Osmotic Stress Responses and Plant Growth Controlled by Potassium Transporters in Arabidopsis

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K⁺ uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K⁺ channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using ⁸⁶radioactive rubidium ion (⁸⁶Rb⁺) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.     

Heteromeric AtKC1��AKT1 Channels in Arabidopsis Roots Facilitate Growth under K+-limiting Conditions

Plant growth and development is driven by osmotic processes. Potassium represents the major osmotically active cation in plants cells. The uptake of this inorganic osmolyte from the soil in Arabidopsis involves a root K⁺ uptake module consisting of the two K⁺ channel α-subunits, AKT1 and AtKC1. AKT1-mediated potassium absorption from K⁺-depleted soil was shown to depend on the calcium-sensing proteins CBL1/9 and their interacting kinase CIPK23. Here we show that upon activation by the CBL·CIPK complex in low external potassium homomeric AKT1 channels open at voltages positive of E_K, a condition resulting in cellular K⁺ leakage. Although at submillimolar external potassium an intrinsic K⁺ sensor reduces AKT1 channel cord conductance, loss of cytosolic potassium is not completely abolished under these conditions. Depending on channel activity and the actual potassium gradients, this channel-mediated K⁺ loss results in impaired plant growth in the atkc1 mutant. Incorporation of the AtKC1 subunit into the channel complex, however, modulates the properties of the K⁺ uptake module to prevent K⁺ loss. Upon assembly of AKT1 and AtKC1, the activation threshold of the root inward rectifier voltage gate is shifted negative by approximately −70 mV. Additionally, the channel conductance gains a hypersensitive K⁺ dependence. Together, these two processes appear to represent a safety strategy preventing K⁺ loss through the uptake channels under physiological conditions. Similar growth retardation phenotypes of akt1 and atkc1 loss-of-function mutants in response to limiting K⁺ supply further support such functional interdependence of AKT1 and AtKC1. Taken together, these findings suggest an essential role of AtKC1-like subunits for root K⁺ uptake and K⁺ homeostasis when plants experience conditions of K⁺ limitation.Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K⁺ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ∼20 μm and a low affinity transport system with K_m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K⁺ channels AKT1·AtKC1 and members of the HAK·KT·KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5–9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K⁺ channel family. AKT1 is a voltage-dependent inward-rectifying K⁺ channel mediating potassium uptake over a wide range of external potassium concentrations (10–15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K⁺ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 μm and less) (11, 12, 15). Rescue of yeast growth on 20 μm K⁺ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K⁺ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with AKT1 (18–20). AtKC1 α-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K⁺ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 α-subunits do not form K⁺ channels per se but modulate the properties of the AKT1·AtKC1 heterocomplex (20–22). Previously, two groups in their ground-breaking studies demonstrated that AKT1 is activated by the CBL²-interacting, serine/threonine kinase, CIPK23, particularly under low K⁺ conditions (23, 24). CIPK23 itself was shown to be activated by the two calcineurin B-like proteins, CBL1 and 9, acting in a Ca²⁺-dependent manner upstream of CIPK23 (25, 26). Genetic disruption of these elements resulted in transgenic plants exhibiting a phenotype comparable with that of the AKT1 loss of function mutant. This regulatory system, based on a calcium sensor, a protein kinase, and a K⁺ channel, was functionally reconstituted in Xenopus oocytes (23, 24, 27), suggesting that these elements are essential and sufficient to operate as a low K⁺-sensitive potassium uptake system. Here we report on the physiological properties of the heteromeric K⁺ uptake module formed by the predominant root potassium uptake channel subunits, AKT1 and AtKC1 and its regulating kinase complex, CBL1 and CIPK23. Our studies show that the physical interaction of the CBL1·CIPK23 complex is specific for AKT1 channels and does not involve the AtKC1 subunit. AKT1 possesses a K⁺ (absence) sensor affecting channel activity at submillimolar K⁺ concentrations by strongly reducing its maximal cord conductance. Despite this K⁺ sensor, upon activation, AKT1 homomeric channels were shown to represent a potassium leak at low external potassium concentrations. Integration of AtKC1 into the K⁺ uptake module, however, prevented potassium loss by modulating both the voltage sensor and conductance in the channel complex. Moreover, activation of the AKT1-like maize channel ZMK1 by CBL1·CIPK23 suggests a conserved interaction and regulation across monocot and dicotyledonous plant species. Our biophysical studies as well as growth assays with plant mutant lines lacking the respective channels underline that acquisition of potassium under limiting K⁺ conditions is mediated via the root AKT1·AtKC1 K⁺ uptake channel complex.     

A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula

Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H⁺-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H⁺-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H⁺-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H⁺-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.     

Calcineurin B‐like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis

Potassium transporters and channels play crucial roles in K⁺ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K⁺ channel in Arabidopsis roots that is involved in K⁺ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B‐like proteins CBL1/CBL9. The present study showed that another calcineurin B‐like protein (CBL10) may also regulate AKT1 activity. The CBL10‐over‐expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low‐K⁺ conditions. In addition, the K⁺ content of both CBL10‐over‐expressing lines and akt1 mutant plants were significantly reduced compared with wild‐type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two‐hybrid, BiFC and co‐immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1‐mediated inward K⁺ currents. Furthermore, the results from the yeast two‐hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL‐interacting protein kinase‐independent regulatory mechanism of calcineurin B‐like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.     

Systems Dynamic Modeling of a Guard Cell Cl? Channel Mutant Uncovers an Emergent Homeostatic Network Regulating Stomatal Transpiration

Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K⁺ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H⁺ load on the cytosol and promoted Ca²⁺ influx to elevate cytosolic pH (pH_i) and free cytosolic Ca²⁺ concentration ([Ca2+]_i), in turn regulating the K⁺ channels. We have confirmed these predictions, measuring pH_i and [Ca2+]_i in vivo, and report that experimental manipulation of pH_i and [Ca2+]_i is sufficient to recover K⁺ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K⁺ channels. Additionally, these findings underscore the importance of H⁺-coupled anion transport for pH_i homeostasis.Guard cells surround stomatal pores in the epidermis of plant leaves and regulate pore aperture to balance the demands for CO₂ in photosynthesis with the need to conserve water by the plant. Transpiration through stomata accounts for much of the 70% of global water usage associated with agriculture, and it has a profound impact on the water and carbon cycles of the world (Gedney et al., 2006; Betts et al., 2007). Guard cells open the pore by transport and accumulation of osmotically active solutes, mainly K⁺ and Cl⁻ and the organic anion malate²⁻ (Mal), to drive water uptake and cell expansion. They close the pore by coordinating the release of these solutes through K⁺ and anion channels at the plasma membrane. The past half-century has generated a wealth of knowledge on guard cell transport, signaling, and homeostasis, resolving the properties of the major transport processes and metabolic pathways for osmotic solute uptake and accumulation, and many of the signaling pathways that control them (Blatt, 2000; Schroeder et al., 2001; McAinsh and Pittman, 2009; Hills et al., 2012). Even so, much of stomatal dynamics remains unresolved, especially how the entire network of transporters in guard cells works to modulate solute flux and how this network is integrated with organic acid metabolism (Wang and Blatt, 2011) to achieve a dynamic range of stomatal apertures.This gap in understanding is most evident in a number of often unexpected observations, many of which have led necessarily to ad hoc interpretations. Among these, recent studies highlighted a diurnal variation in the free cytosolic Ca²⁺ concentration ([Ca2+]_i), high in the daytime despite the activation of primary ion-exporting ATPases, and have been interpreted to require complex levels of regulation (Dodd et al., 2007). Other findings wholly defy intuitive explanation. For example, the tpk1 mutant of Arabidopsis (Arabidopsis thaliana) removes a major pathway for K⁺ flux across the tonoplast and suppresses stomatal closure, yet the mutant has no significant effect on cellular K⁺ content (Gobert et al., 2007). Similarly, the Arabidopsis clcc mutant eliminates the H⁺-Cl⁻ antiporter at the tonoplast; it affects Cl⁻ uptake, reduces vacuolar Cl⁻ content, and slows stomatal opening; however, counterintuitively, it also suppresses stomatal closure (Jossier et al., 2010). In work leading to this study, we observed that the slac1 anion channel mutant of Arabidopsis paradoxically profoundly alters the activities of the two predominant K⁺ channels at the guard cell plasma membrane. The SLAC1 anion channel is a major pathway for anion loss from the guard cells during stomatal closure (Negi et al., 2008; Vahisalu et al., 2008), and its mutation leads to incomplete and slowed closure of stomata in response to physiologically relevant signals of dark, high CO₂, and the water-stress hormone abscisic acid. Guard cells of the slac1 mutant accumulate substantially higher levels of Cl⁻, Mal, and also K⁺ when compared with guard cells of wild-type Arabidopsis (Negi et al., 2008). The latter observation is consistent with additional impacts on K⁺ transport; however, a straightforward explanation for these findings has not been not forthcoming.Quantitative systems analysis offers one approach to such problems. Efforts to model stomatal function generally have been driven by a “top-down” approach (Farquhar and Wong, 1984; Eamus and Shanahan, 2002) and have not incorporated detail essential to understanding the molecular and cellular mechanics that drive stomatal movement. Only recently we elaborated a quantitative systems dynamic approach to modeling the stomatal guard cell that incorporates all of the fundamental properties of the transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and the essential cytosolic pH (pH_i) and [Ca2+]_i buffering characteristics that have been described in the literature (Hills et al., 2012). The model resolved with this approach (Chen et al., 2012b) successfully recapitulated a wide range of known stomatal behaviors, including transport and aperture dependencies on extracellular pH, KCl, and CaCl₂ concentrations, diurnal changes in [Ca2+]_i (Dodd et al., 2007), and oscillations in membrane voltage and [Ca2+]_i thought to facilitate stomatal closure (Blatt, 2000; McAinsh and Pittman, 2009; Chen et al., 2012b). We have used this approach to resolve the mechanism behind the counterintuitive alterations in K⁺ channel activity uncovered in the slac1 mutant of Arabidopsis. Here, we show how anion accumulation in the mutant affects the H⁺ and Ca²⁺ loads on the cytosol, elevating pH_i and [Ca2+]_i, and in turn regulating the K⁺ channels. We have validated the key predictions of the model and, in so doing, have uncovered a previously unrecognized homeostatic network that ameliorates the effects of the slac1 mutant on transpiration from the plant.     

The Role of a Potassium Transporter OsHAK5 in Potassium Acquisition and Transport from Roots to Shoots in Rice at Low Potassium Supply Levels

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A β-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.Potassium (K) is one of the three most important macronutrients and the most abundant cation in plants. As a major osmoticum in the vacuole, K drives the generation of turgor pressure, enabling cell expansion. In the vascular tissue, K is an important participant in the generation of root pressure (for review, see Wegner, 2014 [including his new hypothesis]). In the phloem, K is critical for the transport of photoassimilates from source to sink (Marschner, 1996; Deeken et al., 2002; Gajdanowicz et al., 2011). In addition, enhancing K absorption and decreasing sodium (Na) accumulation is a major strategy of glycophytes in salt stress tolerance (Maathuis and Amtmann, 1999; Munns and Tester, 2008; Shabala and Cuin, 2008).Plants acquire K through K-permeable proteins at the root surface. Since available K concentration in the soil may vary by 100-fold, plants have developed multiple K uptake systems for adapting to this variability (Epstein et al., 1963; Grabov, 2007; Maathuis, 2009). In a classic K uptake experiment in barley (Hordeum vulgare), root K absorption has been described as a high-affinity and low-affinity biphasic transport process (Epstein et al., 1963). It is generally assumed that the low-affinity transport system (LATS) in the roots mediates K uptake in the millimolar range and that the activity of this system is insensitive to external K concentration (Maathuis and Sanders, 1997; Chérel et al., 2014). In contrast, the high-affinity transport system (HATS) was rapidly up-regulated when the supply of exogenous K was halted (Glass, 1976; Glass and Dunlop, 1978).The membrane transporters for K flux identified in plants are generally classified into three channels and three transporter families based on phylogenetic analysis (Mäser et al., 2001; Véry and Sentenac, 2003; Lebaudy et al., 2007; Alemán et al., 2011). For K uptake, it was predicted that, under most circumstances, K transporters function as HATS, while K-permeable channels mediate LATS (Maathuis and Sanders, 1997). However, a root-expressed K channel in Arabidopsis (Arabidopsis thaliana), Arabidopsis K Transporter1 (AKT1), mediates K absorption over a wide range of external K concentrations (Sentenac et al., 1992; Lagarde et al., 1996; Hirsch et al., 1998; Spalding et al., 1999), while evidence is accumulating that many K transporters, including members of the K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) family, are low-affinity K transporters (Quintero and Blatt, 1997; Senn et al., 2001), implying that functions of plant K channels and transporters overlap at different K concentration ranges.Out of the three families of K transporters, cation proton antiporter (CPA), high affinity K/Na transporter (HKT), and KT/HAK/KUP, CPA was characterized as a K⁺(Na⁺)/H⁺ antiporter, HKT may cotransport Na and K or transport Na only (Rubio et al., 1995; Uozumi et al., 2000), while KT/HAK/KUP were predicted to be H⁺-coupled K⁺ symporters (Mäser et al., 2001; Lebaudy et al., 2007). KT/HAK/KUP were named by different researchers who first identified and cloned them (Quintero and Blatt, 1997; Santa-María et al., 1997). In plants, the KT/HAK/KUP family is the largest K transporter family, including 13 members in Arabidopsis and 27 members in the rice (Oryza sativa) genome (Rubio et al., 2000; Mäser et al., 2001; Bañuelos et al., 2002; Gupta et al., 2008). Sequence alignments show that genes of this family share relatively low homology to each other. The KT/HAK/KUP family was divided into four major clusters (Rubio et al., 2000; Gupta et al., 2008), and in cluster I and II, they were further separated into A and B groups. Genes of cluster I or II likely exist in all plants, cluster III is composed of genes from both Arabidopsis and rice, while cluster IV includes only four rice genes (Grabov, 2007; Gupta et al., 2008).The functions of KT/HAK/KUP were studied mostly in heterologous expression systems. Transporters of cluster I, such as AtHAK5, HvHAK1, OsHAK1, and OsHAK5, are localized in the plasma membrane (Kim et al., 1998; Bañuelos et al., 2002; Gierth et al., 2005) and exhibit high-affinity K uptake in the yeast Saccharomyces cerevisiae (Santa-María et al., 1997; Fu and Luan, 1998; Rubio et al., 2000) and in Escherichia coli (Horie et al., 2011). Transporters of cluster II, like AtKUP4 (TINY ROOT HAIRS1, TRH1), HvHAK2, OsHAK2, OsHAK7, and OsHAK10, could not complement the K uptake-deficient yeast (Saccharomyces cerevisiae) but were able to mediate K fluxes in a bacterial mutant; they might be tonoplast transporters (Senn et al., 2001; Bañuelos et al., 2002; Rodríguez-Navarro and Rubio, 2006). The function of transporters in clusters III and IV is even less known (Grabov, 2007).Existing data suggest that some KT/HAK/KUP transporters also may respond to salinity stress (Maathuis, 2009). The cluster I transporters of HvHAK1 mediate Na influx (Santa-María et al., 1997), while AtHAK5 expression is inhibited by Na (Rubio et al., 2000; Nieves-Cordones et al., 2010). Expression of OsHAK5 in tobacco (Nicotiana tabacum) BY2 cells enhanced the salt tolerance of these cells by accumulating more K without affecting their Na content (Horie et al., 2011).There are only scarce reports on the physiological function of KT/HAK/KUP in planta. In Arabidopsis, mutation of AtKUP2 (SHORT HYPOCOTYL3) resulted in a short hypocotyl, small leaves, and a short flowering stem (Elumalai et al., 2002), while a loss-of-function mutation of AtKUP4 (TRH1) resulted in short root hairs and a loss of gravity response in the root (Rigas et al., 2001; Desbrosses et al., 2003; Ahn et al., 2004). AtHAK5 is the only system currently known to mediate K uptake at concentrations below 0.01 mm (Rubio et al., 2010) and provides a cesium uptake pathway (Qi et al., 2008). AtHAK5 and AtAKT1 are the two major physiologically relevant molecular entities mediating K uptake into roots in the range between 0.01 and 0.05 mm (Pyo et al., 2010; Rubio et al., 2010). AtAKT1 may contribute to K uptake within the K concentrations that belong to the high-affinity system described by Epstein et al. (1963).Among all 27 members of the KT/HAK/KUP family in rice, OsHAK1, OsHAK5, OsHAK19, and OsHAK20 were grouped in cluster IB (Gupta et al., 2008). These four rice HAK members share 50.9% to 53.4% amino acid identity with AtHAK5. OsHAK1 was expressed in the whole plant, with maximum expression in roots, and was up-regulated by K deficiency; it mediated high-affinity K uptake in yeast (Bañuelos et al., 2002). In this study, we examined the tissue-specific localization and the physiological functions of OsHAK5 in response to variation in K supply and to salt stress in rice. By comparing K uptake and translocation in OsHAK5 knockout (KO) mutants and in OsHAK5-overexpressing lines with those in their respective wild-type lines supplied with different K concentrations, we found that OsHAK5 not only mediates high-affinity K acquisition but also participates in root-to-shoot K transport as well as in K-regulated salt tolerance.     

Ethylene Responses in Rice Roots and Coleoptiles Are Differentially Regulated by a Carotenoid Isomerase-Mediated Abscisic Acid Pathway

Ethylene and abscisic acid (ABA) act synergistically or antagonistically to regulate plant growth and development. ABA is derived from the carotenoid biosynthesis pathway. Here, we analyzed the interplay among ethylene, carotenoid biogenesis, and ABA in rice (Oryza sativa) using the rice ethylene response mutant mhz5, which displays a reduced ethylene response in roots but an enhanced ethylene response in coleoptiles. We found that MHZ5 encodes a carotenoid isomerase and that the mutation in mhz5 blocks carotenoid biosynthesis, reduces ABA accumulation, and promotes ethylene production in etiolated seedlings. ABA can largely rescue the ethylene response of the mhz5 mutant. Ethylene induces MHZ5 expression, the production of neoxanthin, an ABA biosynthesis precursor, and ABA accumulation in roots. MHZ5 overexpression results in enhanced ethylene sensitivity in roots and reduced ethylene sensitivity in coleoptiles. Mutation or overexpression of MHZ5 also alters the expression of ethylene-responsive genes. Genetic studies revealed that the MHZ5-mediated ABA pathway acts downstream of ethylene signaling to inhibit root growth. The MHZ5-mediated ABA pathway likely acts upstream but negatively regulates ethylene signaling to control coleoptile growth. Our study reveals novel interactions among ethylene, carotenogenesis, and ABA and provides insight into improvements in agronomic traits and adaptive growth through the manipulation of these pathways in rice.     

The K+ battery-regulating Arabidopsis K+ channel AKT2 is under the control of multiple post-translational steps

Potassium (K⁺) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K⁺ in plant homeostasis was shown. It was demonstrated that K⁺ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K⁺ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K⁺ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K⁺ channel.Key words: potassium, channel, potassium channel, AKT2, phloem (re)loading, post-translational modifications, potassium batteryPotassium (K⁺) is the most abundant mineral element in plants, and together with nitrogen and phosphorous, is limiting for plant production in many natural and agricultural habitats. Voltage-gated K⁺ channels are key players in the acquisition of K⁺ ions from the soil and in its redistribution within the plant.¹ Structurally, these channels result from the assembly of four so-called α-subunits. The subunits are encoded by nine genes in Arabidopsis and both homo- and hetero-tetramers are expressed.^2,3 The K⁺ channel α-subunits can be categorized into four different subfamilies, based on the voltage-gating characteristics of the exogenous K⁺ conductance when expressed in an appropriate heterologous expression system. K_in α-subunits form hyperpolarization-activated channels that mediate K⁺ uptake.^4–7 K_out α-subunits form depolarization-activated channels that mediate K⁺ release from cells.^8–10 K_silent subunits appear unable to yield functional homomeric channels, but can combine with K_in subunits and fine-tune the K⁺-uptake properties of the resulting heteromeric channels.^11–14 Finally, K_weak α-subunits form channels with complex voltage-gating; they allow both K⁺ uptake and release.^15–19 In Arabidopsis, a single member is found in this subfamily, AKT2, and this channel can assemble in heteromeric channels with the K_in subunit KAT2.²⁰To date, only scarce and speculative information has been obtained for the function of K_weak channels. When expressed in heterologous expression systems, two different subpopulations of AKT2 channels differing in their sensitivity to voltage were found.²¹ Channels of the first type showed gating properties and currents analogous to that of K_in channels, while the other sort enabled a non-rectified (leak-like) current; they were open over the entire physiological voltage range.A given channel can be converted from one type to the other by post-translational modifications.²¹ A voltage-dependent phosphorylation was found to be an essential step for this switch,^22,23 although the kinase responsible for this conversion still needs to be uncovered.²⁴ In biophysical studies, mutant versions of the Arabidopsis K_weak channel subunit AKT2 have been created that showed impaired gating mode settings.^22,23 Recently, Gajdanowicz et al. generated transgenic Arabidopsis thaliana plants that express these mutant AKT2 channels in the background of the akt2-1 null-allele plant.²⁵ The major conclusion from analyses of these mutants is that the status switching of AKT2 from an inward-rectifying to a non-rectifying channel is crucial for plants to overcome energy-limiting conditions. This function of AKT2 could be correlated to its expression in phloem tissues. Selective expression of AKT2 under the control of the phloem companion cell-specific AtSUC2 promoter rescued the akt2-1 line, but conversely, selective expression of AKT2 under the control of the guard cell-specific GC1 promoter,²⁶ resulted in further impairment of plant growth (Fig. 1). By combining diverse experimental approaches with mathematical simulation methods, an existing model for phloem (re)loading^18,27 was fundamentally improved. This allowed the uncovering of a novel and interesting role of K⁺ in phloem physiology: K⁺ gradients present between the sieve element/companion cell (SE/CC) complex and the apoplast can serve as an energy source in phloem (re)loading processes. This “potassium battery” can be tapped by means of AKT2 regulation. This clarifies the observation of Deeken et al.²⁸ that in AKT2 loss-of-function mutant plants, assimilates leaking away from the sieve tube were not efficiently reloaded into the main phloem stream.Open in a separate windowFigure 1AKT2 expressed only in guard cells delays plant development. (A–C) Representative wild-type, akt2-1 and akt2-1+pGC1:AKT2 complementation plants grown for 7 weeks (A), 9 weeks (B) and 12 weeks (C) under 12-h day/12-h night conditions at normal light intensity (150 µmol m⁻² s⁻¹). (D) akt2-1+pGC1:AKT2 developed a similar number of leaves as the akt2-1 knock out plants, but bolting-time was delayed. (B and E) After 9 weeks, wild-type plants were at an advanced bolting stage, akt2-1 plants had started bolting, but only initial signs of bolting were visible in akt2-1+pGC1:AKT2 plants. (C and F) At 12 weeks, akt2-1 plants had caught up with the wild-type and akt2-1+pGC1:AKT2 was just starting to bolt, although rosette-leaves were showing clear signs of senescence. For the generation of akt2-1+pGC1:AKT2, the AKT2 cDNA was fused to the guard cell-specific GC1 promoter²⁶ kindly provided by J.I. Schroeder, San Diego. The pGC1:AKT2 construct was cloned into pGreen0229-35S by replacing the 35S promoter and then transformed into the akt2-1 knockout plant. All seeds were cold-treated for 24 h at 4°C. Plants were grown on artificial substrate (type GS-90, Einheitserde). After 2 weeks, seedlings were transferred to single pots. Plants were grown in 60% relative humidity at 21°C during the day and 18°C at night. Phenotypical analyses were done in the middle of the day. Data are shown as means ± SD of n ≥ 9 plants. Statistical analyses using Student''s t test: (D, WT/akt2-1: p < 2e-08; D, WT/pGC-AKT2: p < 2e-08; D, akt2-1/pGC-AKT2: p < 5e-03; E, WT/akt2-1: p < 4e-06; E, WT/pGC-AKT2: p < 1e-10; E, akt2-1/pGC-AKT2: p < 5e-04; F, WT/akt2-1: p = 0.51; F, WT/pGC-AKT2: p < 1e-10; F, akt2-1/pGC-AKT2: p < 1e-10).AKT2 expression is especially abundant in phloem tissues and the root stele, both of which are characterized by a poor availability of oxygen.^29,30 This local internal hypoxia impairs respiratory activity of the vascular tissue and concomitantly, respiratory ATP production is reduced.³¹ As a consequence, phloem transport is very susceptible to decreasing oxygen supply to the plant.^29,32 It is therefore comprehensible that the above mentioned support by the K⁺ driving force for sucrose retrieval is especially relevant in the phloem. Indeed Gajdanowicz et al.²⁵ showed that transgenic plants lacking the AKT2 K⁺ channel were severely impaired in growth when exposed to mild hypoxia (10% v:v), whereas growth of wild-type plants was unaffected by this treatment. These observations illustrate the importance of biochemical flexibility in plant cells to cope with the energetic consequences of the steep oxygen concentration gradients that generally occur in plant stems and roots.In fact, the role of K⁺ gradients in driving sugar, amino acid and organic acid transport across plant cell membranes was first suggested several decades ago.^33,34 Experimental evidence for this concept was provided by various tests in which pieces of plant tissue were incubated in solutions with different K⁺ concentrations and pH levels.^33,34 Unfortunately, at that time the lack of genetic information to support this hypothesis (e.g., identifying transporter proteins that could provide a molecular mechanism to explain the working mechanism of substrate transport driven by a K⁺-motive force) resulted in this idea falling into oblivion. Indeed, the unequivocal experimental observation of this new role of K⁺ gradients in phloem reloading is extremely challenging. Under normal experimental conditions, K⁺ fluxes and sucrose fluxes are coupled during phloem loading in source tissues and unloading in sink tissues. Nonetheless, computational simulations predict that under certain conditions, a local K⁺/Suc antiport is also thermodynamically possible. In this antiport system, the energy from the K⁺ gradient is used to transport Suc into the phloem. This process is only transient; flooding the apoplast with K⁺ will decrease the K⁺ gradient. However, the gradient can be maintained for longer if surrounding cells take up the apoplastic K⁺ for their own use. A K⁺/Suc antiport will not occur in obvious sink or source tissues since the energy balances in such cells are fundamentally different. Consequently, in these tissues only the coupled symport of K⁺ and Suc can be observed. However, the computational predictions allowed the identification of the experimental conditions under which the effect of the K⁺/Suc antiport system is empirically observable at the whole plant level.An essential role in the regulation of AKT2 is played by (de)phosphorylation events of serine residues at positions S210 and S329. The replacement of both serines by asparagine (AKT2-S210N-S329N) resulted in a K⁺-selective leak that is locked in a continuously open mode when the channels are expressed in Xenopus oocytes. Under certain conditions, plants expressing the AKT2-S210N-S329N mutation showed growth benefits over wild-type plants; akt2-1+AKT2-S210N-S329N plants reach the generative state faster, possess an increased number of leaves and increased fresh weight (Fig. 2). Intuitively, one would expect a continuously open channel to cause severe problems for the plant, not a benefit as was observed here. We therefore have to postulate that phosphorylation at residues AKT2-S210 and AKT2-S329 is insufficient for converting AKT2 from an inward-rectifying into a non-rectifying channel; other, as yet unknown mechanisms, must contribute to the switch in the AKT2 gating mode. Such a concept would correspond to results that would otherwise be hard to explain. For instance, when both serine residues were replaced by glutamate, the mutant AKT2-S210E-S329E still showed wild-type characteristics.²² The S to E substitution is expected to mimic the phosphorylated state better than the S to N replacement. Furthermore, position AKT2-K197 has a fundamental influence on the AKT2 gating mode.²³ AKT2 mutants with that particular lysine substituted with a serine are far less sensitive towards (de)phosphorylation; they display the characteristics of a pure inward-rectifying K⁺ channel,²³ and transgenic Arabidopsis plants expressing AKT2 channels with this substitution showed the characteristics of akt2-1 knock-out plants.²⁵ Initially, it was proposed that the positive charge is important for sensitizing AKT2 to phosphorylation. However, the charge-conserving mutant AKT2-K197R is similar to the charge inverting mutant AKT2-K197D,²³ a purely inward-rectifying channel (Fig. 3). We therefore need to take into account that in plants, K197 may also be a target of post-translational modification.³⁵ At present, we can explain the beneficial effect of the AKT2-S210N-S329N mutant on plant growth only by a multiple step regulation of AKT2 (Fig. 4). The double-N mutation would then bypass the phosphorylation step, but AKT2-S210N-S329N could still be deregulated into an inward-rectifying channel. Thus, AKT2 can be considered as a highly specialized K_in channel that can be converted into a leak-like channel by a cascade of post-translational modification steps.Open in a separate windowFigure 2Plants expressing the AKT2-S210N-S329N mutant reach the generative state faster than wild-type plants. The mutant channel AKT2-S210N-S329N was expressed under the control of the native AKT2 promoter in the akt2-1 knock-out background. (A) Photos of representative Arabidopsis thaliana plants grown 7 weeks under short day conditions (12-h day/12-h night, light intensity = 150 µE m⁻²s⁻¹). Seven weeks after sowing, plants expressing only AKT2-S210N-S329N mutant channels (n = 22) differed significantly (Student''s t test, p < 4e-05) from wild-type plants (n = 20) in the height of the main inflorescent stalk (B) and fresh weight (C). At later time points, these differences decrease.²⁵Open in a separate windowFigure 3The mutant AKT2-K197R channel is inward-rectifying. Steady-state current-voltage characteristics measured at the end of activation voltage steps. Currents were normalized to the current values measured at −145 mV in 10 mM K⁺ and are shown as means ± SD (n = 6).Open in a separate windowFigure 4Minimal model for AKT2 gating-mode regulation. To switch AKT2 from an inward-rectifying into a non-rectifying channel, at least two post-translational steps are postulated. (1) Phosphorylation at residues AKT2-S210 and AKT2-S329 (transitions [1]→[2] and [3]→[4]) and (2) a yet unknown modification that most likely involves the residue AKT2-K197 (transitions [1]→[3] and [2]→[4]). Only after both modifications will AKT2 allow the efflux of K⁺ (state [4]).     

ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis in the succulent xerophyte Zygophyllum xanthoxylum

The inward‐rectifying K⁺ channel AKT1 constitutes an important pathway for K⁺ acquisition in plant roots. In glycophytes, excessive accumulation of Na⁺ is accompanied by K⁺ deficiency under salt stress. However, in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse environments, K⁺ concentration remains at a relatively constant level despite increased levels of Na⁺ under salinity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K⁺ and Na⁺ homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K⁺‐uptake‐defective phenotype of yeast strain CY162, suppressed the salt‐sensitive phenotype of yeast strain G19, and complemented the low‐K⁺‐sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an inward‐rectifying K⁺ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1‐silenced plants exhibited stunted growth compared to wild‐type Z. xanthoxylum. Further experiments showed that ZxAKT1‐silenced plants exhibited a significant decline in net uptake of K⁺ and Na⁺, resulting in decreased concentrations of K⁺ and Na⁺, as compared to wild‐type Z. xanthoxylum grown under 50 mm NaCl. Compared with wild‐type, the expression levels of genes encoding several transporters/channels related to K⁺/Na⁺ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a ZxAKT1‐silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K⁺ uptake but also functions in modulating Na⁺ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal growth.     

The rectification control and physiological relevance of potassium channel OsAKT2

AKT2 potassium (K⁺) channels are members of the plant Shaker family which mediate dual-directional K⁺ transport with weak voltage-dependency. Here we show that OsAKT2 of rice (Oryza sativa) functions mainly as an inward rectifier with strong voltage-dependency and acutely suppressed outward activity. This is attributed to the presence of a unique K191 residue in the S4 domain. The typical bi-directional leak-like property was restored by a single K191R mutation, indicating that this functional distinction is an intrinsic characteristic of OsAKT2. Furthermore, the opposite R195K mutation of AtAKT2 changed the channel to an inward-rectifier similar to OsAKT2. OsAKT2 was modulated by OsCBL1/OsCIPK23, evoking the outward activity and diminishing the inward current. The physiological relevance in relation to the rectification diversity of OsAKT2 was addressed by functional assembly in the Arabidopsis (Arabidopsis thaliana) akt2 mutant. Overexpression (OE) of OsAKT2 complemented the K⁺ deficiency in the phloem sap and leaves of the mutant plants but did not significantly contribute to the transport of sugars. However, the expression of OsAKT2-K191R overcame both the shortage of phloem K⁺ and sucrose of the akt2 mutant, which was comparable to the effects of the OE of AtAKT2, while the expression of the inward mutation AtAKT2-R195K resembled the effects of OsAKT2. Additionally, OE of OsAKT2 ameliorated the salt tolerance of Arabidopsis.

The presence of a unique K191 residue retains the activity of rice potassium channel OsAKT2 mainly as an inward rectifier (Mode I) that emphasizes its in planta role of phloem K⁺ translocation.     

The H+-ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis

A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H⁺-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.     

Arabidopsis Transporter MGT6 Mediates Magnesium Uptake and Is Required for Growth under Magnesium Limitation

Although magnesium (Mg²⁺) is the most abundant divalent cation in plant cells, little is known about the mechanism of Mg²⁺ uptake by plant roots. Here, we report a key function of Magnesium Transport6 (MGT6)/Mitochondrial RNA Splicing2-4 in Mg²⁺ uptake and low-Mg²⁺ tolerance in Arabidopsis thaliana. MGT6 is expressed mainly in plant aerial tissues when Mg²⁺ levels are high in the soil or growth medium. Its expression is highly induced in the roots during Mg²⁺ deficiency, suggesting a role for MGT6 in response to the low-Mg²⁺ status in roots. Silencing of MGT6 in transgenic plants by RNA interference (RNAi) resulted in growth retardation under the low-Mg²⁺ condition, and the phenotype was restored to normal growth after RNAi plants were transferred to Mg²⁺-sufficient medium. RNAi plants contained lower levels of Mg²⁺ compared with wild-type plants under low Mg²⁺ but not under Mg²⁺-sufficient conditions. Further analysis indicated that MGT6 was localized in the plasma membrane and played a key role in Mg²⁺ uptake by roots under Mg²⁺ limitation. We conclude that MGT6 mediates Mg²⁺ uptake in roots and is required for plant adaptation to a low-Mg²⁺ environment.     

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress

Drought is a major threat to plant growth and crop productivity. Calcium-dependent protein kinases (CDPKs, CPKs) are believed to play important roles in plant responses to drought stress. Here, we report that Arabidopsis thaliana CPK8 functions in abscisic acid (ABA)- and Ca²⁺-mediated plant responses to drought stress. The cpk8 mutant was more sensitive to drought stress than wild-type plants, while the transgenic plants overexpressing CPK8 showed enhanced tolerance to drought stress compared with wild-type plants. ABA-, H₂O₂-, and Ca²⁺-induced stomatal closing were impaired in cpk8 mutants. Arabidopsis CATALASE3 (CAT3) was identified as a CPK8-interacting protein, confirmed by yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation assays. CPK8 can phosphorylate CAT3 at Ser-261 and regulate its activity. Both cpk8 and cat3 plants showed lower catalase activity and higher accumulation of H₂O₂ compared with wild-type plants. The cat3 mutant displayed a similar drought stress-sensitive phenotype as cpk8 mutant. Moreover, ABA and Ca²⁺ inhibition of inward K⁺ currents were diminished in guard cells of cpk8 and cat3 mutants. Together, these results demonstrated that CPK8 functions in ABA-mediated stomatal regulation in responses to drought stress through regulation of CAT3 activity.     

Voltage-Sensor Transitions of the Inward-Rectifying K+ Channel KAT1 Indicate a Latching Mechanism Biased by Hydration within the Voltage Sensor

The Kv-like (potassium voltage-dependent) K⁺ channels at the plasma membrane, including the inward-rectifying KAT1 K⁺ channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K⁺ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K⁺ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 α-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.Plant cells utilize the potassium ion (K⁺) to maintain hydrostatic (turgor) pressure, to drive irreversible cell expansion for growth, and to facilitate reversible changes in cell volume during stomatal movements. Potassium uptake and its circulation throughout the plant relies both on high-affinity, H⁺-coupled K⁺ transport (Quintero and Blatt, 1997; Rubio et al., 2008) and on K⁺ channels to facilitate K⁺ ion transfer across cell membranes. Uptake via K⁺ channels is thought to be responsible for roughly 50% of the total K⁺ content of the plant under most field conditions (Spalding et al., 1999; Rubio et al., 2008; Amtmann and Blatt, 2009). K⁺ channels confer on the membranes of virtually every tissue distinct K⁺ conductances and regulatory characteristics (Véry and Sentenac, 2003; Dreyer and Blatt, 2009). Their characteristics are thus of interest for engineering directed to manipulating K⁺ flux in many aspects of plant growth and cellular homeostasis. The control of K⁺ channel gating has been identified as the most promising target for the genetic engineering of stomatal responsiveness (Lawson and Blatt, 2014; Wang et al., 2014a), based on the recent development of quantitative systems models of guard cell transport and metabolism (Chen et al., 2012b; Hills et al., 2012; Wang et al., 2012). By contrast, modifying the expression and, most likely, the population of native K⁺ channels at the membrane was found to have no substantial effect on stomatal physiology (Wang et al., 2014b).The Kv-like K⁺ channels of the plant plasma membrane (Pilot et al., 2003; Dreyer and Blatt, 2009) share a number of structural features with the Kv superfamily of K⁺ channels characterized in animals and Drosophila melanogaster (Papazian et al., 1987; Pongs et al., 1988). The functional channels assemble from four homologous subunits and surround a central transmembrane pore that forms the permeation pathway (Daram et al., 1997). Each subunit comprises six transmembrane α-helices, designated S1 to S6, and both N and C termini are situated on the cytosolic side of the membrane (Uozumi et al., 1998). The pore or P loop between the S5 and S6 α-helices incorporates a short α-helical stretch and the highly conserved amino acid sequence TxGYGD, which forms a selectivity filter for K⁺ (Uozumi et al., 1995; Becker et al., 1996; Nakamura et al., 1997). The carbonyl oxygen atoms of these residues in all four K⁺ channel subunits face inward to form coordination sites for K⁺ ions between them (Doyle et al., 1998; Jiang et al., 2003; Kuo et al., 2003; Long et al., 2005) and a multiple-ion pore (Thiel and Blatt, 1991) such that K⁺ ions pass through the selectivity filter as if in free solution. The plant channels are also sensitive to a class of neurotoxins that exhibit high specificity in binding around the mouth of the channel pore (Obermeyer et al., 1994).These K⁺ channels also share a common gating mechanism. Within each subunit, the first four α-helices form a quasiindependent unit, the voltage sensor domain (VSD), with the S4 α-helix incorporating positively charged (Arg or Lys) residues regularly positioned across the lipid bilayer and transmembrane electric field. Voltage displaces the S4 α-helix within the membrane and couples rotation of the S5 and S6 α-helices lining the pore, thereby opening or closing the channel (Sigworth, 2003; Dreyer and Blatt, 2009). For outward-rectifying channels, such as the mammalian Kv1.2 and the D. melanogaster Shaker K⁺ channels, an inside-positive electric field drives the positively charged, S4 α-helix outward (the up position), which draws on the S4-S5 linker to open the pore. This simple expedient of a lever and string secures current flow in one direction by favoring opening at positive, but not negative, voltages. This same model applies to the Arabidopsis (Arabidopsis thaliana) Kv-like K⁺ channels, including outward rectifiers that exhibit sensitivity to external K⁺ concentration (Blatt, 1988; Blatt and Gradmann, 1997; Johansson et al., 2006), and it serves equally in the gating of inward-rectifying K⁺ channels such as KAT1, which gates open at negative voltages (Dreyer and Blatt, 2009).Studies of KAT1 gating (Latorre et al., 2003; Lai et al., 2005) have indicated that the S4 α-helix of the channel most likely undergoes very similar conformational changes with voltage as those of the mammalian and Shaker K⁺ channels. These findings conform with the present understanding of the evolution of VSD structure (Palovcak et al., 2014) and the view of a common functional dynamic to its molecular design. It is likely, therefore, that a similar electrostatic network occurs in KAT1 to stabilize the VSD. Crucially, however, experimental evidence in support of such a network has yet to surface. Electrostatic countercharges and the hydration of amino acid side chains between the α-helices within the VSDs of mammalian and Shaker K⁺ channel models are important for the latch-like stabilization of the so-called down and up states of these channels (Tao et al., 2010; Pless et al., 2011). Nonetheless, some studies (Gajdanowicz et al., 2009; Riedelsberger et al., 2010) have pointed to subtle differences in the structure of KAT1 that relate to the VSD.We have explored the electrostatic network of the KAT1 VSD through site-directed mutagenesis to manipulate the voltage dependence of KAT1, combining these studies with molecular dynamic simulations previously shown to accommodate the plant VSDs and their hydration during gating transitions (Gajdanowicz et al., 2009; Garcia-Mata et al., 2010). We report here that gating of KAT1 is sensitive to manipulations affecting a set of electrostatic charge transfer centers. These findings conform in large measure to the mammalian and Shaker models. However, virtually all manipulations affecting a highly conserved, central Phe favor the up state of the VSD and the closed KAT1 channel, whereas mutations affecting the electrostatic networks on either side of this Phe favor the down state of the VSD and the open channel. These and additional observations suggest that hydration within the VSD is a major determinant of KAT1 gating.     

MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression.