant responses to the environment.

Manipulating exudate composition from root apices shapes the microbiome throughout the root system.

Certain soil microorganisms can improve plant growth, and practices that encourage their proliferation around the roots can boost production and reduce reliance on agrochemicals. The beneficial effects of the microbial inoculants currently used in agriculture are inconsistent or short-lived because their persistence in soil and on roots is often poor. A complementary approach could use root exudates to recruit beneficial microbes directly from the soil and encourage inoculant proliferation. However, it is unclear whether the release of common organic metabolites can alter the root microbiome in a consistent manner and if so, how those changes vary throughout the whole root system. In this study, we altered the expression of transporters from the ALUMINUM-ACTIVATED MALATE TRANSPORTER and the MULTIDRUG AND TOXIC COMPOUND EXTRUSION families in rice (Oryza sativa L.) and wheat (Triticum aestivum L.) and tested how the subsequent release of their substrates (simple organic anions, including malate, citrate, and γ-amino butyric acid) from root apices affected the root microbiomes. We demonstrate that these exudate compounds, separately and in combination, significantly altered microbiome composition throughout the root system. However, the root type (seminal or nodal), position along the roots (apex or base), and soil type had a greater influence on microbiome structure than the exudates. These results reveal that the root microbiomes of important cereal species can be manipulated by altering the composition of root exudates, and support ongoing attempts to improve plant production by manipulating the root microbiome.

Enhanced reactive oxygen detoxification occurs in salt-stressed soybean roots expressing GmSALT3.

Soybean (Glycine max) is an important crop globally for food and edible oil production. Soybean plants are sensitive to salinity (NaCl), with significant yield decreases reported under saline conditions. GmSALT3 is the dominant gene underlying a major QTL for salt tolerance in soybean. GmSALT3 encodes a transmembrane protein belonging to the plant cation/proton exchanger (CHX) family, and is predominately expressed in root phloem and xylem associated cells under both saline and non-saline conditions. It is currently unknown through which molecular mechanism(s) the ER-localised GmSALT3 contributes to salinity tolerance, as its localisation excludes direct involvement in ion exclusion. In order to gain insights into potential molecular mechanism(s), we used RNA-seq analysis of roots from two soybean NILs (near isogenic lines); NIL-S (salt-sensitive, Gmsalt3), and NIL-T (salt-tolerant, GmSALT3), grown under control and saline conditions (200 mM NaCl) at three time points (0 h, 6 h, and 3 days). Gene ontology (GO) analysis showed that NIL-T has greater responses aligned to oxidation reduction. ROS were less abundant and scavenging enzyme activity was greater in NIL-T, consistent with the RNA-seq data. Further analysis indicated that genes related to calcium signalling, vesicle trafficking and Casparian strip (CS) development were upregulated in NIL-T following salt treatment. We propose that GmSALT3 improves the ability of NIL-T to cope with saline stress through preventing ROS overaccumulation in roots, and potentially modulating Ca2+ signalling, vesicle trafficking and formation of diffusion barriers.

Evolution of chloroplast retrograde signaling facilitates green plant adaptation to land.

Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells.

MYB77 regulates high-affinity potassium uptake by promoting expression of HAK5.

In Arabidopsis, the high-affinity K+ transporter HAK5 is the major pathway for root K+ uptake when below 100 µM; HAK5 responds to Low-K+ (LK) stress by strongly and rapidly increasing its expression during K+ -deficiency. Therefore, positive regulators of HAK5 expression have the potential to improve K+ uptake under LK. Here, we show that mutants of the transcription factor MYB77 share a LK-induced leaf chlorosis phenotype, lower K+ content, and lower Rb+ uptake of the hak5 mutant, but not the shorter root growth, and that overexpression of MYB77 enhanced K+ uptake and improved tolerance to LK stress. Furthermore, we demonstrated that MYB77 positively regulates the expression of HAK5, by binding to the HAK5 promoter and enhances high-affinity K+ uptake of roots. As such, our results reveal a novel pathway for enhancing HAK5 expression under LK stress, and provides a candidate for increasing the tolerance of plants to LK.

Soybean CHX-type ion transport protein GmSALT3 confers leaf Na+ exclusion via a root derived mechanism, and Cl- exclusion via a shoot derived process.

Soybean (Glycine max) yields are threatened by multiple stresses including soil salinity. GmSALT3 (a cation-proton exchanger protein) confers net shoot exclusion for both Na+ and Cl- and improves salt tolerance of soybean; however, how the ER-localized GmSALT3 achieves this is unknown. Here, GmSALT3's function was investigated in heterologous systems and near isogenic lines that contained the full-length GmSALT3 (NIL-T; salt-tolerant) or a truncated transcript Gmsalt3 (NIL-S; salt-sensitive). GmSALT3 restored growth of K+ -uptake-defective Escherichia coli and contributed towards net influx and accumulation of Na+ , K+ and Cl- in Xenopus laevis oocytes, while Gmsalt3 was non-functional. Time-course analysis of NILs confirmed shoot Cl- exclusion occurs distinctly from Na+ exclusion. Grafting showed that shoot Na+ exclusion occurs via a root xylem-based mechanism; in contrast, NIL-T plants exhibited significantly greater Cl- content in both the stem xylem and phloem sap compared to NIL-S, indicating that shoot Cl- exclusion likely depends upon novel phloem-based Cl- recirculation. NIL-T shoots grafted on NIL-S roots contained low shoot Cl- , which confirmed that Cl- recirculation is dependent on the presence of GmSALT3 in shoots. Overall, these findings provide new insights on GmSALT3's impact on salinity tolerance and reveal a novel mechanism for shoot Cl- exclusion in plants.

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABAA receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat (Triticum aestivum) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux but GABA does not complex Al3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14C-GABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA]i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status.

Plant transporters involved in combating boron toxicity: beyond 3D structures.

Membrane transporters control the movement and distribution of solutes, including the disposal or compartmentation of toxic substances that accumulate in plants under adverse environmental conditions. In this minireview, in the light of the approaching 100th anniversary of unveiling the significance of boron to plants (K. Warington, 1923; Ann. Bot.37, 629) we discuss the current state of the knowledge on boron transport systems that plants utilise to combat boron toxicity. These transport proteins include: (i) nodulin-26-like intrinsic protein-types of aquaporins, and (ii) anionic efflux (borate) solute carriers. We describe the recent progress made on the structure-function relationships of these transport proteins and point out that this progress is integral to quantitative considerations of the transporter's roles in tissue boron homeostasis. Newly acquired knowledge at the molecular level has informed on the transport mechanics and conformational states of boron transport systems that can explain their impact on cell biology and whole plant physiology. We expect that this information will form the basis for engineering transporters with optimised features to alleviate boron toxicity tolerance in plants exposed to suboptimal soil conditions for sustained food production.

Cytosolic GABA inhibits anion transport by wheat ALMT1.

Anion transport by aluminium-activated malate transporter (ALMT) proteins is negatively regulated by gamma-aminobutyric acid (GABA), which increases in concentration during stress. Here, the interaction between GABA and wheat (Triticum aestivum, Ta) TaALMT1 heterologously-expressed in Xenopus laevis oocytes was investigated. GABA inhibited anion transport by TaALMT1 in membrane patches from the cytosolic, not extracellular membrane face, via a reduction in open probability (NPopen ), not an inhibition of channel current magnitude. TaALMT1 currents in patches frequently exhibited rundown with complete removal of cytosolic factors, but were partially sustained by protein kinase C dependent phosphorylation. When applied to whole oocytes a GABA-analogue-BODIPY conjugate inhibited TaALMT1 anion currents from the cytoplasmic face only, whereas free GABA inhibited from both the inside and outside consistent with GABA traversing the TaALMT1 pore then acting from the inside. We propose GABA does not competitively inhibit ALMT conductance through the same pore but rather leads to an allosteric effect, reducing anion channel opening frequency. Across plants GABA is a conserved regulator of anion transport via ALMTs - a family with numerous physiological roles beyond Al3+ tolerance. Our data suggests that a GABA-ALMT interaction from the cytosolic face has the potential to form part of a novel plant signalling pathway.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

Role of TaALMT1 malate-GABA transporter in alkaline pH tolerance of wheat.

Malate exudation through wheat (Triticum aestivum L) aluminium-activated malate transporter 1 (TaALMT1) confers Al3+ tolerance at low pH, but is also activated by alkaline pH, and is regulated by and facilitates significant transport of gamma-aminobutyric acid (GABA, a zwitterionic buffer). Therefore, TaALMT1 may facilitate acidification of an alkaline rhizosphere by promoting exudation of both malate and GABA. Here, the performance of wheat near isogenic lines ET8 (Al+3 -tolerant, high TaALMT1 expression) and ES8 (Al+3 -sensitive, low TaALMT1 expression) are compared. Root growth (at 5 weeks) was higher for ET8 than ES8 at pH 9. ET8 roots exuded more malate and GABA at high pH and acidified the rhizosphere more rapidly. GABA and malate exudation was enhanced at high pH by the addition of aluminate in both ET8 and transgenic barley expressing TaALMT1. Xenopus laevis oocytes expressing TaALMT1 acidified an alkaline media more rapidly than controls corresponding to higher GABA efflux. TaALMT1 expression did not change under alkaline conditions but key genes involved in GABA turnover changed in accordance with a high rate of GABA synthesis. We propose that TaALMT1 plays a role in alkaline tolerance by exuding malate and GABA, possibly coupled to proton efflux.

A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in bread wheat.

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+ ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.

Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH, Ca2+ and GABA.

We investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters.

A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform.

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na(+) ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na(+)-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na(+) ion binding site. Our data enhance the understanding of the permeation functions of Bot1.