1. Essential functions and beneficial roles of chloride

Chlorine is a micronutrient for higher plants, which is required in trace amounts. The chloride ion (Cl^―), a monovalent anion, is delivered from the soil to the root primarily via mass flow. Thus, its uptake is closely associated with the movement of water through the soil. The critical deficiency concentration of Cl^― varies across non-halophytic plants: rice (Oryza sativa) requires approximately 3 mg g⁻¹ shoot dry weight (DW), while barley (Hordeum vulgare) and wheat (Triticum aestivum) need 4 mg g⁻¹ DW. Lettuce (Lactuca sativa) and maize (Zea mays), on the other hand, have much lower demands, showing deficiency symptoms at concentrations around 0.14 mg g⁻¹ DW and 0.1 mg g⁻¹ DW, respectively (Geilfus, Reference Geilfus2018b). Kiwifruit (Actinidia deliciosa) stands out for its relatively high Cl^― requirement, with leaf concentrations of 225–550 μmol g⁻¹ DW needed for optimal growth (Smith et al., Reference Smith, Clark and Holland1987). Species with lower Cl^― demands, such as lettuce and maize, often receive sufficient amounts from rainfall, which typically supplies 4–8 kg ha⁻¹ annually in non-coastal regions (Geilfus, Reference Geilfus2018b). When Cl^― concentrations fall below these thresholds, plants tend to exhibit a reduction in photosynthesis (Terry, Reference Terry1977) and leaf area, the latter being often associated with decreased cell division rates (Franco-Navarro et al., Reference Franco-Navarro, Brumós, Rosales, Cubero-Font, Talón and Colmenero-Flores2016).

Foliar Cl^― concentrations vary between species depending on Cl^― fertilization levels. For example, Arabidopsis thaliana can accumulate around 25 mg Cl^― g⁻¹ DW (Cubero-Font, Reference Cubero-Font2017), while the Solanaceae species tomato (Solanum lycopersicum) (Rosales et al., Reference Rosales, Vázquez-Rodríguez, Franco-Navarro, Cubero-Font and Colmenero-Flores2012), and tobacco (Nicotiana tabacum) can accumulate up to 50 mg Cl^― g⁻¹ DW (Franco-Navarro et al., Reference Franco-Navarro, Brumós, Rosales, Cubero-Font, Talón and Colmenero-Flores2016). Cl^― is essential for chloroplast function and is the preferred substrate of VCCN1, a Cl^― channel in plastids that finetunes the electrical potential across the thylakoid membrane (Herdean et al., Reference Herdean, Teardo, Nilsson, Pfeil, Johansson, Ünnep and Lundin2016). Cl^― stabilizes the oxygen-evolving complex of photosystem II (PSII-OEC), initiating the electron flux from the stroma to the thylakoid lumen. When Cl^― is deficient, electron transfer is impeded by the formation of a salt bridge between lys317 of D2 and asp61 of D1 (Kawakami et al., Reference Kawakami, Umena, Kamiya and Shen2009; Raven, Reference Raven2017). For its catalytic role in PSII-OEC, Cl^― is enriched by the extrinsic proteins PsbP and PsbQ. These extrinsic proteins possess high-affinity Cl^― binding sites that enhance the Cl^― binding affinity of the Mn–Ca–Cl complex (Kakiuchi et al., Reference Kakiuchi, Uno, Ido, Nishimura, Noguchi, Ifuku and Sato2012; Nishimura et al., Reference Nishimura, Uno, Ido, Nagao, Noguchi, Sato and Ifuku2014; Seidler, Reference Seidler1996) and thus aid in saturating the Cl^― PSII-OEC requirement at chloroplastic Cl^― concentrations that are, for example, 40 mM in non-halophytes such as Triticum and Hordeum vulgare (Bose et al., Reference Bose, Munns, Shabala, Gilliham, Pogson and Tyerman2017). At higher chloroplastic Cl^― levels, this Cl^― enrichment is dispensable for maintaining PSII activity (Seidler, Reference Seidler1996). This is consistent with some halophytes, which typically have higher chloroplastic Cl^― levels, for example, 100 mM in Beta vulgaris and Sueda australis, where PsbQ is often absent and PsbP levels are reduced, while PSII operation remains functional (Bose et al., Reference Bose, Munns, Shabala, Gilliham, Pogson and Tyerman2017). In addition to the catalytic role of Cl^― in the fully assembled PSII-OEC, Cl^― is required for OEC synthesis and resynthesis after photodamage (Vinyard et al., Reference Vinyard, Badshah, Riggio, Kaur, Fanuy and Gunner2019). This Cl^― requirement is significantly higher than that required to operate already assembled OEC, as demonstrated by a doubling of the oxygen evolution rate after a 5-min assembly with 100 mM Cl^― instead of 20 mM in an in vitro experiment in the non-halophyte spinach (Vinyard et al., Reference Vinyard, Badshah, Riggio, Kaur, Fanuy and Gunner2019). The higher estimated Cl^― requirement to saturate the (re-)assembly of PSII-OEC compared to PSII-OEC operation with 40 mM and 2 mM Cl^―, respectively, could provide a rationale for the positive effects of supplying Cl^― to crops beyond micronutrient levels (as described below), as it might enhance the rate of OEC assembly and the repair of photodamaged thylakoids, thereby supporting a more efficient photosynthesis (Raven, Reference Raven2020).

Another potential essential function is related to the activity of the vacuolar proton-pumping ATPase (V-H⁺-ATPase) that depends on Cl^― for maximal activity, as described by Churchill and Sze (Reference Churchill and Sze1984). Beyond these essential roles, Cl^― can fulfil additional, less specific functions that can also be fulfilled by other anions. Among them are processes like the regulation of enzyme activities such as the asparagine synthetase (Rognes, Reference Rognes1980) and α-amylases (α-1,4-glucan-4-glucanohydrolase) (Metzler, Reference Metzler1979; D’amico et al., Reference D’Amico, Gerday and Feller2000). Cl^― also contributes to turgor maintenance and osmoregulation, which are additional non-essential but beneficial functions (Nieves-Cordones et al., Reference Nieves-Cordones, García-Sánchez, Pérez-Pérez, Colmenero-Flores, Rubio and Rosales2019; Shelke et al., Reference Shelke, Nikalje, Nikam, Maheshwari, Punita, Rao and Suprasanna2019). Here, Cl^― might have an advantage over nitrate, as Cl^― has a tighter bound hydration shell compared to nitrate, which might prevent further water loss (Wege et al., Reference Wege, Gilliham and Henderson2017). However, once Cl^― reaches higher levels (toxicity), molecular crowding might lead to the disruption of many cellular processes, particularly in chloroplasts. In addition, influx of Cl^― into guard cells is relevant for the opening of leaf pores (Humble & Hsiao, Reference Humble and Hsiao1969). For these beneficial functions, Cl^― is required in much greater quantities than for its essential role in chloroplasts, which necessitates only 0.1–0.2 mg g⁻¹ (~2.8–5.5 μmol) based on shoot DW (Geilfus, Reference Geilfus2018a; Marschner, Reference Marschner2011). To support these additional functions, Cl^― concentrations in the millimolar (mM) range are often necessary. When Cl^― is given in concentrations typical for a macronutrient, Franco-Navarro et al. (Reference Franco-Navarro, Rosales, Cubero-Font, Calvo, Álvarez and Díaz-Espejo2019) demonstrated that Cl^― treatments ranging from 0.075 to 5.075 mM in the growth medium increased biomass production and improved water relations in tobacco plants. Their studies suggest that Cl^― regulates leaf osmotic potential and turgor, enhances cell growth and increases drought tolerance in tobacco. These effects were also observed in other species tested, including lettuce, spinach and tomato. Koch et al. (Reference Koch, Pawelzik and Kautz2021) investigated the effects of Cl^― on potato (Solanum tuberosum L.) using four different Cl^― doses: 0, 200, 400 and 800 mg Cl^― kg⁻¹ soil. They found that Cl^― application significantly altered soil–plant water relations, with the highest dose (800 mg Cl^― kg⁻¹ soil) improving plant hydration status without reducing tuber yield or dry matter. Cl^― was suggested to play a crucial role in improving water-use efficiency (WUE) by reducing transpiration through lowered stomatal conductance. Overall, these authors discuss that Cl^― doses above those of a micronutrient but below toxicity thresholds enhance biomass production and improve WUE. This review focusses on Cl^―; for sodium homeostasis, see the article by Munns, Tyerman and Bose in this special issue.

2. Chloride toxicity and responses

Regions particularly affected by Cl^― salinity include coastal areas, arid and semi-arid zones, and irrigated agricultural lands in countries such as Australia, the United States and parts of the Middle East (Munns & Tester, Reference Munns and Tester2008). Cl^― concentrations in soil solutions can vary widely: from 1 to 250 mg L⁻¹ in freshwater streams and lakes, 500 to 5,000 mg⁻¹ in brackish water and up to 19,400 mg⁻¹ in seawater (Flowers, Reference Flowers2004). Cl^― salinity is a common issue driven by both natural and human activities. Natural sources include the weathering of salt-rich materials and wind-borne salt depositions from oceans and lakes (Rengasamy, Reference Rengasamy2006), while human activities mainly involve irrigation with Cl^―-rich water and the use of Cl^―-containing fertilizers (Flowers & Yeo, Reference Flowers and Yeo2012). In detail, fertilization with Cl^―-containing fertilizers can lead to high Cl^― concentrations in the soil, particularly under conditions of limited water percolation that reduce leaching. Common fertilizers contain varying levels of Cl^― by weight, with magnesium Cl^― (MgCl₂) containing 74% Cl^― by weight, ammonium Cl^― (NH₄Cl) containing 66% Cl^― by weight and potassium Cl^― (KCl) containing 47% Cl^― by weight (Turhan, Reference Turhan2021). Organic fertilizers like pig slurries also add substantial amounts of Cl^―. For example, applying 210 kg N ha⁻¹ year⁻¹ via pig slurry can add 282–458 kg Cl^― ha⁻¹ year⁻¹ (Moral et al., Reference Moral, Perez-Murcia, Perez-Espinosa, Moreno-Caselles, Paredes and Rufete2008), while chicken and pigeon manure contribute 7.1 and 6.1 g Cl^― per kg DW, respectively (Li-Xian et al., Reference Li-Xian, Guo-Liang, Shi-Hua, Gavin and Zhao-Huan2007). If there is insufficient rainfall to leach Cl^― deeper into the soil, these high Cl^― loads can accumulate in the root zone, leading to osmotic stress, ion toxicity and nutrient imbalances in plants, negatively impacting crop yield and quality (Teakle & Tyerman, Reference Teakle and Tyerman2010).

However, Cl^― can be toxic to plants at excessive concentrations, with critical toxicity estimated to be 4–7 mg g⁻¹ for Cl^―-sensitive species and 15–35 mg g⁻¹ DW for Cl^―-tolerant species (Colmenero-Flores et al., Reference Colmenero-Flores, Franco-Navarro, Cubero-Font, Peinado-Torrubia and Rosales2019; Xu et al., Reference Xu, Magen, Tarchitzky and Kafkafi2000). Differences in tolerance to Cl^― salinity are often observed not only between species but also within a single species. Under salt stress conditions, woody perennial plants exhibit toxicity symptoms due to Cl^―, rather than sodium accumulation in their leaves. This association exists not because Cl^― is metabolically more harmful than sodium to these species but because these plants are able to secrete a greater amount of sodium into their woody organs (Gong et al., Reference Gong, Blackmore, Clingeleffer, Sykes, Jha, Tester and Walker2011; Shelke et al., Reference Shelke, Nikalje, Nikam, Maheshwari, Punita, Rao and Suprasanna2019). This limits sodium transport to the leaves to reduce its impact upon cellular metabolism within photosynthetic organs (Li, Tester, & Gilliham, Reference Li, Tester and Gilliham2017b). The high level of Cl^― accumulation leads to decreased transpiration, photosynthesis, crop yield and quality, and ultimately to plant death (Teakle & Tyerman, Reference Teakle and Tyerman2010). This phenomenon has been documented in fruit tree species grown on rootstocks, such as citrus (Citrus) (Storey & Walker, Reference Storey and Walker1999; Tadeo et al., Reference Tadeo, Cercos and Colmenero-Flores2008), grapevine (Vitis) (Sykes, Reference Sykes1992) and avocado (Persea Americana) (Bar et al., Reference Bar, Apelbaum and Goren1997). In citrus, the ability to exclude Cl^― is a critical factor in determining salt tolerance. Tolerant citrus rootstocks, such as C. macrophylla and Poncirus trifoliata, exclude Cl^― primarily by reducing xylem loading and retaining Cl^― in distal root tissues, resulting in leaf concentrations of 5–13 mg g⁻¹ DW. In contrast, sensitive rootstocks like C. aurantium accumulate up to 30 mg g⁻¹ DW, due to higher xylem Cl^― loading and thus limited retention ability. This difference is partly regulated by genes like CcICln, which modulate Cl^― transport and homeostasis (Brumós et al., Reference Brumós, Talon, Bouhlal and Colmenero-Flores2010). Similarly, Sykes (Reference Sykes1992) reported that grapevine rootstocks exhibit varying efficiencies in Cl^― exclusion. Rootstocks such as Ramsey and 1103 Paulsen are particularly effective, maintaining leaf Cl^― concentrations below harmful thresholds (less than 10 mg g⁻¹ DW) in saline conditions. In contrast, rootstocks such as Salt Creek may accumulate more Cl^―, which cause symptoms of toxicity (Sykes, Reference Sykes1992). Genetic variation in Cl^― accumulation was also found for rice, with sensitive cultivars like IR29 accumulating above 30 mg Cl^― g⁻¹ DW, while the tolerant cultivar Pokkali maintained leaf Cl^― concentrations below 15 mg g⁻¹ DW (Shannon et al., Reference Shannon, Rhoades, Draper, Scardaci and Spyres1998).

Control of Cl^― transport and Cl^― ‘exclusion’ from shoots is correlated with salt tolerance in many species, particularly for legumes (Franzisky et al., Reference Franzisky, Geilfus, Kränzlein, Zhang and Zörb2019 ). Luo et al. (Reference Luo, Yu and Liu2005) evaluated seedlings of two Glycine max cultivars (salt-tolerant Nannong 1138-2 and salt-sensitive Zhongzihuangdou-yi) and two Glycine soja populations (BB52 and N23232) under iso-osmotic conditions of 150 mM sodium, Cl^― and sodium chloride. They showed that Glycine max cultivars suffered more damage from Cl^― than from sodium, with leaf sensitivity linked to their ability to restrict Cl^― accumulation in roots and stems. The ability of soybean plants to exclude Cl^―, as well as sodium, from leaves is related to the activity of the putative cation proton exchanger GmSALT3 through a yet-unknown mechanism; plants without a functional GmSALT3 gene show an increase in foliar Cl^― before sodium increases (Qu et al., Reference Qu, Guan, Bose, Henderson, Wege, Qiu and Gilliham2020). A functional GmSALT3 protein is also present in the G. max relative, Glycine soja. Different G. soja populations are more susceptible to sodium toxicity than others, indicating that their salt tolerance relies on preventing leaf sodium accumulation. This may also be because wild soybean has a very high Cl^― exclusion capacity, making sodium toxicity more apparent compared to domesticated soybean.

In addition to soy, Teakle et al. (Reference Teakle, Flowers, Real and Colmer2007) studied another leguminous species under salinity and waterlogging conditions, Lotus corniculatus. They demonstrated that salinity and waterlogging conditions hinder growth in sensitive species by increasing sodium and Cl^― transfer to shoots. L. corniculatus had 50% higher Cl^― levels in xylem and shoots than L. tenuis in 200 mM aerated sodium Cl^―, but sodium and potassium levels were comparable. Under conditions of waterlogging and salt stress, L. corniculatus had double the xylem Cl^― and sodium concentrations as L. tenuis. L. tenuis roots had increased inherent porosity due to the production of aerenchyma, which is important for oxygen supply and ion ‘exclusion’. This aerenchyma not only supports processes like compartmentalization and ion ‘exclusion’, but also limits the accumulation of Cl^― in roots. While the precise mechanisms remain to be elucidated, Cl^― exclusion is pivotal for salinity tolerance (White & Broadley, Reference White and Broadley2001). Franzisky et al. (Reference Franzisky, Geilfus, Kränzlein, Zhang and Zörb2019) tested the toxicity of Cl^― on thirteen non-salt-tolerant Vicia faba genotypes. Plants were exposed to 100 mM sodium Cl^― hydroponically until necrotic leaf patches formed. At this time, Cl^― concentrations in the affected leaves were similar among genotypes, but sodium levels varied, suggesting that Cl^― toxicity predominantly caused the lesions (Slabu et al., Reference Slabu, Zörb, Steffens and Schubert2009; Tavakkoli et al., Reference Tavakkoli, Rengasamy and McDonald2010). Higher Cl^― accumulation in photosynthetic tissues negatively influenced growth and photosynthesis in sensitive genotypes. Tolerant cultivars, such as Scoop and Nebraska, likely limited Cl^― transfer to leaves, reducing damage and extending photosynthetic activity.

Grass species are also affected by Cl^― toxicity. Tavakkoli et al. (Reference Tavakkoli, Rengasamy and McDonald2010) evaluated four barley genotypes (Barque73, Clipper, Sahara and Tadmor) under different Cl^― levels in culture solution (control, 1.9 mM Cl^―; and high-Cl^― treatment, 125 mM Cl^―). They reported that the high-Cl^― treatment reduced photosynthetic capacity due to non-stomatal effects, causing chlorophyll degradation and a decrease in the actual quantum yield of electron transport in PSII. These effects were associated with both photochemical quenching and excitation energy capture efficiency. In addition, Khare et al. (Reference Khare, Kumar and Kishor2015) evaluated two rice genotypes, Panvel-3 (salt-tolerant) and Sahyadri-3 (salt-sensitive), under varying Cl^― levels in a hydroponic solution (15 mM CaCl₂, 15 mM MgCl₂ and 40 mM KCl). They found a significant decrease in DW in both cultivars, which was linked to higher levels of reactive oxygen species (ROS), such as H₂O₂ and O₂ ^―, and enhanced cell death. An accumulation of ROS causes oxidative damage, which in turn disrupts metabolic functions and hinders growth. Furthermore, as a defence mechanism, high ROS contents cause programmed cell death, which further reduces biomass.

3. Chloride uptake at low chloride conditions

Cl^― has minimal affinity for soil components because most soil particles, such as clay and organic matter, are negatively charged. This negative charge tends to repel negatively charged ions like Cl^―. Consequently, Cl^― remains in the aqueous phase, where its movement is largely governed by water flow. This enables Cl^― to follow the path of water, making its transport within the soil highly dependent on moisture dynamics and mass flow (Geilfus, Reference Geilfus2019; Moya et al., Reference Moya, Gomez-Cadenas, Primo-Millo and Talon2003). Cl^― uptake in plant roots can occur via an apoplastic, a symplastic or a transcellular pathway, with Casparian bands playing a crucial role in selectively regulating ion transport by limiting passive flow through the apoplast (Abbaspour et al., Reference Abbaspour, Kaiser and Tyerman2014; Barberon, Reference Barberon2017; Karahara et al., Reference Karahara, Ikeda, Kondo and Uetake2004). Casparian bands are key structural components of plant roots, situated at the innermost cortex cell layer, the endodermis. Casparian strips develop here in the radial and tangential walls of endodermal cells and prevent the unselective flux of ions from the soil to the vasculature (Abbaspour et al., Reference Abbaspour, Kaiser and Tyerman2014; Barberon, Reference Barberon2017; Karahara et al., Reference Karahara, Ikeda, Kondo and Uetake2004). Casparian bands therefore prevent ions, most likely including Cl^―, from passively reaching the xylem via simple diffusion.

Cl^― concentrations in non-saline soil solutions typically range from 1 to 4 mM (Tavakkoli et al., Reference Tavakkoli, Rengasamy and McDonald2010; Wada et al., Reference Wada, Odahara, Gunjikake and Takada2006), while cytosolic Cl^― concentrations in the root cells of A. thaliana are around 10 mM (Lorenzen et al., Reference Lorenzen, Aberle and Plieth2004). Similarly, in barley root cells, cytosolic Cl^― levels range from 8 to 10 mM under external Cl^― concentrations of 0.1 mM (Britto et al., Reference Britto, Ruth, Lapi and Kronzucker2004). Teakle and Tyerman (Reference Teakle and Tyerman2010) found that cytosolic Cl^― concentrations between 5 and 20 mM are typical for many glycophytic plants not under excessive Cl^― stress. Additionally, Bazihizina et al. (Reference Bazihizina, Colmer, Cuin, Mancuso and Shabala2019) reported cytosolic Cl^― concentrations ranging from 2 to 50 mM under non-saline conditions in three halophytic plant species: Suaeda maritima (Hajibagheri & Flowers, Reference Hajibagheri and Flowers1989), Atriplex amnicola (Jeschke et al., Reference Jeschke, Aslam and Greenway1986) and Atriplex spongiosa (Storey et al., Reference Storey, Pitman and Stelzer1983). Due to the negative potential across the plasma membrane (PM) of root cells, the uptake of Cl^― via anion channels is energetically unfavourable (Maathuis & Sanders, Reference Maathuis and Sanders1993; Wang et al., Reference Wang, Dindas, Rienmuller, Krebs, Waadt, Schumacher, Wu, Hedrich and Roelfsema2015). Thus, under low or moderate external Cl^― concentrations when [Cl^―]_out < [Cl^―]_cyt, energy is required to move Cl^― into the cytoplasm; i.e. it is an active transport (Felle, Reference Felle1994; White & Broadley, Reference White and Broadley2001). Here, adenosine triphosphate (ATP) is consumed by H⁺ pumps, and the term to be referred to is secondary active transport (Saleh & Plieth, Reference Saleh and Plieth2013). With stoichiometry, the overall transport has a surplus of one positive elementary charge and thus is supported by the pH gradient, as well as the membrane potential (negative on the cytosolic side).

The transport proteins that enable the uptake of Cl^― are likely to be members of the nitrate transporter 1/peptide transporter family (NPF) (Léran et al., Reference Léran, Varala, Boyer, Chiurazzi, Crawford, Daniel-Vedele, David, Dickstein, Fernandez, Forde, Gassmann, Geiger, Gojon, Gong, Halkier, Harris, Hedrich, Limami, Rentsch and Lacombe2014). The first identified member of this group of transporters was NPF6.3 (NRT1.1), which facilitates the uptake of NO₃ ^― across the PM (Tsay et al., Reference Tsay, Schroeder, Feldmann and Crawford1993). However, the homologous maize ZmNPF6.4 protein acts as a high-affinity Cl^―-selective pH-dependent transporter, whereas ZmNPF6.6 has a lower Cl^― affinity (Wen et al., Reference Wen, Tyerman, Dechorgnat, Ovchinnikova, Dhugga and Kaiser2017). Single-point mutations in these transporters had a strong impact on NO₃ ^―/ Cl^― selectivity, which suggests that genes in the NPF6 group encode H⁺-coupled transporters with a variable selectivity of Cl^― versus NO₃ ^―. The ability of these proteins to transport both anions would also explain why high NO₃ ^― concentrations compete with Cl^― uptake from the soil (Bar et al., Reference Bar, Apelbaum and Goren1997).

Pitman (Reference Pitman1982) estimated that the diffusion coefficient of monovalent ions, such as Cl^―, in the apoplast is approximately 10⁻¹⁰ m² s⁻¹. Cl^― transport by the symplastic pathway provides a more selective and regulated mechanism for ion movement. Once Cl^― enters the root symplast, it moves from cell to cell through plasmodesmata towards the cortex. According to Zhang et al. (Reference Zhang, Ren, Tan, Qi, Yao, Wu and Wang2016), this intracellular movement distributes Cl^― throughout the root and may involve short-term storage in vacuoles, mediated by transporters of the chloride channel (CLC) family of plant nitrate and putative Cl^― proton antiporters (not channels as the name might suggest), which helps in maintaining osmotic pressure balance under stress. Additionally, during abiotic stress, signals like ATP, abscisic acid (ABA) and ROS affect the activity of important transporters, which in turn affects Cl^― transport within roots (Colmenero-Flores et al., Reference Colmenero-Flores, Franco-Navarro, Cubero-Font, Peinado-Torrubia and Rosales2019).

The transcellular pathway adds an additional layer of control, facilitating ion transport across cell membranes. This process requires transporter proteins, which mediate consecutive influx and efflux steps as Cl^― crosses various root cell layers, including the exodermis, hypodermis, cortex and endodermis, before reaching the stele (Andersen et al., Reference Andersen, Naseer, Ursache, Wybouw, Smet, De Rybel and Geldner2018). The exodermis, a specialized hypodermis found in certain plant species, forms the second outermost root layer and shares structural similarities with the endodermis. Both layers act as selective barriers equipped with Casparian strips and suberin depositions that restrict the apoplastic flow (Damus et al., Reference Damus, Peterson, Enstone and Peterson1997). However, passage cells, which are unsuberized cells scattered throughout the exodermis and endodermis, allow for selective transcellular transport (Holbein et al., Reference Holbein, Shen and Andersen2021). These passage cells, first described in the 19th century (Kroemer, Reference Kroemer1903 reviewed in Holbein et al., Reference Holbein, Shen and Andersen2021), are assumed to play distinct but overlapping roles in Cl^― transport. Endodermal passage cells, in particular, frequently co-localize with xylem poles, highlighting their role in ion transport (Hose et al., Reference Hose, Clarkson, Steudle, Schreiber and Hartung2001). As Cl^― moves radially towards the stele, its transport through the transcellular pathway bypasses the hydrophobic barriers of suberized cell walls. This mechanism becomes particularly relevant in older root regions, where apoplastic diffusion is hindered. Instead, transcellular transport depends on PM-localized transporter proteins, whose expression may be concentrated in parenchyma cells (Ramakrishna & Barberon, Reference Ramakrishna and Barberon2019).

4. Chloride uptake during salt stress

When seedlings of A. thaliana are exposed to an external Cl^― concentration of 100 mM, Cl^― rapidly accumulates in root cells to cytosolic concentrations that exceed 50 mM (Lorenzen et al., Reference Lorenzen, Aberle and Plieth2004). However, the accumulation of Cl^― is inhibited by high extracellular Ca²⁺ concentrations, as well as by the application of 2 mM of the broad-range cation channel blocker La³⁺. Roots thus seem to have an uptake system that enables rapid sequestering of Cl^― and is regulated by a Ca²⁺-dependent mechanism.

It is likely that the same NPF transporters that are essential for the uptake of Cl^― at low concentrations (as explained above) also cause the rapid accumulation of Cl^― during salt stress. A main function of NPF transporters is the uptake of NO₃ ^―, which is essential for protein synthesis. Because of the limiting ability of NPF transporters to discriminate between Cl^― and NO₃ ^―, it is likely that Cl^― uptake is inevitable. Consequently, Cl^― toxicity can be reduced by high levels of NO₃ ^― (Bar et al., Reference Bar, Apelbaum and Goren1997) or by breeding plants with transporters that have a higher specificity for NO₃ ^― over Cl^― (Bazihizina et al., Reference Bazihizina, Colmer, Cuin, Mancuso and Shabala2019).

The importance of NPF transporters in Cl^― tolerance was confirmed in a study on soybean (Wu et al., Reference Wu, Yuan, Shen, Li, Li, Cao, Zhu, Liu, Sun, Jia, Chen, Wang, Kudla, Zhang, Gai and Zhang2025). In a genome-wide association study, the GmNPF7.5 gene was found to be of major importance for salt tolerance. A point mutation in GmNPF7.5 reduced the Cl^― permeability of the transporter, while keeping the NO₃ ^― transport activity high and thus enhancing the ability to grow at high-Cl^― conditions.

5. Xylem loading of Cl ^―

Once Cl^― has entered the root epidermal cells, or cortex cells, it is likely to move from cell to cell via plasmodesmata into the endodermis. The apoplastic movement of Cl^― into the endodermis is blocked by the Casparian strip, whose formation is enhanced during salt stress in maize plants (Karahara et al., Reference Karahara, Ikeda, Kondo and Uetake2004). In line with this important role, a lower salt tolerance has been found for mutants with defects in Casparian strip development (Wang et al., Reference Wang, Cao, Liang, Zhuang, Wang, Qin and Jiang2022).

Cl^― that has entered the endodermis is loaded into xylem vessels via a mechanism in which anion channels play a major role. The activation of anion channels depolarizes the PM of xylem parenchyma cells and enables the extrusion of both Cl^― and potassium into xylem vessels (Hmidi et al., Reference Hmidi, Muraya, Fizames, Véry and Roelfsema2024). Slow (S)-type anion channels are found to be active in xylem parenchyma cells (Köhler & Raschke, Reference Köhler and Raschke2000; Wegner & Raschke, Reference Wegner and Raschke1994), and in A. thaliana, the S-type channels SLAH2 and SLAH3 exert this function (Cubero-Font et al., Reference Cubero-Font, Maierhofer, Jaslan, Rosales, Espartero, Díaz-Rueda and Geiger2016; Maierhofer et al., Reference Maierhofer, Lind, Hüttl, Scherzer, Papenfuß, Simon and Geiger2014). The SLAH3 channel interacts with the non-function SLAH1-subunit, which enhances the activity and Cl^― conductivity of SLAH3 (Cubero-Font et al., Reference Cubero-Font, Maierhofer, Jaslan, Rosales, Espartero, Díaz-Rueda and Geiger2016). It is thus very likely that the SLAH3/SLAH1 heteromeric channels are important for the long-distance transport of Cl^― through the xylem. In addition, in maize, a rapid (R)-type anion channel was found to be prevalent, which suggests that these channels may also contribute to the translocation of Cl^― from roots to shoots (Gilliham & Tester, Reference Gilliham and Tester2005).

In addition to anion channels, NPF transporters have also been found to play a role in xylem transport, with their activity regulated by salt stress (Shelke et al., Reference Shelke, Nikalje, Nikam, Maheshwari, Punita, Rao and Suprasanna2019). The transporter AtNPF2.4 is localized to the PM of cells in the root stele, and its overexpression increases shoot Cl^― levels by approximately 23%, underscoring its essential function (Geilfus, Reference Geilfus2018a; Li et al., Reference Li, Byrt, Qiu, Baumann, Hrmova, Evrard and Roy2016), while silencing of AtNPF2.4 in A. thaliana reduces shoot Cl^― by 20–30% (Li et al., Reference Li, Byrt, Qiu, Baumann, Hrmova, Evrard and Roy2016). A similar function was found for AtNPF2.5, closely related to AtNPF2.4 (Li, Qiu, et al., Reference Li, Qiu, Jayakannan, Xu, Li, Mayo and Roy2017a). While the authors suggest a role of AtNPF2.4 and 2.5 in Cl^― extrusion (Li et al., Reference Li, Byrt, Qiu, Baumann, Hrmova, Evrard and Roy2016; Li, Tester, et al., Reference Li, Tester and Gilliham2017b), alternatively these transporters may facilitate Cl^― uptake in root cells, as suggested above. In this scenario, the high activity of NPF proteins would ensure high cytosolic Cl^― concentrations in root cells and an efficient loading of Cl^― into the root xylem. In maize, ZmNPF6.4 has been identified as encoding a potential Cl^―-selective transporter. This protein is localized in root cell membranes and likely employs H⁺-coupled active transport. This process depends on an ExxER/K motif containing three proton-binding, chargeable residues in its first transmembrane helix. Wen et al. (Reference Wen, Tyerman, Dechorgnat, Ovchinnikova, Dhugga and Kaiser2017) reported that increasing the external Cl^― concentration from 0 to 10 mM enhanced Cl^― uptake by ZmNPF6.4.

6. Stomatal movements

The R- and S-type of anion channels were first identified in patch clamp experiments with guard cells (Keller et al., Reference Keller, Hedrich and Raschke1989; Linder & Raschke, Reference Linder and Raschke1992; Schroeder & Hagiwara, Reference Schroeder and Hagiwara1989) and later also in other cell types. In A. thaliana, the S-type anion channels are encoded by SLAC/SLAH genes (Geiger et al., Reference Geiger, Maierhofer, Al-Rasheid, Scherzer, Mumm, Liese, Ache, Wellmann, Marten and Grill2011; Guzel et al., Reference Guzel, Scherzer, Nuhkat, Kedzierska, Kollist, Brosche, Unyayar, Boudsocq, Hedrich and Roelfsema2015; Negi et al., Reference Negi, Matsuda, Nagasawa, Oba, Takahashi, Kawai-Yamada and Iba2008; Vahisalu et al., Reference Vahisalu, Kollist, Wang, Nishimura, Chan, Valerio, Lamminmaki, Brosche, Moldau, Desikan, Schroeder and Kangasjarvi2008) and play a major role in regulating stomatal movements (Merilo et al., Reference Merilo, Laanemets, Hu, Xue, Jakobsen, Tulva, Gonzales-Guzman, Rodriguez, Schroeder, Brosche and Kollist2013). The S-type channels differ in the selectivity for the anions they conduct; SLAC1 conducts both NO₃ ^― and Cl^― (Geiger et al., Reference Geiger, Maierhofer, Al-Rasheid, Scherzer, Mumm, Liese, Ache, Wellmann, Marten and Grill2011), SLAH3 has a preference for NO₃ ^― (Geiger et el., 2011; Hedrich & Geiger, Reference Hedrich and Geiger2017), while SLAH2 is selective for nitrate (Maierhofer et al., Reference Maierhofer, Lind, Hüttl, Scherzer, Papenfuß, Simon and Geiger2014). So far, no Cl^―-selective SLAC1/SLAH channel has been identified, and it is thus likely that the extrusion of Cl^― by these channels is always linked to that of NO₃ ^―.

Stimuli that provoke stomatal closure, such as darkness, high atmospheric CO₂ concentrations, ABA and microbe-associated molecular patterns (MAMPs), all activate S-type anion channels (Guzel et al., Reference Guzel, Scherzer, Nuhkat, Kedzierska, Kollist, Brosche, Unyayar, Boudsocq, Hedrich and Roelfsema2015; Pei et al., Reference Pei, Kuchitsu, Ward, Schwarz and Schroeder1997; Roelfsema et al., Reference Roelfsema, Hanstein, Felle and Hedrich2002; Roelfsema et al., Reference Roelfsema, Levchenko and Hedrich2004). Upon activation of these channels, Cl^― is released, causing a depolarization of the PM (Hmidi et al., Reference Hmidi, Muraya, Fizames, Véry and Roelfsema2024), which also supports K⁺ release via the GORK K⁺ channel (Ache et al., Reference Ache, Becker, Ivashikina, Dietrich, Roelfsema and Hedrich2000; Hosy et al., Reference Hosy, Vavasseur, Mouline, Dreyer, Gaymard, Poree, Boucherez, Lebaudy, Bouchez, Very, Simonneau, Thibaud and Sentenac2003). The simultaneous efflux of Cl^― and K⁺ lowers the osmotic content of guard cells, which leads to efflux of water and closure of stomata (Kollist et al., Reference Kollist, Nuhkat and Roelfsema2014).

In A. thaliana, the loss of the aluminium-activated malate transporter 12 (ALMT12) stimulates stomatal opening (Jalakas et al., Reference Jalakas, Nuhkat, Vahisalu, Merilo, Brosché and Kollist2021) and inhibits several stomatal closure responses (Meyer et al., Reference Meyer, Mumm, Imes, Endler, Weder, Al-Rasheid, Geiger, Marten, Martinoia and Hedrich2010; Sasaki et al., Reference Sasaki, Mori, Furuichi, Munemasa, Toyooka, Matsuoka, Murata and Yamamoto2010). It is likely that the ALMT12-encoded protein is related to R-type channels, since the loss of ALMT12 reduced the activity of these channels in guard cells (Meyer et al., Reference Meyer, Mumm, Imes, Endler, Weder, Al-Rasheid, Geiger, Marten, Martinoia and Hedrich2010). Although the ALMT12 channel mainly seems to transport malate (Jaslan et al., Reference Jaslan, Marten, Jakobson, Arjus, Deeken, Sarmiento, De Angeli, Brosché, Kollist and Hedrich2023), R-type channels in Vicia faba guard cells also conduct Cl⁻ and NO₃ ^―, in addition to small organic anions like acetate and malate (Dietrich & Hedrich, Reference Dietrich and Hedrich1998), and thus provide an alternative efflux pathway to S-type anion channels. Despite in-depth insights into the structure of ALMT12 (Qin et al., Reference Qin, Tang, Xu, Zhang, Zhu, Zhang, Wang, Liu, Li, Sun, Su, Zhai and Chen2022), the properties of R-type channels are still under discussion, and these channels may be influenced by so-far-unidentified components (Jaslan et al., Reference Jaslan, Marten, Jakobson, Arjus, Deeken, Sarmiento, De Angeli, Brosché, Kollist and Hedrich2023).

7. Storage of Cl ⁻ in the vacuole and other organelles

Vacuolar Cl^― accumulation in non-halophytes depends on external Cl^― availability, with vacuolar concentrations easily reaching 40 mM (Barbier-Brygoo et al., Reference Barbier-Brygoo, Vinauger, Colcombet, Ephritikhine, Frachisse and Maurel2000; Fricke et al., Reference Fricke, Leigh and Deri Tomos1994; Geilfus, Reference Geilfus2018a). In root cortical cells of barley, vacuolar Cl^― concentrations under non-saline conditions range from approximately 2–9 mM, but increase significantly upon exposure to saline conditions, with concentrations of up to 290 mM (Flowers & Hajibagheri, Reference Flowers and Hajibagheri2001; Huang & Van Steveninck, Reference Huang and Van Steveninck1989). Similarly, in maize, salinity induces an increase in the vacuolar Cl^― concentration from approximately 40 mM to as high as 1300 mM (Harvey, Reference Harvey1985). In the vacuoles of leaf epidermal cells of barley, Cl^― concentrations reach approximately 100 mM in response to salt exposure (Fricke et al., Reference Fricke, Hinde, Leigh and Tomos1995), but also substantially higher levels of up to 400 mM are reported in the vacuoles of epidermal and mesophyll cells of barley and wheat following salt exposure (James et al., Reference James, Munns, Von Caemmerer, Trejo, Miller and Condon2006).

The vacuolar membrane has an electrical potential that is negative at the cytosolic side, just as the PM. Anion channels thus can support the uptake of Cl^―, but because of the low potential (approximately −30 mV, Wang et al., Reference Wang, Dindas, Rienmuller, Krebs, Waadt, Schumacher, Wu, Hedrich and Roelfsema2015) it can only support an accumulation of up to threefold of the concertation of the cytosol. This mechanism is supported by a group of ALMT proteins that are localized to the vacuolar membrane (Sharma et al., Reference Sharma, Dreyer, Kochian and Piñeros2016). ALMT9 is shown to encode a Cl^―-permeable channel, which supports light-induced stomatal opening, suggesting that it enables the accumulation of Cl^― in vacuoles. The activation of cation channels, such as two-pore potassium (TPK) channels (Dabravolski & Isayenkov, Reference Dabravolski and Isayenkov2023) and two-pore channel 1 (TPC1) (Jaslan et al., Reference Jaslan, Dreyer, Lu, O'Malley, Dindas, Marten and Hedrich2019), is likely to depolarize the vacuolar membrane and thereby enable the backflow of Cl^―, from the vacuole into the cytosol (Eisenach et al., Reference Eisenach, Baetz, Huck, Zhang, De Angeli, Beckers and Martinoia2017).

CLC proteins in plants, such as AtCLCa, are major contributors to nitrate storage in vacuoles (Geelen et al., Reference Geelen, Lurin and Bouchez2000). In A. thaliana leaves, vacuoles serve as reservoirs, accumulating nitrate to concentrations as high as 30 mM, a significant enrichment compared to the 2 mM in the cytosol (Cookson et al., Reference Cookson, Williams and Miller2005). Barley root cells show a similar pattern, with cytosolic nitrate concentrations remaining relatively stable around 4 mM, while vacuolar concentrations increase significantly, up to 20-fold, depending on the external nitrate supply (van der Leij et al., Reference van der Leij, Smith and Miller1998). A single amino acid modification in CLC proteins can shift selectivity from nitrate to Cl^―. The proline residue at position 160 (P160) in the selectivity filter of AtCLCa is critical for its ability to preferentially transport nitrate over Cl^―, a function essential for nitrate accumulation in planta (Wege et al., Reference Wege, Jossier, Filleur, Thomine, Barbier-Brygoo, Gambale and De Angeli2010). Mutation of this amino acid shifts the selectivity of the transporter and turns the preferentially nitrate-transporting AtCLCa into an efficient Cl^― transporter (Bergsdorf et al., Reference Bergsdorf, Zdebik and Jentsch2009; Wege et al., Reference Wege, Jossier, Filleur, Thomine, Barbier-Brygoo, Gambale and De Angeli2010). Whole-vacuole patch-clamp analysis demonstrated that the mutation does not affect the stoichiometry of the anion–proton coupling, an uncoupling effect that is observed in animal CLCs (Wege et al., Reference Wege, Jossier, Filleur, Thomine, Barbier-Brygoo, Gambale and De Angeli2010). The selectivity motif (containing either a proline or a serine) of other CLC family members suggests that they likely function as 2Cl^―/H⁺ antiporters, such as AtCLCc and AtCLCd; this is supported by the phenotypic defects observed in CLCc loss-of-function mutants (Jossier et al., Reference Jossier, Kroniewicz, Dalmas, Le Thiec, Ephritikhine, Thomine and Leonhardt2010). Plants lacking functional CLCs show reduced Cl^― uptake, decreased salinity tolerance and impaired stomata aperture regulation (Hu et al., Reference Hu, Zhu, Wei, Chen, Shi, Shen and Zhang2017; Jossier et al., Reference Jossier, Kroniewicz, Dalmas, Le Thiec, Ephritikhine, Thomine and Leonhardt2010). CLC proteins reside in endomembranes and are not present in the PM; the importance of endomembrane-localized Cl^― transporters strongly suggests that the anion has an important role here. This is also demonstrated by the severe phenotypic defects observed in plants lacking a different endomembrane-localized Cl^―-permeable channel, cation chloride cotransporter (CCC) (McKay et al., Reference McKay, McFarlane, Qu, Situmorang, Gilliham and Wege2022).

The first plant CCC characterized was from A. thaliana (AtCCC1) (Colmenero-Flores et al., Reference Colmenero-Flores, Martínez, Gamba, Vázquez, Iglesias, Brumós and Talón2007). The CCC genes encode putative Cl^― and potassium transporters (Negi et al., Reference Negi, Matsuda, Nagasawa, Oba, Takahashi, Kawai-Yamada and Iba2008). When expressed in Xenopus oocytes, AtCCC1 facilitated the cotransport of Cl^―, sodium and potassium. AtCCC1 is, however, localized in the trans-Golgi-network/early endosome (TGN/EE), where it also remains upon salt or osmotic shock exposure (McKay et al., Reference McKay, McFarlane, Qu, Situmorang, Gilliham and Wege2022). Additionally, AtCCC1 is expressed in all cell types, and CCC1 loss-of-function plants show severe phenotypic defects under control non-stressed conditions, demonstrating a role of CCCs outside of salinity tolerance in both A. thaliana and rice (Chen et al., Reference Chen, Yamaji, Fujii-Kashino and Ma2016; McKay et al., Reference McKay, McFarlane, Qu, Situmorang, Gilliham and Wege2022). Saleh and Plieth (Reference Saleh and Plieth2013) discovered that internal calcium impacted the transport of Cl^―. A calcium-activated Cl^― channel (CaCC) blocker, anthracene-9-carboxylic acid (A9C), causes Cl^― accumulation (Saleh & Plieth, Reference Saleh and Plieth2013) in root cells of A. thaliana during salt stress and is controlled by both internal and external calcium levels.

In some halophytes, epidermal bladder cells have been proposed to contribute to salt tolerance by sequestering excess Cl^― and sodium (Kiani-Pouya et al., Reference Kiani-Pouya, Roessner, Jayasinghe, Lutz, Rupasinghe, Bazihizina and Shabala2017; Shabala et al., Reference Shabala, Bose and Hedrich2014). However, another study challenges this. While wild-type epidermal bladder cells store substantial amounts of Cl^― under saline conditions, the ebcf mutant shows no such enrichment, indicating that Cl^― retention in EBCs is not essential for salt tolerance (Moog et al., Reference Moog, Trinh, Nørrevang, Bendtsen, Wang, Østerberg and Palmgren2022).

8. The role of chloride transport in ion homeostasis

In recent years, it has become more and more clear that the investigation of individual transport events is insufficient for explaining the complex interconnectivity of ions and therefore nutrient homeostasis. Looking at transporter networks is a more promising approach (Blatt, Reference Blatt2024; Dreyer, Reference Dreyer2021). The smallest unit of a transporter network is a so-called homeostat, as described, for example, for potassium by Dreyer et al. (Reference Dreyer, Hernández-Rojas, Bolua-Hernández, de los Angeles Tapia-Castillo, Astola-Mariscal, Díaz-Pico and Michard2024). Potassium, similar to Cl^―, is a monovalent ion that is not metabolized, and it functions primarily as an osmoticum and, importantly, as a positive charge that can be shuttled across membranes without major disturbances of metabolic reactions. This potassium shuttling is crucial for maintaining cellular function and does not constitute futile cycling (Dreyer, Reference Dreyer2021).

A similar situation might exist for Cl^― transport and might explain further the preference of plants for Cl^― over nitrate as a non-metabolized anion. A constant cycling of the anion across cellular membranes might be crucial for the capacity of cells to dynamically respond to external changes, as well as maintaining internal nutrient concentrations. Nitrate, as a similarly sized monovalent anion, can replace Cl^― in this role; a constant cycling of the metabolized ion might yet have several disadvantages, and plants that show a defect in vacuolar nitrate storage show increased amino acid catabolism (Hodin et al., Reference Hodin, Lind, Marmagne, Espagne, Bianchi, De Angeli and Filleur2023). Cycling across the PM holds the potential risk of nitrate loss to the environment. This loss has been observed, for example, in rice (Kurimoto et al., Reference Kurimoto, Day, Lambers and Noguchi2004), where up to 40% of the nitrate taken up by the plants is effluxed back in media without added Cl^―. Interestingly, in carrot (Daucus carota), Cl^― uptake from external media is faster than nitrate uptake in root tissue starved for both ions (Cram, Reference Cram1973). This very interesting observation might be explained by the need for a ‘cycling’ anion. Cl^― is taken up faster and first, compared to nitrate, so that the subsequent potential loss of nitrogen can be minimized and nitrate can be directly and efficiently imported into the vacuole for storage and subsequent nitrate reduction. Higher cytosolic Cl^― concentrations compared to nitrate also point in this direction.

Cl^― transport across intracellular membranes might play equally important roles. CCC proteins in the TGN/EE are part of the pH regulatory circuit in this major cellular trafficking hub, which likely explains the multitude of phenotypic defects observed in CCC loss-of-function plants in rice and A. thaliana. These plants show defects in almost all parts, including reduced root and root hair lengths, malformed leaves, disturbed phyllotaxis and a strongly reduced fertility, in both rice and A. thaliana (Chen et al., Reference Chen, Yamaji, Fujii-Kashino and Ma2016; Colmenero-Flores et al., Reference Colmenero-Flores, Martínez, Gamba, Vázquez, Iglesias, Brumós and Talón2007; McKay et al., Reference McKay, McFarlane, Qu, Situmorang, Gilliham and Wege2022). Similarly, double knockout plants lacking the activity of both TGN/EE-localized AtCLCd and AtCLCf cannot be isolated (Scholl et al., Reference Scholl, Hillmer, Krebs and Schumacher2021), suggesting that putative 2Cl^―/H⁺ antiporters are crucial for plants to complete their life cycle. CLCs might fulfil similar yet different roles as H⁺/K⁺ antiporters at endomembranes. Different from the large group of likely electroneutral K⁺/H⁺ antiporters in plants (NHXs, KEAs, CHXs), CLCs mediate electrogenic transport with a stoichiometry of 2Cl^―:1H⁺ (De Angeli et al., Reference De Angeli, Monachello, Ephritikhine, Frachisse, Thomine, Gambale and Barbier-Brygoo2006; Wege et al., Reference Wege, Jossier, Filleur, Thomine, Barbier-Brygoo, Gambale and De Angeli2010). This has a major impact on Cl^― homeostats and indicates that the homeostats either function differently and/or that not all components are yet known.

In addition, Cl^― transport across the PM has been shown to directly impact potassium transport, further strengthening a connection of the homeostats of the two non-metabolized ions. Plants with the loss of the Cl^― channel SLAC1 also show decreased potassium movement across the PM, although SLAC1 does not transport potassium (Jezek & Blatt, Reference Jezek and Blatt2017; Wang et al., Reference Wang, Papanatsiou, Eisenach, Karnik, Williams, Hills and Blatt2012).