Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

ABA INSENSITIVE 2 promotes flowering by inhibiting OST1/ABI5-dependent FLOWERING LOCUS C transcription in Arabidopsis.

The plant hormone abscisic acid (ABA) is an important regulator of plant growth and development and plays a crucial role in both biotic and abiotic stress responses. ABA modulates flowering time, but the precise molecular mechanism remains poorly understood. Here we report that ABA INSENSITIVE 2 (ABI2) is the only phosphatase from the ABA-signaling core that positively regulates the transition to flowering in Arabidopsis. Loss-of-function abi2-2 mutant shows significantly delayed flowering both under long day and short day conditions. Expression of floral repressor genes such as FLOWERING LOCUS C (FLC) and CYCLING DOF FACTOR 1 (CDF1) was significantly up-regulated in abi2-2 plants while expression of the flowering promoting genes FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was down-regulated. Through genetic interactions we further found that ost1-3 and abi5-1 mutations are epistatic to abi2-2, as both of them individually rescued the late flowering phenotype of abi2-2. Interestingly, phosphorylation and protein stability of ABA INSENSITIVE 5 (ABI5) were enhanced in abi2-2 plants suggesting that ABI2 dephosphorylates ABI5, thereby reducing protein stability and the capacity to induce FLC expression. Our findings uncovered the unexpected role of ABI2 in promoting flowering by inhibiting ABI5-mediated FLC expression in Arabidopsis.

Structure-Guided Identification of Critical Residues in the Vacuolar Cation/Proton Antiporter NHX1 from Arabidopsis thaliana.

Cation/Proton Antiporters (CPA) acting in all biological membranes regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here, we present an updated and improved structural model of the tonoplast-localized K+, Na+/H+ antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is essential for the accumulation of K+ into plant vacuoles. Conserved residues that were judged as functionally important were mutated, and the resulting protein variants were tested for activity in the yeast Saccharomyces cerevisiae. The results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences for their activity. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with a differential ability to control the vacuolar pH of the yeast.

A salt stress-activated GSO1-SOS2-SOS1 module protects the Arabidopsis root stem cell niche by enhancing sodium ion extrusion.

Soil salinity impairs plant growth reducing crop productivity. Toxic accumulation of sodium ions is counteracted by the Salt Overly Sensitive (SOS) pathway for Na+ extrusion, comprising the Na+ transporter SOS1, the kinase SOS2, and SOS3 as one of several Calcineurin-B-like (CBL) Ca2 + sensors. Here, we report that the receptor-like kinase GSO1/SGN3 activates SOS2, independently of SOS3 binding, by physical interaction and phosphorylation at Thr16. Loss of GSO1 function renders plants salt sensitive and GSO1 is both sufficient and required for activating the SOS2-SOS1 module in yeast and in planta. Salt stress causes the accumulation of GSO1 in two specific and spatially defined areas of the root tip: in the endodermis section undergoing Casparian strip (CS) formation, where it reinforces the CIF-GSO1-SGN1 axis for CS barrier formation; and in the meristem, where it creates the GSO1-SOS2-SOS1 axis for Na+ detoxification. Thus, GSO1 simultaneously prevents Na+ both from diffusing into the vasculature, and from poisoning unprotected stem cells in the meristem. By protecting the meristem, receptor-like kinase-conferred activation of the SOS2-SOS1 module allows root growth to be maintained in adverse environments.

Salinity-Induced Cytosolic Alkaline Shifts in Arabidopsis Roots Require the SOS Pathway.

Plants have evolved elaborate mechanisms to sense, respond to and overcome the detrimental effects of high soil salinity. The role of calcium transients in salinity stress signaling is well established, but the physiological significance of concurrent salinity-induced changes in cytosolic pH remains largely undefined. Here, we analyzed the response of Arabidopsis roots expressing the genetically encoded ratiometric pH-sensor pHGFP fused to marker proteins for the recruitment of the sensor to the cytosolic side of the tonoplast (pHGFP-VTI11) and the plasma membrane (pHGFP-LTI6b). Salinity elicited a rapid alkalinization of cytosolic pH (pHcyt) in the meristematic and elongation zone of wild-type roots. The pH-shift near the plasma membrane preceded that at the tonoplast. In pH-maps transversal to the root axis, the epidermis and cortex had cells with a more alkaline pHcyt relative to cells in the stele in control conditions. Conversely, seedlings treated with 100 mM NaCl exhibited an increased pHcyt in cells of the vasculature relative to the external layers of the root, and this response occurred in both reporter lines. These pHcyt changes were substantially reduced in mutant roots lacking a functional SOS3/CBL4 protein, suggesting that the operation of the SOS pathway mediated the dynamics of pHcyt in response to salinity.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

The Na+/H+ antiporter SALT OVERLY SENSITIVE 1 regulates salt compensation of circadian rhythms by stabilizing GIGANTEA in Arabidopsis.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.

The phosphoinositide PI(3,5)P2 inhibits the activity of plant NHX proton/potassium antiporters: Advantages of a novel electrophysiological approach.

In the present work, we discuss the way in which the parallel application of the patch-clamp technique and the 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence detection for recording luminal proton changes allows the functional characterization of nonelectrogenic potassium/proton vacuolar antiporters of the NHX (Na+/H+ exchanger) family. Moreover, we review the functional role of the tonoplast-specific phosphoinositide PI(3,5)P2, able to simultaneously inhibit the activity of NHXs and CLC-a transporters, whose coordinated action can play an important role in the water balance of plant cells.

Non-Expresser of PR-Genes 1 Positively Regulates Abscisic Acid Signaling in Arabidopsis thaliana.

The plant hormone, abscisic acid (ABA), is not only important for promoting abiotic stress responses but also plays a versatile and crucial role in plant immunity. The pathogen infection-induced dynamic accumulation of ABA mediates the degradation of non-expresser of PR genes 1 (NPR1) through the CUL3NPR3NPR4 proteasome pathway. However, the functional significance of NPR1 degradation by other E3 ligases in response to ABA remains unclear. Here, we report that NPR1 is induced transcriptionally by ABA and that npr1-1 mutation results in ABA insensitivity during seed germination and seedling growth. Mutants lacking NPR1 downregulate the expression of ABA-responsive transcription factors ABA INSENSITIVE4 (ABI4) and ABA INSENSITIVE5 (ABI5), and that of their downstream targets EM6, RAB18, RD26, and RD29B. The npr1-1 mutation also affects the transcriptional activity of WRKY18, which activates WRKY60 in the presence of ABA. Furthermore, NPR1 directly interacts with and is degraded by HOS15, a substrate receptor for the DDB1-CUL4 ubiquitin E3 ligase complex. Collectively, our findings demonstrate that NPR1 acts as a positive regulator of ABA-responsive genes, whereas HOS15 promotes NPR1 degradation in a proteasome-dependent manner.

Negative regulation of floral transition in Arabidopsis by HOS15-PWR-HDA9 complex.

Arabidopsis HOS15/PWR/HDA9 repressor complex, which is similar to the TBL1/NcoR1/HDAC complex in animals, plays a well-known role in epigenetic regulation. PWR and HDA9 have been reported to interact with each other and modulate the flowering time by repressing AGL19 expression, whereas HOS15 and HDA9, together with the photoperiodic evening complex, regulate flowering time through repression of GI transcription. However, the role of the HOS15/PWR/HDA9 core repressor complex as a functional unit in the regulation of flowering time is yet to be explored. In this study, we reported that the loss-of-function hos15-2/pwr/hda9 triple mutant accumulates higher transcript levels of AGL19 and exhibits an early flowering phenotype similar to those of hos15, pwr, and hda9 single mutants. Interestingly, the accumulation of HOS15 in the nucleus was drastically reduced in pwr and hda9 mutants. As a result, HOS15 could not perform its role in histone deacetylation or interaction with H3 in the nucleus. Furthermore, HOS15 is also associated with the same region of the AGL19 promoter known for PWR-HDA9 binding. The acetylation level of the AGL19 promoter was increased in the hos15-2 mutant, similar to the pwr and hda9 mutants. Therefore, our findings reveal that the HOS15/PWR/HDA9 repressor complex deacetylates the promoter region of AGL19, thereby negatively regulating AGL19 transcription, which leads to early flowering in Arabidopsis.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3.

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7-11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

The Arabidopsis protein NPF6.2/NRT1.4 is a plasma membrane nitrate transporter and a target of protein kinase CIPK23.

Nitrate and potassium nutrition is tightly coordinated in vascular plants. Physiological and molecular genetics studies have demonstrated that several NPF/NRT1 nitrate transporters have a significant impact on both uptake and the root-shoot partition of these nutrients. However, how these traits are biochemically connected remain controversial since some NPF proteins, e.g. NPF7.3/NRT1.5, have been suggested to mediate K+/H+ exchange instead of nitrate fluxes. Here we show that NPF6.2/NRT1.4, a protein that gates nitrate accumulation at the leaf petiole of Arabidopsis thaliana, also affects the root/shoot distribution of potassium. We demonstrate that NPF6.2/NRT1.4 is a plasma membrane nitrate transporter phosphorylated at threonine-98 by the CIPK23 protein kinase that is a regulatory hub for nitrogen and potassium nutrition. Heterologous expression of NPF6.2/NRT1.4 and NPF7.3/NRT1.5 in yeast mutants with altered potassium uptake and efflux systems showed no evidence of nitrate-dependent potassium transport by these proteins.

Insights into the mechanisms of transport and regulation of the arabidopsis high-affinity K+ transporter HAK51.

The high-affinity K+ transporter HAK5 from Arabidopsis (Arabidopsis thaliana) is essential for K+ acquisition and plant growth at low micromolar K+ concentrations. Despite its functional relevance in plant nutrition, information about functional domains of HAK5 is scarce. Its activity is enhanced by phosphorylation via the AtCIPK23/AtCBL1-9 complex. Based on the recently published three-dimensionalstructure of the bacterial ortholog KimA from Bacillus subtilis, we have modeled AtHAK5 and, by a mutational approach, identified residues G67, Y70, G71, D72, D201, and E312 as essential for transporter function. According to the structural model, residues D72, D201, and E312 may bind K+, whereas residues G67, Y70, and G71 may shape the selective filter for K+, which resembles that of K+shaker-like channels. In addition, we show that phosphorylation of residue S35 by AtCIPK23 is required for reaching maximal transport activity. Serial deletions of the AtHAK5 C-terminus disclosed the presence of an autoinhibitory domain located between residues 571 and 633 together with an AtCIPK23-dependent activation domain downstream of position 633. Presumably, autoinhibition of AtHAK5 is counteracted by phosphorylation of S35 by AtCIPK23. Our results provide a molecular model for K+ transport and describe CIPK-CBL-mediated regulation of plant HAK transporters.

The role of PQL genes in response to salinity tolerance in Arabidopsis and barley.

While soil salinity is a global problem, how salt enters plant root cells from the soil solution remains underexplored. Non-selective cation channels (NSCCs) are suggested to be the major pathway for the entry of sodium ions (Na+), yet their genetic constituents remain unknown. Yeast PQ loop (PQL) proteins were previously proposed to encode NSCCs, but the role of PQLs in plants is unknown. The hypothesis tested in this research is that PQL proteins constitute NSCCs mediating some of the Na+ influx into the root, contributing to ion accumulation and the inhibition of growth in saline conditions. We identified plant PQL homologues, and studied the role of one clade of PQL genes in Arabidopsis and barley. Using heterologous expression of AtPQL1a and HvPQL1 in HEK293 cells allowed us to resolve sizable inwardly directed currents permeable to monovalent cations such as Na+, K+, or Li+ upon membrane hyperpolarization. We observed that GFP-tagged PQL proteins localized to intracellular membrane structures, both when transiently over-expressed in tobacco leaf epidermis and in stable Arabidopsis transformants. Expression of AtPQL1a, AtPQL1b, and AtPQL1c was increased by salt stress in the shoot tissue compared to non-stressed plants. Mutant lines with altered expression of AtPQL1a, AtPQL1b, and AtPQL1c developed larger rosettes in saline conditions, while altered levels of AtPQL1a severely reduced development of lateral roots in all conditions. This study provides the first step toward understanding the function of PQL proteins in plants and the role of NSCC in salinity tolerance.

Beyond the patch-clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides.

We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. Upon application of a steep cytosol-directed potassium ion (K+ ) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+ ) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P2 , which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof-of-principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

HKT sodium and potassium transporters in Arabidopsis thaliana and related halophyte species.

High salinity induces osmotic stress and often leads to sodium ion-specific toxicity, with inhibitory effects on physiological, biochemical and developmental pathways. To cope with increased Na+ in soil water, plants restrict influx, compartmentalize ions into vacuoles, export excess Na+ from the cell, and distribute ions between the aerial and root organs. In this review, we discuss our current understanding of how high-affinity K+ transporters (HKT) contribute to salinity tolerance, focusing on HKT1-like family members primarily involved in long-distance transport, and in the recent research in the model plant Arabidopsis and its halophytic counterparts of the Eutrema genus. Functional characterization of the salt overly sensitive (SOS) pathway and HKT1-type transporters in these species indicate that they utilize similar approaches to deal with salinity, regardless of their tolerance.

HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity.

Transcriptional regulation is a complex and pivotal process in living cells. HOS15 is a transcriptional corepressor. Although transcriptional repressors generally have been associated with inactive genes, increasing evidence indicates that, through poorly understood mechanisms, transcriptional corepressors also associate with actively transcribed genes. Here, we show that HOS15 is the substrate receptor for an SCF/CUL1 E3 ubiquitin ligase complex (SCFHOS15) that negatively regulates plant immunity by destabilizing transcriptional activation complexes containing NPR1 and associated transcriptional activators. In unchallenged conditions, HOS15 continuously eliminates NPR1 to prevent inappropriate defense gene expression. Upon defense activation, HOS15 preferentially associates with phosphorylated NPR1 to stimulate rapid degradation of transcriptionally active NPR1 and thus limit the extent of defense gene expression. Our findings indicate that HOS15-mediated ubiquitination and elimination of NPR1 produce effects contrary to those of CUL3-containing ubiquitin ligase that coactivate defense gene expression. Thus, HOS15 plays a key role in the dynamic regulation of pre- and postactivation host defense.

The Histone-Modifying Complex PWR/HOS15/HD2C Epigenetically Regulates Cold Tolerance.

Cold stress is a major environmental stress that severely affects plant growth and crop productivity. Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15) is a substrate receptor of the CULLIN4-based CLR4 ubiquitin E3 ligase complex, which epigenetically regulates cold tolerance by degrading HISTONE DEACETYLASE2C (HD2C) to switch from repressive to permissive chromatin structure in response to cold stress. In this study, we characterized a HOS15-binding protein, POWERDRESS (PWR), and analyzed its function in the cold stress response. PWR loss-of-function plants (pwr) showed lower expression of cold-regulated (COR) genes and sensitivity to freezing. PWR interacts with HD2C through HOS15, and cold-induced HD2C degradation by HOS15 is diminished in the pwr mutant. The association of HOS15 and HD2C to promoters of cold-responsive COR genes was dependent on PWR. Consistent with these observations, the high acetylation levels of histone H3 by cold-induced and HOS15-mediated HD2C degradation were significantly reduced in pwr under cold stress. PWR also interacts with C-repeat element-binding factor transcription factors to modulate their cold-induced binding to the promoter of COR genes. Collectively, our data signify that the PWR-HOS15-HD2C histone-modifying complex regulates the expression of COR genes and the freezing tolerance of plants.