ABSTRACT
The maintenance of sodium/potassium (Na+ /K+ ) homeostasis in plant cells is essential for salt tolerance. Plants export excess Na+ out of cells mainly through the Salt Overly Sensitive (SOS) pathway, activated by a calcium signal; however, it is unknown whether other signals regulate the SOS pathway and how K+ uptake is regulated under salt stress. Phosphatidic acid (PA) is emerging as a lipid signaling molecule that modulates cellular processes in development and the response to stimuli. Here, we show that PA binds to the residue Lys57 in SOS2, a core member of the SOS pathway, under salt stress, promoting the activity and plasma membrane localization of SOS2, which activates the Na+ /H+ antiporter SOS1 to promote the Na+ efflux. In addition, we reveal that PA promotes the phosphorylation of SOS3-like calcium-binding protein 8 (SCaBP8) by SOS2 under salt stress, which attenuates the SCaBP8-mediated inhibition of Arabidopsis K+ transporter 1 (AKT1), an inward-rectifying K+ channel. These findings suggest that PA regulates the SOS pathway and AKT1 activity under salt stress, promoting Na+ efflux and K+ influx to maintain Na+ /K+ homeostasis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Protein Serine-Threonine Kinases , Salt Stress , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Homeostasis , Phosphatidic Acids/metabolism , Potassium/metabolism , Protein Serine-Threonine Kinases/metabolism , Salt Stress/genetics , Sodium/metabolismABSTRACT
Membrane properties are emerging as important cues for the spatiotemporal regulation of hormone signaling. Lysophosphatidic acid (LPA) evokes multiple biological responses by activating G protein-coupled receptors in mammals. In this study, we demonstrated that LPA derived from the mitochondrial glycerol-3-phosphate acyltransferases GPAT1 and GPAT2 is a critical lipid-based cue for auxin-controlled embryogenesis and plant growth in Arabidopsis thaliana. LPA levels decreased, and the polarity of the auxin efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane (PM) was defective in the gpat1 gpat2 mutant. As a consequence of distribution defects, instructive auxin gradients and embryonic and postembryonic development are severely compromised. Further cellular and genetic analyses revealed that LPA binds directly to PIN1, facilitating the vesicular trafficking of PIN1 and polar auxin transport. Our data support a model in which LPA provides a lipid landmark that specifies membrane identity and cell polarity, revealing an unrecognized aspect of phospholipid patterns connecting hormone signaling with development.
Subject(s)
Arabidopsis , Indoleacetic Acids , Animals , Lysophospholipids , Arabidopsis/genetics , Plant Development , MammalsABSTRACT
Salt stress impairs nutrient metabolism in plant cells, leading to growth and yield penalties. However, the mechanism by which plants alter their nutrient metabolism processes in response to salt stress remains elusive. In this study, we identified and characterized the rice (Oryza sativa) rice salt tolerant 1 (rst1) mutant, which displayed improved salt tolerance and grain yield. Map-based cloning revealed that the gene RST1 encoded an auxin response factor (OsARF18). Molecular analyses showed that RST1 directly repressed the expression of the gene encoding asparagine synthetase 1 (OsAS1). Loss of RST1 function increased the expression of OsAS1 and improved nitrogen (N) utilization by promoting asparagine production and avoiding excess ammonium (NH4+) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1Hap III decreased its transcriptional repression activity and contributed to salt tolerance and grain weight. Together, our findings unravel a synergistic regulator of growth and salt tolerance associated with N metabolism and provide a new strategy for the development of tolerant cultivars.
Subject(s)
Aspartate-Ammonia Ligase , Oryza , Salt Tolerance/genetics , Oryza/genetics , Aspartate-Ammonia Ligase/genetics , Gene ExpressionABSTRACT
High-affinity K+ transporters/K+ uptake permeases/K+ transporters (HAK/KUP/KT) are important pathways mediating K+ transport across cell membranes, which function in maintaining K+ homeostasis during plant growth and stress response. An increasing number of studies have shown that HAK/KUP/KT transporters play crucial roles in root K+ uptake and root-to-shoot translocation. However, whether HAK/KUP/KT transporters also function in phloem K+ translocation remain unclear. In this study, we revealed that a phloem-localized rice HAK/KUP/KT transporter, OsHAK18, mediated cell K+ uptake when expressed in yeast, Escherichia coli and Arabidopsis. It was localized at the plasma membrane. Disruption of OsHAK18 rendered rice seedlings insensitive to low-K+ (LK) stress. After LK stress, some WT leaves showed severe wilting and chlorosis, whereas the corresponding leaves of oshak18 mutant lines (a Tos17 insertion line and two CRISPR lines) remained green and unwilted. Compared with WT, the oshak18 mutants accumulated more K+ in shoots but less K+ in roots after LK stress, leading to a higher shoot/root ratio of K+ per plant. Disruption of OsHAK18 does not affect root K+ uptake and K+ level in xylem sap, but it significantly decreases phloem K+ concentration and inhibits root-to-shoot-to-root K+ (Rb+ ) translocation in split-root assay. These results reveal that OsHAK18 mediates phloem K+ loading and redistribution, whose disruption is in favor of shoot K+ retention under LK stress. Our findings expand the understanding of HAK/KUP/KT transporters' functions and provide a promising strategy for improving rice tolerance to K+ deficiency.
Subject(s)
Arabidopsis , Oryza , Oryza/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Potassium/metabolism , Phloem/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Plant Roots/metabolism , Gene Expression Regulation, PlantABSTRACT
Ammonium (NH4+) is a key inorganic nitrogen source in cellular amino acid biosynthesis. The coupling of transcriptional and posttranslational regulation of AMMONIUM TRANSPORTER (AMT) ensures that NH4+ acquisition by plant roots is properly balanced, which allows for rapid adaptation to a variety of nitrogen conditions. Here, we report that phospholipase D (PLD)-derived phosphatidic acid (PA) interacts with AMT1;1 to mediate NH4+ uptake in Arabidopsis (Arabidopsis thaliana). We examined pldα1 pldδ-knockout mutants and found that a reduced PA level increased seedling growth under nitrogen deficiency and inhibited root growth upon NH4+ stress, which was consistent with the enhanced accumulation of cellular NH4+. PA directly bound to AMT1;1 and inhibited its transport activity. Mutation of AMT1;1 R487 to Gly (R487G) resulted in abolition of PA suppression and, subsequently, enhancement of ammonium transport activity in vitro and in vivo. Observations of AMT1;1-GFP showed suppressed endocytosis under PLD deficiency or by mutation of the PA-binding site in AMT1;1. Endocytosis was rescued by PA in the pldα1 pldδ mutant but not in the mutant AMT1;1R487G-GFP line. Together, these findings demonstrated PA-based shutoff control of plant NH4+ transport and point to a broader paradigm of lipid-transporter function.
Subject(s)
Ammonium Compounds , Arabidopsis Proteins , Arabidopsis , Ammonium Compounds/pharmacology , Ammonium Compounds/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Membrane Transport Proteins/metabolism , Nitrogen/metabolism , Phosphatidic Acids/metabolism , Plant Roots/metabolismABSTRACT
Potassium (K+) is an essential element for growth and development in both animals and plants, while high levels of environmental sodium (Na+) represent a threat to most plants. The uptake of K+ from high-saline environments is an essential mechanism to maintain intracellular K+/Na+ homeostasis, which can help reduce toxicity caused by Na+ accumulation, thereby improving the salt tolerance of plants. However, the mechanisms and regulation of K+-uptake during salt stress remain poorly understood. In this study, we identified an endoplasmic reticulum-localized cytochrome b5 (OsCYB5-2) that interacted with a high-affinity K+ transporter (OsHAK21) at the plasma membrane. The association of OsCYB5-2 with the OsHAK21 transporter caused an increase in transporter activity by enhancing the apparent affinity for K+-binding but not Na+-binding. Heme binding to OsCYB5-2 was essential for the regulation of OsHAK21. High salinity directly triggered the OsHAK21-OsCYB5-2 interaction, promoting OsHAK21-mediated K+-uptake and restricting Na+ entry into cells; this maintained intracellular K+/Na+ homeostasis in rice cells. Finally, overexpression of OsCYB5-2 increased OsHAK21-mediated K+ transport and improved salt tolerance in rice seedlings. This study revealed a posttranslational regulatory mechanism for HAK transporter activity mediated by a cytochrome b5 and highlighted the coordinated action of two proteins to perceive Na+ in response to salt stress.
Subject(s)
Cytochromes b/metabolism , Gene Expression Regulation, Plant/drug effects , Oryza/drug effects , Oryza/metabolism , Plant Proteins/metabolism , Sodium/toxicity , Cytochromes b/genetics , Plant Proteins/genetics , Plant Roots , Plant Shoots , Salinity , Salt Stress , SeedlingsABSTRACT
The phosphatidylinositol-specific phospholipase Cs (PI-PLCs) catalyze the hydrolysis of phosphatidylinositols, which play crucial roles in signaling transduction during plant development and stress response. However, the regulation of PI-PLC is still poorly understood. A previous study showed that a rice PI-PLC, OsPLC1, was essential to rice salt tolerance. Here, we identified a 14-3-3 protein, OsGF14b, as an interaction partner of OsPLC1. Similar to OsPLC1, OsGF14b also positively regulates rice salt tolerance, and their interaction can be promoted by NaCl stress. OsGF14b also positively regulated the hydrolysis activity of OsPLC1, and is essential to NaCl-induced activation of rice PI-PLCs. We further discovered that OsPLC1 was degraded via ubiquitin-proteasome pathway, and OsGF14b could inhibit the ubiquitination of OsPLC1 to protect OsPLC1 from degradation. Under salt stress, the OsPLC1 protein level in osgf14b was lower than the corresponding value of WT, whereas overexpression of OsGF14b results in a significant increase of OsPLC1 stability. Taken together, we propose that OsGF14b can interact with OsPLC1 and promote its activity and stability, thereby improving rice salt tolerance. This study provides novel insights into the important roles of 14-3-3 proteins in regulating protein stability and function in response to salt stress.
Subject(s)
Oryza , Salt Tolerance , Salt Tolerance/physiology , 14-3-3 Proteins/metabolism , Oryza/physiology , Sodium Chloride/metabolism , Phosphatidylinositols/metabolism , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Stress, PhysiologicalABSTRACT
KEY MESSAGE: OsCYBDOMG1 positively regulates salt tolerance, plant growth, and grain yield by affecting ascorbate biosynthesis and redox state. Soil salinity is a major abiotic stress affecting rice growth and productivity. Many genes involved in the salt stress response have been identified, but the precise mechanisms underlying salt tolerance remain unclear. In this study, we isolated a salt-sensitive mutant of rice, rss5, which exhibited more severe wilting and chlorosis with a significant increase in lipid peroxidation, electrolyte leakage, and shoot Na+ concentration compared to wild-type plants. Map-based cloning, MutMap analysis, and genetic complementation revealed that a single-nucleotide mutation in a gene encoding a cytochrome b561 domain-containing protein (OsCYBDOMG1) was responsible for the mutant phenotype of rss5. The OsCYBDOMG1 gene was mainly expressed in young shoots and nodes, and the encoded protein was principally located in the plasma membrane and endoplasmic reticulum. Mutations of OsCYBDOMG1 resulted in decreased ascorbic acid (AsA) content and AsA/DHA (dehydroascorbate) ratio, which led to increased H2O2 accumulation and reduced salt tolerance. Moreover, plant growth and grain yield of rss5 and the OsCYBDOMG1 knockout mutant (cr-1) were significantly decreased compared to wild-type plants under normal conditions. The elite haplotype of OsCYBDOMG1 associated with higher salt tolerance and grain width and weight was mainly existed in japonica varieties. These results suggest that OsCYBDOMG1 plays an important role in the regulation of salt tolerance, plant growth, and grain yield in rice.
Subject(s)
Oryza , Salt Tolerance , Salt Tolerance/genetics , Hydrogen Peroxide/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/metabolism , Edible Grain/genetics , Edible Grain/metabolism , Gene Expression Regulation, PlantABSTRACT
Remodeling of auxin distribution during the integration of plant growth responses with the environment requires the precise control of auxin influx and efflux transporters. The plasma membrane-localized PIN-FORMED (PIN) proteins facilitate auxin efflux from cells, and their activity is regulated by reversible phosphorylation. How PIN modulates plant cellular responses to external stresses and whether its activity is coordinated by phospholipids remain unclear. Here, we reveal that, in Arabidopsis (Arabidopsis thaliana), the phosphatidic acid (PA)-regulated PINOID (PID) kinase is a crucial modulator of PIN2 activity and auxin redistribution in response to salt stress. Under salt stress, loss of phospholipase D function impaired auxin redistribution and resulted in markedly reduced primary root growth; these effects were reversed by exogenous PA. The phospholipase D-derived PA interacted with PID and increased PID-dependent phosphorylation of PIN2, which activated auxin efflux and altered auxin accumulation, promoting root growth when exposed to salt stress. Ablation of the PA binding motif not only diminished PID accumulation at the plasma membrane but also abolished PA-promoted PID phosphorylation of PIN2 and its function in coping with salt stress; however, this ablation did not affect inflorescence and cotyledon development or PIN2-dependent gravitropic and halotropic responses. Our data indicate a role for PA in coupling extracellular salt signaling to PID-directed PIN2 phosphorylation and polar auxin transport, highlighting the importance of lipid-protein interactions in the spatiotemporal regulation of auxin signaling.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Phosphatidic Acids/pharmacology , Plant Roots/metabolism , Arabidopsis/drug effects , Indoleacetic Acids/metabolism , Phosphorylation/drug effects , Plant Roots/drug effects , Salt Stress , Signal Transduction/drug effectsABSTRACT
The plant Shaker K+ channel AtAKT2 has been identified as a weakly rectifying channel that can stabilize membrane potentials to promote photoassimilate phloem loading and translocation. Thus, studies on functional characterization and regulatory mechanisms of AtAKT2-like channels in crops are highly important for improving crop production. Here, we identified the rice OsAKT2 as the ortholog of Arabidopsis AtAKT2, which is primarily expressed in the shoot phloem and localized at the plasma membrane. Using an electrophysiological assay, we found that OsAKT2 operated as a weakly rectifying K+ channel, preventing H+ /sucrose-symport-induced membrane depolarization. Three critical amino acid residues (K193, N206, and S326) are essential to the phosphorylation-mediated gating change of OsAKT2, consistent with the roles of the corresponding sites in AtAKT2. Disruption of OsAKT2 results in delayed growth of rice seedlings under short-day conditions. Interestingly, the lipid second messenger phosphatidic acid (PA) inhibits OsAKT2-mediated currents (both instantaneous and time-dependent components). Lipid dot-blot assay and liposome-protein binding analysis revealed that PA directly bound with two adjacent arginine residues in the ANK domain of OsAKT2, which is essential to PA-mediated inhibition of OsAKT2. Electrophysiological and phenotypic analyses also showed the PA-mediated inhibition of AtAKT2 and the negative correlation between intrinsic PA level and Arabidopsis growth, suggesting that PA may inhibit AKT2 function to affect plant growth and development. Our results functionally characterize the Shaker K+ channel OsAKT2 and reveal a direct link between phospholipid signaling and plant K+ channel modulation.
Subject(s)
Arabidopsis/genetics , Oryza/genetics , Phosphatidic Acids/metabolism , Potassium Channels/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Membrane/metabolism , Oryza/metabolism , Potassium Channels/genetics , Seedlings/genetics , Seedlings/metabolismABSTRACT
Rice leaf angle is an important agronomic trait determining plant architecture and crop yield. Brassinosteroids (BRs) play crucial roles in controlling rice leaf angle, thus an increasing number of researches were focused on the BR signaling pathway in rice. However, the orthologs of some important components in Arabidopsis BR signaling have not yet been characterized in rice. In this study, we identified a rice bHLH transcription factor named OsBIM1, as the closest rice homolog of AtBIM1 (BES1-Interacting MYC-like Protein1). Overexpression of OsBIM1 significantly increases rice leaf angles, whereas the T-DNA knock-out mutant osbim1 and wide type (WT) showed similar leaf inclination. OsBIM1 overexpression enhances the sensitivity and response to BR treatment in rice. Gene expression analysis showed that the overexpression of OsBIM1 significantly increased the transcripts of INCREASED LEAF INCLINATION1 (OsILI1) that functions as a key transcription factor promoting BR signaling and response. Meanwhile, OsBIM1 inhibited the expression of DWARF2 (OsD2, a key enzyme in BR biosynthesis pathway). OsBIM1 can bind with OsILI1 promoter and enhance OsILI1 expression in response to BR treatment. The promoting effect of OsBIM1 overexpression on leaf angle can still be observed at harvest stage, but overexpression of OsBIM1 resulted in smaller grain size and reduced yield. These results indicate that OsBIM1 functions as a positive regulator in BR signaling, and its overexpression increases rice lamina inclination by promoting BR sensitivity and response.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Brassinosteroids/metabolism , Oryza/growth & development , Plant Leaves/growth & development , Plant Proteins/metabolism , Genetic Techniques , Oryza/genetics , Oryza/metabolism , Phenotype , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Signal TransductionABSTRACT
Maintaining Na+ /K+ homeostasis is a critical feature for plant survival under salt stress, which depends on the operation of Na+ and K+ transporters. Although some K+ transporters mediating root K+ uptake have been reported to be essential to the maintenance of Na+ /K+ homeostasis, the effect of K+ long-distance translocation via phloem on plant salt tolerance remains unclear. Here, we provide physiological and genetic evidence of the involvement of phloem-localized OsAKT2 in rice salt tolerance. OsAKT2 is a K+ channel permeable to K+ but not to Na+ . Under salt stress, a T-DNA knock-out mutant, osakt2 and two CRISPR lines showed a more sensitive phenotype and higher Na+ accumulation than wild type. They also contained more K+ in shoots but less K+ in roots, showing higher Na+ /K+ ratios. Disruption of OsAKT2 decreases K+ concentration in phloem sap and inhibits shoot-to-root redistribution of K+ . In addition, OsAKT2 also regulates the translocation of K+ and sucrose from old leaves to young leaves, and affects grain shape and yield. These results indicate that OsAKT2-mediated K+ redistribution from shoots to roots contributes to maintenance of Na+ /K+ homeostasis and inhibition of root Na+ uptake, providing novel insights into the roles of K+ transporters in plant salt tolerance.
Subject(s)
Edible Grain/genetics , Oryza/metabolism , Plant Proteins/metabolism , Potassium Channels/metabolism , Potassium/metabolism , Salt Tolerance , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Gene Editing , Gene Knockdown Techniques , Oryza/genetics , Oryza/growth & development , Oryza/physiology , Phloem/metabolism , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/physiology , Plant Roots/metabolism , Plant Shoots/metabolism , Potassium Channels/genetics , Potassium Channels/physiology , Salt Tolerance/genetics , Salt Tolerance/physiologyABSTRACT
BACKGROUND: The anionic toxicity of plants under salt stress is mainly caused by chloride (Cl-). Thus Cl- influx, transport and their regulatory mechanisms should be one of the most important aspects of plant salt tolerance studies, but are often sidelined by the focus on sodium (Na+) toxicity and its associated adaptations. Plant chloride channels (CLCs) are transport proteins for anions including Cl- and nitrate (NO3-), and are critical for nutrition uptake and transport, adjustment of cellular turgor, stomatal movement, signal transduction, and Cl- and NO3- homeostasis under salt stress. RESULTS: Among the eight soybean CLC genes, the tonoplast-localized c2 has uniquely different transcriptional patterns between cultivated soybean N23674 and wild soybean BB52. Using soybean hairy root transformation, we found that GsCLC-c2 over-expression contributed to Cl- and NO3- homeostasis, and therefore conferred salt tolerance, through increasing the accumulation of Cl- in the roots, thereby reducing their transportation to the shoots where most of the cellular damages occur. Also, by keeping relatively high levels of NO3- in the aerial part of the plant, GsCLC-c2 could reduce the Cl-/NO3- ratio. Wild type GsCLC-c2, but not its mutants (S184P, E227V and E294G) with mutations in the conserved domains, is able to complement Saccharomyces cerevisiae â³gef1 Cl- sensitive phenotype. Using two-electrode voltage clamp on Xenopus laevis oocytes injected with GsCLC-c2 cRNA, we found that GsCLC-c2 transports both Cl- and NO3- with slightly different affinity, and the affinity toward Cl- was pH-independent. CONCLUSION: This study revealed that the expression of GsCLC-c2 is induced by NaCl-stress in the root of wild soybean. The tonoplast localized GsCLC-c2 transports Cl- with a higher affinity than NO3- in a pH-independent fashion. GsCLC-c2 probably alleviates salt stress in planta through the sequestration of excess Cl- into the vacuoles of root cells and thus preventing Cl- from entering the shoots where it could result in cellular damages.
Subject(s)
Chloride Channels/metabolism , Chlorides/metabolism , Glycine max/genetics , Biological Transport , Chloride Channels/genetics , Nitrates/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Salinity , Salt Tolerance , Glycine max/physiologyABSTRACT
In Arabidopsis (Arabidopsis thaliana), the Shaker K(+) channel AKT1 conducts K(+) uptake in root cells, and its activity is regulated by CBL1/9-CIPK23 complexes as well as by the AtKC1 channel subunit. CIPK23 and AtKC1 are both involved in the AKT1-mediated low-K(+) (LK) response; however, the relationship between them remains unclear. In this study, we screened suppressors of low-K(+) sensitive [lks1 (cipk23)] and isolated the suppressor of lks1 (sls1) mutant, which suppressed the leaf chlorosis phenotype of lks1 under LK conditions. Map-based cloning revealed a point mutation in AtKC1 of sls1 that led to an amino acid substitution (G322D) in the S6 region of AtKC1. The G322D substitution generated a gain-of-function mutation, AtKC1(D), that enhanced K(+) uptake capacity and LK tolerance in Arabidopsis. Structural prediction suggested that glycine-322 is highly conserved in K(+) channels and may function as the gating hinge of plant Shaker K(+) channels. Electrophysiological analyses revealed that, compared with wild-type AtKC1, AtKC1(D) showed enhanced inhibition of AKT1 activity and strongly reduced K(+) leakage through AKT1 under LK conditions. In addition, phenotype analysis revealed distinct phenotypes of lks1 and atkc1 mutants in different LK assays, but the lks1 atkc1 double mutant always showed a LK-sensitive phenotype similar to that of akt1 This study revealed a link between CIPK-mediated activation and AtKC1-mediated modification in AKT1 regulation. CIPK23 and AtKC1 exhibit distinct effects; however, they act synergistically and balance K(+) uptake/leakage to modulate AKT1-mediated LK responses in Arabidopsis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Potassium Channels/metabolism , Potassium/pharmacology , Protein Serine-Threonine Kinases/metabolism , Shaker Superfamily of Potassium Channels/metabolism , Stress, Physiological/drug effects , Amino Acid Sequence , Arabidopsis/drug effects , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Cloning, Molecular , Ethyl Methanesulfonate , Genetic Complementation Test , Germination/drug effects , Mutagenesis , Mutation/genetics , Phenotype , Plant Shoots/drug effects , Plant Shoots/metabolism , Potassium Channels/chemistry , Potassium Channels/genetics , Sequence AlignmentABSTRACT
Sodium transporters play key roles in plant tolerance to salt stress. Here, we report that a member of the High-Affinity K(+) Transporter (HKT) family, OsHKT1;1, in rice (Oryza sativa 'Nipponbare') plays an important role in reducing Na(+) accumulation in shoots to cope with salt stress. The oshkt1;1 mutant plants displayed hypersensitivity to salt stress. They contained less Na(+) in the phloem sap and accumulated more Na(+) in the shoots compared with the wild type. OsHKT1;1 was expressed mainly in the phloem of leaf blades and up-regulated in response to salt stress. Using a yeast one-hybrid approach, a novel MYB coiled-coil type transcription factor, OsMYBc, was found to bind to the OsHKT1;1 promoter. In vivo chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that OsMYBc binds to AAANATNC(C/T) fragments within the OsHKT1;1 promoter. Mutation of the OsMYBc-binding nucleotides resulted in a decrease in promoter activity of OsHKT1;1. Knockout of OsMYBc resulted in a reduction in NaCl-induced expression of OsHKT1;1 and salt sensitivity. Taken together, these results suggest that OsHKT1;1 has a role in controlling Na(+) concentration and preventing sodium toxicity in leaf blades and is regulated by the OsMYBc transcription factor.
Subject(s)
Cation Transport Proteins/metabolism , Oryza/physiology , Plant Proteins/metabolism , Potassium/metabolism , Salt Tolerance , Transcription Factors/metabolism , Agrobacterium/physiology , Base Sequence , Cation Transport Proteins/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation, Plant/drug effects , Gene Knockout Techniques , Molecular Sequence Data , Mutation/genetics , Oryza/drug effects , Oryza/genetics , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Shoots/drug effects , Plant Shoots/genetics , Plant Shoots/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Salt Tolerance/drug effects , Salt Tolerance/genetics , Sodium/metabolism , Sodium Chloride/pharmacology , Stress, Physiological/drug effects , Nicotiana/microbiologyABSTRACT
Potassium (K(+)) influx into pollen tubes via K(+) transporters is essential for pollen tube growth; however, the mechanism by which K(+) transporters are regulated in pollen tubes remains unknown. Here, we report that Arabidopsis thaliana Ca(2+)-dependent protein kinase11 (CPK11) and CPK24 are involved in Ca(2+)-dependent regulation of the inward K(+) (K(+)in) channels in pollen tubes. Using patch-clamp analysis, we demonstrated that K(+)in currents of pollen tube protoplasts were inhibited by elevated [Ca(2+)]cyt. However, disruption of CPK11 or CPK24 completely impaired the Ca(2+)-dependent inhibition of K(+)in currents and enhanced pollen tube growth. Moreover, the cpk11 cpk24 double mutant exhibited similar phenotypes as the corresponding single mutants, suggesting that these two CDPKs function in the same signaling pathway. Bimolecular fluorescence complementation and coimmunoprecipitation experiments showed that CPK11 could interact with CPK24 in vivo. Furthermore, CPK11 phosphorylated the N terminus of CPK24 in vitro, suggesting that these two CDPKs work together as part of a kinase cascade. Electrophysiological assays demonstrated that the Shaker pollen K(+)in channel is the main contributor to pollen tube K(+)in currents and acts as the downstream target of the CPK11-CPK24 pathway. We conclude that CPK11 and CPK24 together mediate the Ca(2+)-dependent inhibition of K(+)in channels and participate in the regulation of pollen tube growth in Arabidopsis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Pollen Tube/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Protein Kinases/metabolism , Shaker Superfamily of Potassium Channels/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Calcium/metabolism , Mutation , Patch-Clamp Techniques , Phosphorylation , Pollen Tube/genetics , Pollen Tube/growth & development , Protein Kinases/genetics , Protoplasts/metabolism , Shaker Superfamily of Potassium Channels/geneticsABSTRACT
The intracellular potassium (K(+) ) homeostasis, which is crucial for plant survival in saline environments, is modulated by K(+) channels and transporters. Some members of the high-affinity K(+) transporter (HAK) family are believed to function in the regulation of plant salt tolerance, but the physiological mechanisms remain unclear. Here, we report a significant inducement of OsHAK21 expression by high-salinity treatment and provide genetic evidence of the involvement of OsHAK21 in rice salt tolerance. Disruption of OsHAK21 rendered plants sensitive to salt stress. Compared with the wild type, oshak21 accumulated less K(+) and considerably more Na(+) in both shoots and roots, and had a significantly lower K(+) net uptake rate but higher Na(+) uptake rate. Our analyses of subcellular localizations and expression patterns showed that OsHAK21 was localized in the plasma membrane and expressed in xylem parenchyma and individual endodermal cells (putative passage cells). Further functional characterizations of OsHAK21 in K(+) uptake-deficient yeast and Arabidopsis revealed that OsHAK21 possesses K(+) transporter activity. These results demonstrate that OsHAK21 may mediate K(+) absorption by the plasma membrane and play crucial roles in the maintenance of the Na(+) /K(+) homeostasis in rice under salt stress.
Subject(s)
Cation Transport Proteins/metabolism , Gene Expression Regulation, Plant , Oryza/genetics , Potassium/metabolism , Arabidopsis/genetics , Arabidopsis/physiology , Cation Transport Proteins/genetics , Cell Membrane/metabolism , Homeostasis , Ion Transport , Mutation , Oryza/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/physiology , Plant Shoots/genetics , Plant Shoots/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Salinity , Salt Tolerance , Sodium/metabolism , Sodium Chloride/metabolism , Xylem/genetics , Xylem/physiologyABSTRACT
Although there has been long-standing recognition that stimuli-induced cytosolic pH alterations coincide with changes in calcium ion (Ca2+) levels, the interdependence between protons (H+) and Ca2+ remains poorly understood. We addressed this topic using the light-gated channelrhodopsin HcKCR2 from the pseudofungus Hyphochytrium catenoides, which operates as a H+ conductive, Ca2+ impermeable ion channel on the plasma membrane of plant cells. Light activation of HcKCR2 in Arabidopsis guard cells evokes a transient cytoplasmic acidification that sparks Ca2+ release from the endoplasmic reticulum. A H+-induced cytosolic Ca2+ signal results in membrane depolarization through the activation of Ca2+-dependent SLAC1/SLAH3 anion channels, which enabled us to remotely control stomatal movement. Our study suggests a H+-induced Ca2+ release mechanism in plant cells and establishes HcKCR2 as a tool to dissect the molecular basis of plant intracellular pH and Ca2+ signaling.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Calcium Signaling , Calcium , Channelrhodopsins , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Calcium/metabolism , Channelrhodopsins/genetics , Channelrhodopsins/metabolism , Plant Stomata/metabolism , Protons , Rhinosporidium , Hydrogen-Ion ConcentrationABSTRACT
Phosphatidic acid (PA) forms part of plant lipid metabolism and is a signalling molecule used in response to various external stresses. Guanine nucleotide exchange factors (GEFs) activate small GTPase ROPs, serving as molecular switches in a wide range of signalling pathways. However, the interaction between PA and GEFs in plants has not yet been reported. Here we show that PA bound specifically to GEF8 by using fat-Western blot and isothermal titration calorimetry assays. A C-terminal truncation of GEF8 exhibited strong PA binding, and mutation of lysines 13 and 18 in GEF8 PRONE domain caused a total loss of binding to PA. Two ROPs, ROP7 and ROP10, were identified as preferred substrates of GEF8 by pull-down and bimolecular fluorescence complementation assays. GEF8 activity towards ROP7, but not ROP10, was stimulated by PA both in vitro and in cells. Moreover, the PA- or ABA-induced activation of GEF8 was completely lost in the mutant GEF8, which did not bind to PA. Together, these findings identify a direct interconnection between PA-mediated GEFs activity and small GTPase signalling in plants and provide evidence for a synergistic activation of GEF8 by direct PA-binding to its PRONE domain.