Ion transporters and their molecular regulation mechanism in plants

With the global population predicted to grow by at least 25% by 2050, the need for sustainable production of nutritious foods is important for human and environmental health. Recent progress demonstrate that membrane transporters can be used to improve yields of staple crops, increase nutrient content and resistance to key stresses, including salinity, which in turn could expand available arable land. Exposure to salt stress affects plant water relations and creates ionic stress in the form of the cellular accumulation of Na⁺ and Cl⁻ ions. However, salt stress also impacts heavily on the homeostasis of other ions such as Ca²⁺, K⁺, and NO₃^- and therefore requires insights into how transport and compartmentation of these nutrients are altered during salinity stress. Since Na⁺ interferes with K⁺ homeostasis, maintaining a balanced cytosolic Na⁺/K⁺ ratio has become a key salinity tolerance mechanism. Achieving this homeostatic balance requires the activity of Na⁺ and K⁺ transporters and/or channels. The aim of this review is to seek answers to this question by examining the role of major ions transporters and channels in ions uptake, translocation and intracellular homeostasis in plants.

The beginning of 21^st century is marked by global scarcity of water resources, environmental pollution and increased salinization of soil and water [1]. Increasing human population and reduction in land available for cultivation are two threats for agricultural sustainability [2]. A saline soil is generally defined as one in which the electrical conductivity (EC) of the saturation extract (EC_e) in the root zone exceeds 4 dS m⁻¹ (approximately 40 mM NaCl) at 25 °C and has an exchangeable sodium of 15%. The yield of most crop plants is reduced at this EC_e, though many crops exhibit yield reduction at lower EC_es [3,4]. It has been estimated that worldwide 20% of total cultivated and 33% of irrigated agricultural lands are afflicted by high salinity. Furthermore, the salinized areas are increasing at a rate of 10% annually for various reasons, including low precipitation, high surface evaporation, weathering of native rocks, irrigation with saline water, and poor cultural practices. It has been estimated that more than 50% of the arable land would be salinized by the 2050 [4]. To meet the projected food demand of 9.3 billion people by 2050, global agricultural production must be increased by 60 % from its 2005–2007 levels [5]. This urgent need requires a large effort to improve agricultural production. One feasible way to cope with this challenge is to breed robustly salt-tolerant crops. Understanding the mechanisms underlying plant salt tolerance would be of benefit for breeding such crops and mitigating future food shortages. Accumulation of high Na⁺ in the cytosol can not only cause K⁺ deficiency and thus disrupt various enzymatic processes, but also impose an energetic burden on the cell owing to the requirement of organic solute synthesis to compensate for the export of Na⁺ for osmotic adjustment [6]. Thus, understanding how Na⁺ is sensed and transported in plants under saline conditions could help researchers or breeders breed crops with robust salt tolerance. The present review is focused on the main processes that contribute to the overall homeostasis of the main ionic constituents of salinity and also analyses which specific membrane transporters are believed to be involved in uptake, extrusion, long distance transport and compartmentalization of salt at the cellular and tissue level. Figure 1 gives an overview of the main classes of monovalent ion transporters that totals hundreds of isoforms, often derived from large gene families. In the following sections, we will analyze the potential roles of transporter classes and specific proteins regarding uptake, efflux translocation and compartmentation of salt. In addition, these sections will also evaluate which of these provide promising targets in the quest to improve crop salt tolerance.

Na⁺ sensing in plants

Possible salt sensors for perception of Na⁺: Unlike in animal cells, no specific salt sensors have been identified in plant cells to date. Thus, our knowledge of how plants perceive salt stress and thus decode the corresponding signals remains limited. Cramer, et al. [7] found that Ca²⁺ can mitigate the loss of membrane integrity and minimize cytosolic K⁺ leakage and proposed that displacement of Ca²⁺ by Na⁺ from the root cell plasmalemma is a primary response to salt stress. However, Kinraide [8] showed that the Ca²⁺-displacement hypothesis is often of minor importance to salt stress response. SOS1 (salt overly sensitive 1) Na⁺/H⁺ antiporters [9], histidine kinases [10], and AHK1/ATHK1 [11] have also been suggested to be potential salt sensors or osmo-sensors. Shabala, et al. [12] suggested some putative salt stress sensors/proteins involved in early signaling events, including exchangers and transporters such as SOS1 Na⁺/H⁺ antiporters, NCX Na⁺/Ca²⁺ exchangers, NSCC/NADPH oxidase tandem, mechanosensory channels and transporters, cyclic nucleotide receptors, purino-receptors, annexins, and H⁺-ATPase/GORK tandem. The binding of salt stress-induced increases of cyclic nucleotides to their receptors, e.g. CNGCs, can activate this CNGC Ca²⁺-permeable channels, and thus the increase of cyclic nucleotides could be translated into a massive cytosolic Ca²⁺ uptake, which can affect Ca²⁺ signaling [12]. Similarly, sensing of salt-induced eATP (extracellular ATP) by plasma membrane purinoceptors can be translated into other signaling events, such as ROS (reactive oxygen species) and cytosolic Ca²⁺ signature [13].

Root meristem zone: a tissue harboring salt sensors?: Root is the first plant organ that encounters salinity. Thus, Na⁺ enters first into roots and is then transported to shoots. Wu, et al. [14] found that salt-tolerant bread wheat varieties had significantly higher cytosolic Na⁺ in the root meristem zone than salt-sensitive varieties; although no difference in vacuolar Na⁺ fluorescence intensity was found in the root meristem zone. This finding suggests that salt-tolerant wheats could have more ability to buffer or tolerate increased Na⁺ in the cell cytosol in root meristem zone than salt-sensitive wheats. Further, by removal of the root meristem zone from salt-tolerant wheat varieties, Na⁺ distribution in mesophyll cells was altered and a salt-sensitive phenotype resulted [15]. Taken together, these findings suggest that the root meristem zone can act as a salt stress sensor, or at least a tissue that harbors salt stress-sensor components.

Transporters and channels involved in na+ transport in plants under salt stress

The importance of Na⁺ exclusion in plant salt tolerance: The importance of Na⁺ exclusion in protecting plants against salinity stress is widely accepted. Under salt stress, net Na⁺ accumulation in plant cells is determined by the ion-exchange activity of Na⁺ influx and efflux. Na⁺ influx occurs mainly through ion channels such as the high-affinity K⁺ transporter HKT and non-selective cation channels (NSCC), and Na⁺ efflux is known to be mediated by SOS1, a Na⁺/H⁺ antiporter. In the presence of elevated levels of external Na⁺, under saline conditions, Na⁺ efflux from plant cells is an active process [16]. To date, SOS1, expressed mainly in the root apex in Arabidopsis [17], is the only transporter that has been characterized in Na⁺ export from the cytosol to the apoplast. Loss of SOS1 function resulted in a hyper-salt-sensitive phenotype in the halophytic Arabidopsis relative Thellungiella salsuginea [18]. This finding further confirmed the important role of the SOS1 Na⁺/H⁺ antiporter in Na⁺ exclusion and overall plant salt tolerance. Moreover, to date, studies showing the important role of Na⁺ exclusion in overall salt tolerance have been based mostly on shoot/leaf or even whole-plant Na⁺ content [19-24]. Whether this restricted Na⁺ accumulation in shoot/leaves is achieved mainly by root Na⁺ export or shoots Na⁺ exclusion, or by both of these processes with tight regulation/coordination at different growth stages and time scales, however, has remained unclarified.

The importance of vacuolar Na⁺ sequestration in plant salt tolerance: SOS1-mediated Na⁺ export from cytosol to apoplast (against Na⁺ gradient) is an energy-consuming process. Given that most of the cell volume is occupied by vacuole and most metabolisms occurs in the cytoplasm, one way for plants to alleviate Na⁺ toxicity in the cytosol is to store Na⁺ in the vacuole. Vacuolar Na⁺ sequestration is a common and important mechanism in plant salt tolerance, and is mediated by Na⁺/H⁺ antiporters [25-27]. Prevention of cytoplasmic Na⁺ elevation, maintenance of the cytosolic K⁺/Na⁺ ratio, and control of vacuolar osmotic potential in plants under salt stress can be achieved by, or is associated with, vacuolar Na⁺ sequestration [28]. To date, the best-known transporter for vacuolar Na⁺ sequestration is the NHX1 Na⁺, K⁺/H⁺ exchanger. Overexpression of NHX1 improves salt tolerance in many species including Arabidopsis [25], tomato [29], rice [30], and tobacco [31], showing the importance of vacuolar Na⁺ sequestration in plant overall salt tolerance. Salt-tolerant wheat varieties showed significantly higher vacuolar Na⁺ fluorescence intensity in mature root cells than did sensitive varieties [14,32]. Under overexpression of OsNHX1, transgenic rice cells survived better under saline condition and showed significantly higher growth rate and total Na⁺ content than the wild type (WT) [33]. Taken together, these findings show clearly that vacuolar Na⁺ sequestration is an important trait contributing to plant overall salt tolerance. After sequestration of Na⁺ in vacuoles, another important concern is to prevent Na⁺ leakage from vacuole to cytosol. Loss of control of this step could result in futile Na⁺ cycling between vacuole and cytosol, imposing a high energy burden on the plant. FV (fast-activating) and SV (slow-activating) channels are tonoplast Na⁺ and K⁺-permeable channels that control Na⁺ leakage from vacuole to cytosol. Negative control of FV and SV channel activity has been shown in the salt-stressed halophyte quinoa to reduce such leakage [34], suggesting that efficient control of Na⁺ leakage from vacuole to cytosol could be an important mechanism in plant overall salt stress tolerance (Figurer 2).

Control of xylem Na⁺ loading and unloading: Roots absorb ions and then transfer them to shoots via xylem loading, so that control of xylem Na⁺ loading is important in plant overall salt tolerance. To date, SOS1 Na⁺/H⁺ antiporter [16,35,36], CCC co-transporter [37], and SKOR channel (if xylem loading of Na⁺ is passive) [38] have been shown to be involved in xylem Na⁺ loading (Figure 2). Shi, et al. [16] suggested that SOS1 plays a role in xylem Na⁺ loading in Arabidopsis under mild salt stress. Yadav, et al. [39] showed that enhanced xylem Na⁺ loading and higher overall salt tolerance was achieved in tobacco by overexpression of SbSOS1. Recently, a reduction in overall net xylem Na⁺ loading and accumulation in the shoot and thus improved salt tolerance were observed in wheat Nax (locus for Na⁺ exclusion) lines following down regulation of SOS1-like Na⁺/H⁺ antiporter [40]. Besides SOS1, a CCC co-transporter that is preferentially expressed at the xylem/symplast boundary has also been suggested to play a role in xylem Na⁺ loading [37] (Figure 2). With respect to Na⁺ transport in xylem, besides Na⁺ loading into xylem, Na⁺ unloading from xylem is another important mechanism. HKT transporters play a main role in this process. Sunarpi, et al. [41] showed that the AtHKT1 transporter located on the plasma membrane in xylem parenchyma cells in leaves played a role in Na⁺ unloading from xylem vessels to parenchyma cells. Huang, et al. [42] suggested that TmHKT7-A2, which is associated with Nax1 locus, could control xylem Na⁺ unloading in roots and sheaths. Also, Byrt, et al. [20] showed that HKT1;5 is strongly associated with Nax2 locus in durum wheat and its orthologous locus Kna1 in bread wheat removes Na⁺ from xylem in roots and leads to a high K⁺/Na⁺ ratio in leaves. Jaime-Perez, et al. [43] showed that the SlHKT1;2 Na⁺-selective transporter plays an important role in Na⁺ unloading from xylem in tomato shoots and thus modulates its Na⁺ homeostasis under salinity (Figure 2).

Na⁺ recirculation from shoot to root via phloem: Na⁺ recirculation from shoots to roots has been suggested as an efficient way to protect leaf cells from Na⁺ toxicity [44]. Because leaf vacuolar Na⁺ sequestration ability is poor, Na⁺ recirculation from shoots to roots via phloem sap is probably the main mechanism involved in prevention of Na⁺ delivery to leaf cells in most salt-sensitive plants [45]. Apart from shoot growth rate, the rate of recirculation of Na⁺ to the roots via phloem has been suggested as an important factor affecting Na⁺ concentrations in shoots [46]. In several species, such as lupine, clover, sweet pepper, and maize, recirculation of Na⁺ to roots via phloem played a role in overall salt tolerance [47]. Berthomieu, et al. [48] showed that expression of the AtHKT1 gene was restricted to phloem tissues in all organs in Arabidopsis, and that the AtHKT1 gene was involved in Na⁺ recirculation from shoots to roots probably by mediating Na⁺ loading into phloem sap in the shoots and unloading it in roots. However, in Arabidopsis, a role of AtHKT1 in control of both Na⁺ accumulation in roots and retrieval of Na⁺ from xylem, without involvement in root influx or recirculation in the phloem, was suggested by Davenport, et al. [46]. Ren, et al. [49] showed that HKT-type transporter encoded by SKC1 (shoot K⁺ concentration 1) gene might be involved in the recirculation of Na⁺ by unloading it from the xylem in rice. Kobayashi, et al. [50] found that an OsHKT1;5 Na⁺ selective transporter associated with the SKC1 locus is localized in cells adjacent to the xylem in roots, and is involved in mediating Na⁺ exclusion in phloem to protect young leaf blades of rice under salt stress (Figure 2).

Na⁺ transporters: To date, most members of the cation/proton antiporter (CPA) family have been identified as Na⁺/H⁺antiporters (subclass 1), but a few are K⁺/H⁺ antiporters, including CHX13, CHX17, CHX20, and CHX23 in the CPA2 family [51]. Besides vacuolar Na⁺ sequestration, another important pathway for controlling Na⁺ distribution in plant cells is Na⁺ exclusion/export. To date, SOS1 Na⁺/H⁺ antiporter is the only reported antiporter responsible for Na⁺ export from plant cells [52,53]. SOS1 activity is regulated by SOS2, a serine/threonine protein kinase (CIPK24) and SOS3, a myristoylated calcium-binding protein (CBL4) [54-56]. SOS3 recruits SOS2 to the plasma membrane, and then this CBL-CIPK complex activates SOS1 by phosphorylation, dramatically increasing Na⁺/H⁺ exchange activity (Figure 3) [16]. Moreover, the existence of an ATP-driven Na⁺ transport mediated by a Na⁺-ATPase at the plasma membrane has been shown in lower plants, such as the marine alga Heterosigma akashiwo [57] and the moss Physcomitrella patens [58].

The role of HKT1: X transporters in Na⁺ unloading and recirculation in salt stressed plants was mentioned in the previous sections. For example, Kobayashi, et al. [50] found that the OsHKT1;5 Na⁺ selective transporter, which is associated with the SKC1 locus, is localized in cells adjacent to the xylem in roots, and is involved in mediating Na⁺ exclusion in phloem to protect young leaf blades of rice under salt stress.

Na⁺ channels: NSCCs are a large family of channels that lack selectivity for cations. They are typically permeable to wide range of monovalent cations [59] and are located on both the plasma membrane and the tonoplast (Figure 2). They can be divided into voltage-dependent NSCCs (depolarization-activated, hyper-polarization-activated), voltage-independent NSCCs, ROS-activated NSCCs, amino acid-activated NSCCs, cyclic nucleotide-gated NSCCs, etc. Electrophysiological studies suggest that Na⁺ influx across the plasma membrane occurs via NSCC/VIC in root cortical cells [16,60,61]. Maathuis and Sanders [62] found that cyclic nucleotide-regulated VIC (voltage-independent cation channels) channels showed no selectivity among monovalent cations in Arabidopsis root cells.

Molecular regulation of Na⁺ trans Moreover, the existence of an porters/channels in response to salt stress: To date, SOS1 is the only known anti-transporter responsible for Na⁺ export from cytosol to apoplast. Usually, expression of the SOS1 gene is up-regulated in salt stressed plants [52,63,64]. The functional activity of SOS1 mediated Na⁺ export could be influenced by SOS2 [54], SOS3 [56], the assembly of SOS2-SOS3 complex [65], and H⁺-ATPase, which can increase H⁺ efflux to energize Na⁺ efflux through SOS1 antiporters [66]. SOS1 activity could also be influenced by ROS or ROS signaling-associated components. SOS1 mRNA stability is increased in Arabidopsis under H₂O₂ treatment, and NADPH oxidase is also involved in the up-regulation of SOS1 mRNA stability [67]. Besides, SOS1 interacts with RCD1 (radical-induced cell death), a regulator of oxidative stress responses, and functions in oxidative stress tolerance in Arabidopsis [68]. Reduced ROS production and increased SOS1 expression was found in pao1pao5 (polyamine oxidase, PAO) Arabidopsis mutants than in the WT under salt stress [69]. As with SOS1, overexpression of NHX1 to increase plant salt tolerance has been shown in many plant species [25,70,71]. Although the role of AtNHX1 in K⁺ accumulation in the vacuole was discovered in recent years [72-74], this finding cannot completely rule out the involvement of NHX1 in vacuolar Na⁺ sequestration, especially under high salinity [36,75]. Usually, the NHX1 gene is up-regulated in salt-stressed plants, including Arabidopsis [76], barley [77], and alfalfa [78]. However, a clear decrease in the transcript level of NHX1 in wheat roots was observed under salt stress, while almost no change in the NHX1 transcript level was found in leaves [79]. Moroever, in contrast to the successfully improved salt stress tolerance in tomato [29], rice [30], and tobacco [31], overall salt tolerance was not enhanced in Arabidopsis [74] and barley [78] by expression of the NHX1 Na⁺/H⁺ exchanger gene. These conflicting results raise the questions of the importance of tissue specificity in plant salt-stress tolerance. NHX1 is known to be fueled by an H⁺ gradient across the tonoplast that is maintained by vacuolar H⁺-ATPase and vacuolar PPase [80]. Expressing a halophyte vacuolar H⁺-ATPase subunit c1 (SaVHAc1) in rice plants resulted in higher chlorophyll content and yield than in its WT [81]. Overexpression of vacuolar PPase AVP1 improved salt tolerance in transgenic Arabidopsis relative to the WT, showing a healthy growth of transgenic Arabidopsis in the presence of 250 mmol L⁻¹ NaCl compared with the WT, which died after 10 days [82]. These results suggest that manipulating vacuolar H⁺-ATPase and PPase could allow regulating NHX1 activity and eventually plant overall salt tolerance. Other known factors in the regulation of NHX1 activity are SOS2 [83] and CaM15 [84]. Also, CBL10 can interact with SOS2 to protect Arabidopsis shoots from salt stress [85]. Tang, et al. [86] showed that PtCBL10A and PtCBL10B interact with PtSOS2 in the vacuolar membrane to regulate shoot salt tolerance in poplar. Thus, CBL10 is also proposed to regulate NHX1 activity [87]. Two recent reviews have also focused on molecular regulation of Na⁺ transporters/channels in response to salt stress [36,88].

Transporters involved in cl- uptake

Cl⁻ is a major solute in plant vacuoles, particularly during salt stress, and is involved in both turgor and osmoregulation [89]. Although there is a substantial amount of information regarding K⁺ and Na⁺ transport in plants, very little is known about the molecular mechanism behind the substantial Cl⁻ influx that results from salinization [90]. Plants contain CLC type anion channels which are believed to participate in turgor regulation, stomatal movement and anionic nutrient transport such as NO₃^- [91]. Although the transcript abundance of several CLCs is affected by salinity [92], they are unlikely to contribute to root Cl⁻ uptake: Firstly, plant CLCs have only been detected at endomembranes which appears to exclude a role in Cl⁻ uptake and secondly the thermodynamics of Cl⁻ uptake rule out passive channel type mechanisms. A second class of potential Cl⁻ transporters is formed by the cation chloride cotransporters (CCCs) encoding one gene in Arabidopsis and two genes in rice. AtCCC, expressed in root and shoot tissues, probably functions as a 2Cl⁻:K⁺:Na⁺ cotransporter. Loss of function of AtCCC in Arabidopsis led to a changed root: shoot Cl⁻ ratio but also to a large increase in net Cl⁻ uptake arguing against a role of AtCCC in the uptake of this ion [37].

In addition to Na⁺, Cl⁻ compartmentation is also important for salt tolerance, as elevated levels of Cl⁻ in the cytosol may be harmful, particularly in the case of citrus [93]. Since the vacuole is moderately positive with reference to the cytoplasm, part of the vacuolar Cl⁻ sequestration could proceed through ion channels and several voltage-gated anion channels of the CLC family have been detected in the tonoplast of various species. In Arabidopsis, CLCa was recently shown to function primarily as a H⁺ coupled antiporter to drive vacuolar nitrate accumulation [94], whereas CLCc may also be involved in NO₃^- homeostasis rather than vacuolar Cl⁻ sequestration. However, CLC transcription has been found to respond to salinity: In rice, OsCLCa was significantly upregulated in salt sensitive cultivars in response to salinity stress and OsCLCc, which is expressed in both leaves and roots, showed transcript reduction in the chloride accumulating salt tolerant Pokkali variety [95]. Diédhiou and Golldack [92] showed a coordinated regulation of anion and cation homeostasis in salt-treated rice and suggested a function for OsCLCc in osmotic adjustment at high salinity. A similar co-regulation was recorded in soybean for NHX1 and CLC1 [96]. Nakamura, et al. [97] showed that the same CLC channels partially complemented the yeast gef1 mutant which lacks the yeast CLC channel. In conclusion, these findings suggest that CLC type anion channels are important in mediating Cl⁻ sequestration in the vacuole (Figure 2).

Potassium transporters in plants: involvement in k+ acquisition, redistribution and homeostasis

Potassium is a major plant nutrient which has to be accumulated in great quantity by roots and distributed throughout the plant and within plant cells. Membrane transport of potassium can be mediated by potassium channels and secondary potassium transporters. Uptake and distribution of K⁺ in plant cells is carried out by a variety of transporter proteins categorized into several families with varied structures and transport mechanisms that comprise the channel families Shaker-like voltage-dependent, the tandem-pore (TPK), and the two-pore channels (TPC) [98], the carrier-like families KT/HAK/KUP [99,100], HKT uniporters and symporters [101], and cation-proton antiporters (CPA). The CPA family is the largest one and includes the NHX, CHX, and KEA antiporters [102] (Figure 2).

K⁺-Selective Channels: The first K⁺ transporter with a role in nutrient uptake was the Shaker-like, voltage-gated, and K⁺-selective channel AKT1 [103]. Plant voltage-gated K⁺ channels are divided into three subfamilies regarding their response to the membrane potential [104]: (1) Inward-rectifying (Kin) channels that in Arabidopsis include AKT1, AKT6, KAT1, and KAT2; they open at hyperpolarized membrane potentials allowing the uptake of K⁺. (2) Outward-rectifying (Kout) channels that mediate K⁺ release because they open at depolarized membrane potentials; this group is composed of SKOR and GORK channels. (3) Weakly rectifying (Kweak) channels that can mediate both K⁺ uptake and release, and whose Arabidopsis representative is AKT2. In addition, the Arabidopsis KC1 (KAT3) is an electrically silent Shaker-like protein that interacts with and regulates functionality of the Kin channels AKT1, KAT1, KAT2, and AKT2, but not the Kout channels [105].

K⁺ non-Selective Channels: Electrophysiological recordings of channel activities in the tonoplast have identified fast vacuolar (FV), slow vacuolar (SV), and K⁺-selective vacuolar (VK) cation channels that mediate the release of vacuolar K⁺ [98]. The VK currents have been assigned to two-pore K⁺ (TPK) channels [106]. TPK1, 2, 3, and 5 of Arabidopsis are located in the tonoplast, while TPK4 is in plasma membrane. TPK1 currents are independent of the membrane voltage but sensitive to cytosolic Ca²⁺ and regulated by calcium-dependent protein kinases (CDPKs) and 14-3-3 protein binding (Figure 3) [107]. In Arabidopsis, the TPC1 channel accounts for the SV current [108]. TPC1 is voltage-dependent and non-selective, allowing K⁺ and Na⁺ to permeate toward the cytosol. Whether TPC1 also permeates Ca²⁺ or Ca²⁺ is only an effector of TPC1 gating is a matter of controversy [109]. TPC channels are activated by a decrease in transmembrane potential and increased cytosolic Ca²⁺, and inhibited by low luminal pH and Ca²⁺. The ubiquitous nature of TPC channels and the magnitude of the SV/TPC currents are such that TPC channels are capable of contributing substantially to cellular K⁺ homeostasis. However, plants lacking TPC1 function are not impaired in growth and development. This may indicate that the TPC1 channel is closed most of the time and opens upon specific inputs or under stress. Current thinking is that TPC1 is part of a Ca²⁺/ROS relay that propagates stress signals [110,111].

KT/HAK/KUP transporters: Proteins of the KT/HAK/KUP family are present in plants, fungi, bacteria, and even viruses [112,113], and they are often associated with K⁺ transport across membranes and K⁺ supply. Members of this family have been widely associated with high-affinity K⁺ uptake from the soil, while others may function in both low-affinity and/or high-affinity transport [59,114] and other roles related, for example, to K⁺ translocation, control of water mouvement at the plant level, salt tolerance, osmotic/ drought responses, transport of other alkali cations, and developmental processes in plants, such as root hair growth and auxin distribution [100,113]. These diverse functions of KT/HAK/KUP transporters may all result from their critical roles in cellular K⁺ homeostasis (Figure 2).

HKT uniporters and symporters: The High affinity K⁺ Transporters (HKTs) facilitate Na⁺-selective uniport or Na⁺-K⁺ symport with a channel-like activity [115] (Figure 2).

Phylogenetic and functional analyses distinguished two HKT subfamilies [116]. Members of subfamily 1 (HKT1) are ubiquitous in plants, Na⁺-selective, and mostly involved in Na⁺ recirculation through vascular tissues, as best exemplified by AtHKT1;1 [41]. Members of subfamily 2 (HKT2) have been found only in monocotyledonous species. Although they are all K⁺-permeable, mechanistically HKT2s can operate as either Na⁺-K⁺ symporters or K⁺-selective uniporters (reviewed by Benito, et al. [115]). HKT2-like proteins of cereals have been involved in K⁺ nutrition.

Cation-proton antiporters (CPA): The recent meta-analysis of a large number of publications reporting tolerance phenotypes imparted by exchangers of the Cation/Proton Antiporter Family 1 (CPA1, which includes NHX proteins) concluded that the effect on K⁺ status was generally more pronounced than on Na⁺ content [117]. An informative work showed that overexpression of the AtNHX1 in tomato induced K⁺-deficiency symptoms despite transgenic plants having greater K⁺ contents than controls [72]. The intense sequestration of K⁺ in NHX1-overexpressing plants reduced cytosolic K⁺ activity, primed the induction of the high-affinity K⁺ uptake system, and elicited an array of metabolic and hormonal disorders related to K⁺ deprivation [72,118]. Notwithstanding these unintended effects resulting from NHX overexpression, NHX proteins do increase salt tolerance, presumably because retention of cellular K⁺ is a requisite for adaptation to a saline environment [119]. Deletion of NHX1 and NHX2 genes encoding the two majors vacuolar NHX isoforms resulted in the inability to compartmentalize K⁺ and, surprisingly, in sensitivity to K⁺ supply at concentrations that did not compromise the growth of control plants [73,74]. Moreover, nhx1 nhx2 mutant lines showed dysfunctional stomatal activity, with impaired opening and closure [74,120].

Long-distance transport and inter-organ K⁺ partitioning: Potassium absorbed by peripheral root cells and not compartmentalized in vacuoles must be transported to the upper parts of the plant through the xylem [121]. This step is critical in the long-distance distribution of K⁺ from roots to the upper parts of the plant, and is driven by negative pressure (pulling) created by evaporation of water from leaves. The osmotic water uptake that is caused by nutrient absorption in the root also provides a positive force, known as root pressure, from roots to xylem vessels. Under regular K⁺ supply, symplastic K⁺ diffusion to the xylem through the stele may contribute sufficiently to K⁺ transport from root to shoot [122]. Moreover, K⁺ is highly mobile within plants, exhibiting cycling between roots and shoots via xylem and phloem [121]. Potassium channels SKOR and AKT2 play an important role in K⁺ translocation via xylem and phloem. SKOR (Stelar K⁺ Outward Rectifier), being an outward-rectifying channel, is expressed in root stele cells (pericycle and xylem parenchyma cells) of Arabidopsis, where it mediates K⁺ secretion by xylem parenchyma cells of roots and toward the xylem vessels [123] (Figure 2). SKOR opens upon membrane depolarization to allow cytosolic K⁺ efflux. In the presence of ample external K⁺, the channel opens at less negative membrane voltages, thereby minimizing the risk to serve as an undesirable K⁺- influx pathway [124]. Upon acute depolarization of plasma membrane induced by salinity, SKOR in xylem parenchyma cells can be rapidly activated to mediate K⁺ loading into the xylem. After the plasma membrane potential is restored by increased H⁺-ATPase activity, SKOR-dependent K⁺ release from root stelar cells to the xylem by membrane depolarization is suppressed. Then, accumulated ROS under salinity could, in turn, activate SKOR channels to allow xylem K⁺ loading. This may require a highly coordinated mechanism to ensure efficient xylem K⁺ loading in salt-stressed plants (Figure 2).

Large quantities of K⁺ recirculate from roots to shoots via the xylem and subsequently return to the roots via the phloem [125,126]. The magnitude of the K⁺ flux recirculated from the shoots to the roots would constitute a signal by which the growing shoots could communicate to roots for their K⁺ requirement and regulate K⁺ secretion into the xylem sap (and eventually root K⁺ uptake). AKT2 is mainly expressed in the phloem both in leaves and roots [127,128], where the AKT2 channel protein plays a dual role by loading K⁺ in source tissues and unloading K⁺ in sink organs [129]. AKT2 is the only weak inward-rectifier characterized in Arabidopsis [130,131]. The protein phosphatase PP2CA interacts with AKT2 to induce both inhibition of the channel current and enhancement of its inward rectification [132]. AKT2 can modulate the membrane voltage by switching between its modes of an inward or a non-rectifying channel, respectively [127,133]. Depending on the cellular context, the phosphorylation status of the AKT2 channels may change, enabling them to drive either inward or outward K⁺ fluxes [134].

Members of the KT/HAK/KUP family, e.g. AtKUP7 and OsHAK5, have been proposed to facilitate long-distance K⁺ transport from root to shoot, presumably by mediating K⁺ uptake into the xylem parenchyma cells [122,135] (Figure 2). This function of KT/HAK/KUP transporters would be relevant under K⁺ deprivation, when apoplastic K⁺ levels could be below the operational range of channels.

As mentioned earlier, HKT channel-like proteins are primarily involved in Na⁺ fluxes both in roots (monocots) and vascular bundles (monocots and dicots) [101]. However, they often have a significant impact on maintaining high K⁺/Na⁺ ratio in aerial parts during salinity stress and genetic diversity in HKT proteins meditating long-distance transport of Na⁺ and K⁺ have a great impact on the salt tolerance of cereals (Figure 2) [23,49,136].

Co-regulation of k+ and nitrogen uptake

Plants take up numerous mineral nutrients from the soil; some of them are essential (as K⁺ or NO₃^{−), while others can be toxic at high concentrations (as Na+ or NH₄+). Adaptive responses to varying mineral nutrient conditions in the soil, particularly low-nutrient environments, involve multiple signaling pathways whose integration allows plants to grow and adjust their development to each specific nutritional situation [137]. Thus, changes in the concentration of one nutrient trigger a signaling cascade that modify not only the amount, localization, and/or activity of this nutrient-specific transporter/channel, but also transporters/ channels related with other nutrients. N-K interactions are important for root architecture [137].}

K⁺ is the preferred counter ion for root-to-shoot translocation of NO₃^{− in the xylem of crops and Arabidopsis [64,138,139]. NRT1.5, a member of the Nitrate Transporter 1/Peptide Transporter Family (NPF7.3), is important for the NO₃−-dependent K+ translocation in Arabidopsis [140-141]. Lack of NRT1.5 resulted in K+ deficiency in shoots under low NO₃− availability, whereas the root elemental composition was unchanged [140,141]. Mutant analyses revealed that both NRT1.5 and SKOR contributed additively to K+ translocation; SKOR activity was dominant under high NO₃− and low K+ supply, and NRT1.5 was required under low NO₃− [142-,43]. Together, these data indicate that NRT1.5 facilitates K+ release out of root parenchyma cells and loading into xylem vessels (Figure 2). NRT1.5 is a plasma membrane protein that in Xenopus oocytes behaved as a low-affinity, pH-dependent bidirectional nitrate transporter [140]. Surprisingly, NRT1.5 has also been shown to release K+ from Xenopus oocytes and yeast in a pH-dependent manner, and has been proposed to function as a K+/H+ antiporter [143]. Recent knowledge gained about the coordinated regulation of K+ and NO₃− uptake and nutrition. In fact, K+ starvation is required for triggering high-affinity HAK5-mediated K+ uptake in roots of Arabidopsis and tomato. However, limitation of K+, N, or P, induces hyperpolarization of the plasma membrane of root cells and enhanced HAK5 transcription [144], a response that could be due to maintenance of electrical balance since single N and P starvation, probably resulting in lower NO₃− and PO₄3− contents, and led to a concomitant reduction of the K+ content [137]. Alternatively, the transport of a nutrient could become inhibited if another nutrient is limiting [145]. In line with this, NO₃−, PO₄3−, and SO₄2− deficiencies reduced root K+ uptake [139]. Furthermore, comparison of the transcriptional responses to single or multiple nutrient deprivations showed that N starvation had a dominant effect over P and K starvation. In other words, the transcriptional landscape of combined K+ and N limitation was mainly driven by the N-starvation response.}

The CIPK23/CBL1,9 protein kinase complex is key factor in the coordination of plant nutrition, regulating iron, NO₃^{−, and K+ uptakes [146-149]. The transport and regulatory protein AtNRT1.1 (Nitrate Transporter 1) is involved in both high-affinity and low-affinity nitrate uptake. Unphosphorylated AtNRT1.1 is a low-affinity nitrate transporter working as a dimer, and its phosphorylation by CIPK23/CBL1,9 leads to dimer dissociation. Phosphorylated AtNRT1.1 monomer shows a higher nitrate affinity than the dimers [146,150]. On the other hand, AtAMT1, an ammonium transporter, works as trimers and the phosphorylation by CIPK23/CBL1 (and not CBL9) of a single monomer exhibits an allosteric effect, leading to the cooperative closure of all three pores in the trimer [148]. Together, these data indicate that CIPK23 and CBL1 are major regulators of NO₃−, K+, and NH₄+ homeostasis in Arabidopsis.}

Genetic engineering of specific transporters modifies salinity tolerance

Several obvious ways to achieve salinity tolerance include: (1) decreasing sodium conductance and increasing potassium/sodium selectivity of plasma membrane of root epidermal cells; (2) increasing sodium efflux by root epidermal cells; (3) increasing sodium accumulation in vacuoles; (4) altering sodium and potassium loading and unloading to xylem and phloem depending on plant strategy to cope with salinity. Successful attempts to overexpress or knockout genes of vacuolar proton pump H⁺-PPase, NHX, HKT, or SOS1-like transporters and to modulate the salinity tolerance of plants had already been reported. Overexpression of the vacuolar H⁺-PPase would enhance the proton pumping activity at vacuolar membrane and thus permit to accumulate more Na⁺ in vacuoles due to activity of Na⁺(cation)/H⁺ antiporters NHX. The choice of H⁺-pyrophosphatase is explained by a single gene required for the protein, while the other vacuolar H⁺-ATPase is composed of several subunits and needs correct overexpression of several genes [80]. Overexpression of vacuolar H⁺-PPase under control of strong non-specific viral 35S promoter sharply increased salinity tolerance in Arabidopsis, to 250 mM of NaCl [82]. Further attempts to overexpress vacuolar H⁺-PPases from different plant species increased salinity tolerance in tobacco [151-153].

Other candidates for overexpression are vacuolar NHX genes. Overexpression of AtNHX1 increased salinity tolerance in Arabidopsis to 200 mM NaCl. The overexpressing plants accumulated more Na⁺ compared to wild type and demonstrated higher Na⁺/H⁺ exchange activity in isolated leaf vacuoles [25]. The approach of overexpressing AtNHX1 to improve salinity tolerance proved to be successful for tomato; the transgenic plants accumulated more sodium in leaves but not in fruits at 200 mM NaCl [29]. Cotton plants with AtNHX1 from Arabidopsis [154], rice overexpressing SsNHX1 from halophyte Suaeda salsa [155], tomato with heterologous NHX from Pennisetum glaucum [156] also showed increased salinity tolerance. Overexpression of NHX did not influence the phenotype of plants under control conditions [25,29,153-157]. The results with heterologous expression or overexpression of NHX transporters lead to conclusions that the gene is among determinants and potential candidates for engineering salinity tolerance (e.g., [155,158] with more references for successful overexpression of NHX to increase salinity tolerance in sugar beet, wheat, maize and the other plants). The overexpression of NHX was not tissue-specific and under the control of strong promoters, however, a report could not confirm increase in salinity tolerance in Arabidopsis overexpressing AtNHX1 [74]. Expression in a tissue-specific manner could be the next step for using NHX to increase salinity tolerance.

The amazing simplicity of the idea to play with the expression of known and functionally well characterized transporters and get salt tolerant or salt sensitive plants is applied to plasma membrane SOS1 Na⁺/H⁺ antiporters and Na⁺ or Na⁺/K⁺ HKT transporters. SOS1 is expressed in (1) epidermal root cells where it participates in sodium efflux and in (2) xylem parenchyma cells where SOS1 may load Na⁺ to xylem under moderate salinity and unloads Na⁺ under high salinity or has more complex mode of xylem loading/unloading [17,18,56,159-161]. Arabidopsis mutants with defects in gene of SOS1 exhibited strong growth inhibition under salt treatment [162], which was rescued in SOS1 mutant by overexpression of SOS1 gene under 35S promoter [56]. Overexpression of SOS1 gene in wild type plants under 35S promoter enhanced salinity tolerance of Arabidopsis at 100–200 mM NaCl [74,163], reduced sodium accumulation in shoots and sodium concentration in xylem sap [160]. Further, overexpression of SOS1 from A. thaliana increased salinity tolerance in transgenic tobacco [75]. SOS1 gene from durum wheat conferred salinity tolerance to SOS1 mutant of Arabidopsis [164]. Interestingly, the effects of overexpression were observed under salt treatment, while in the absence of stress no differences were observed in growth or morphology between wild-type plants and the transgenic lines. Disruption of SOS1 activity by RNA interference in Thellungiella on the opposite resulted in the loss of tolerance of the halophyte indicating importance of Na⁺ efflux and essential role of SOS1 in salinity tolerance [18]. RNA interference of SOS1 significantly changed the whole transcriptome of Thellungiella [158] and vacuolar pH under salt treatment [67]. A more complicated situation emerges due to tissue-specific expression. SOS1 is important for long-distance ion transport and xylem loading/unloading in Arabidopsis ([17] discussed in: de Boer and Volkov, [165], sodium partioning between plant organs in tomato [161] and ion fluxes in root meristem zone [166], therefore attempts to express it in specific tissues could increase salinity tolerance to a higher extent.

Genetic modification of salinity tolerance using HKT transporters was also successful. Analysis of Arabidopsis plants with mutated HKT gene revealed higher salt sensitivity of the mutants under long term stress, higher sodium accumulation in their shoots under mild salinity treatment [167] and suggested that HKT is involved in recirculation of sodium within plants [48]. Further study confirmed increased sodium in the shoots of Arabidopsis hkt1;1 mutant and clarified that HKT is important for root accumulation of Na⁺ and Na⁺ uptake from xylem in Arabidopsis [46]. The next step was to create plants overexpressing HKT [168]. Arabidopsis plants overexpressing AtHKT under the control of 35S promoter were compared with plants specifically overexpressing HKT in cells of root stele. Pro35S:HKT1;1 plants were salt sensitive probably due to higher Na⁺ uptake by roots while tissue specific overexpression of HKT in stele increased salinity tolerance and reduced sodium accumulation in shoots [168]. The approach was applied to rice where gene from Arabidopsis AtHKT1;1 was heterologously expressed in root cortex. It resulted in lower shoot Na⁺ concentrations, improved salinity tolerance and involved up- and down-regulation of several membrane transport genes including vacuolar H⁺-pyrophosphatases [169]. Overexpression of HKT had none [169-171] or slight inhibiting pleiotropic effect on growth without NaCl depending on type of promotor for expression and on plant line studied [168,169]. HKT transporters proved to be important for Na⁺ exclusion in wheat and were transferred from durum wheat to bread wheat by interspecific crossing; the genes gave beneficial effects including higher K⁺/Na⁺ ratio in leaves under saline conditions [21]. Remarkably, the recent introgression of an ancestral form of the HKT1;5 gene from the more Na⁺-tolerant wheat relative Triticum monococcum into susceptible commercial durum wheat (Triticum turgidum ssp durum) increased grain yields on saline soil by 25% in the field, illustrating the immense potential of this mechanism [23]. Some plants including barley accumulate Na⁺ in shoots; overexpression of barley HvHKT2;1 under 35S promoter in barley increased salinity tolerance at 100 mM NaCl, but opposite to Arabidopsis increased Na⁺ concentration in xylem and Na⁺ accumulation in barley leaves [170]. Taken together the results set HKT transporters to potential candidates for engineering salinity tolerance and among the determinants of the trait (reviewed in: [171-173].

The application of nitrogen (N) fertilizers has greatly increased crop yields. Therefore, enhancing crop nitrogen utilization efficiency is an important goal [174]. For most crops, nitrate is the primary nitrogen source and so enhancing nitrate uptake is one strategy for improving nitrogen utilization efficiency. Multiple nitrate uptake transporters of the NRT1 and NRT2 families work together to enable nitrogen uptake in plants [175,176]. Therefore, nitrate transporters and other proteins that regulate nitrate uptake and sensing provide potential tools for engineering crops with tailored N uptake activity, N metabolism and improved root growth for enhanced nitrogen-use efficiency and reduced-N-fertilizer requirements [177-179].

Although plant salt tolerance at the level of Na⁺ transport is well characterized, the initial plant perception of salt stress and its transduction to subsequent signaling cascades is still obscure. Many genes targets involved in salt tolerance have been identified through various approaches, particularly through transcriptomics studies. Moreover, it appears that forward genetics and yeast complementation strategies have so far been the most successful approaches to identify relevant targets. The accumulative data show importance of two particular classes of transporters: HKTs which function in both Na⁺ uptake and long-distance translocation and NHXs in their capacity as H⁺: Na⁺ antiport or by maintaining K⁺ homeostasis. The significance of these systems is often isoform dependent and may be further complicated by allelic variation between cultivars. Manipulation of several of the genes discussed above has been shown to alter uptake, efflux, translocation and compartmentation of Na⁺. Although in some of these cases improved tolerance can be observed in controlled conditions it has not yet resulted in plants with significantly improved tolerance in field conditions. Simultaneous upregulation of extruding mechanisms through overexpression of systems such as vacuolar pumps, NHXs and SOS1 and loss of function in uptake pathways such as non-selective ion channels and HKTs promises large degrees of additive or synergistic benefits. This is technically challenging but becoming more and more feasible. For instance, salinity tolerance that operates by removal of toxic sodium ions from the xylem sap could be combined with traits that enhance sequestration of sodium into vacuoles, to confer additional salt tolerance. More work will be needed to determine whether or not traits will be compatible when combined. Moreover, many fundamental mechanisms for essential transport processes remain to be uncovered and many essential transporters undoubtedly remain to be discovered. Therefore, knowledge-targeted pyramiding of traits will require future advances in fundamental research into plant membrane transport processes.

This work was supported through funding by the Ministry of Higher Education and Scientific Research of Tunisia.

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