The spatiotemporal regulation of polar auxin transport by PIN-FORMED (PIN) efflux carriers is essential for coordinating plant development with environmental cues. However, whether and how osmotic stress signaling affects auxin transport to regulate plant stress adaptation remain largely unclear. In this study, we identify SnRK2.5, an abscisic acid-independent member of the SNF1-related protein kinase family, as a key molecular link between osmotic stress signaling and auxin transport regulation in Arabidopsis. Osmotic stress activates SnRK2.5, which directly phosphorylates PIN2 at Ser237 and Ser259. Genetic and cell biological analyses demonstrate that these phosphorylation events govern PIN2 vesicular trafficking, vacuolar targeting, and auxin transport activity. Disruption of these phosphorylation sites impairs PIN2-dependent auxin redistribution, thereby compromising root tropic responses and reducing osmotic stress tolerance. Our findings uncover a regulatory mechanism by which SnRK2.5-mediated phosphorylation of PIN2 dynamically adjusts auxin flux in response to water availability, representing a critical adaptive strategy that optimizes plant growth under osmotic stress.

During the early stages of seed development, the small embryo receives large amounts of sugar from the liquid endosperm of the developing seed. A sugar deficit can lead to severe seed abortion and yield loss. However, the key factors influencing sugar transport and crop yield remain largely unknown. In this work, we identified a plasma membrane-localized sugar transporter, GmSWEET48, that was highly expressed in developing soybean seeds. Heterologous expression showed that GmSWEET48 transported fructose and glucose in yeast systems and exhibited 14C-labeled sucrose influx and efflux activities in Xenopus oocytes. Overexpression of GmSWEET48 decreased the levels of sucrose and oil but increased protein levels in seeds and promoted seed yield. In conclusion, GmSWEET48 regulates sugar transport during early seed development and ultimately regulates seed yield and composition.

The spatiotemporal regulation of polar auxin transport by PIN-FORMED (PIN) efflux carriers is essential for coordinating plant development with environmental cues. However, whether and how osmotic stress signaling affects auxin transport to regulate plant stress adaptation remain largely unclear. In this study, we identify SnRK2.5, an abscisic acid-independent member of the SNF1-related protein kinase family, as a key molecular link between osmotic stress signaling and auxin transport regulation in Arabidopsis. Osmotic stress activates SnRK2.5, which directly phosphorylates PIN2 at Ser237 and Ser259. Genetic and cell biological analyses demonstrate that these phosphorylation events govern PIN2 vesicular trafficking, vacuolar targeting, and auxin transport activity. Disruption of these phosphorylation sites impairs PIN2-dependent auxin redistribution, thereby compromising root tropic responses and reducing osmotic stress tolerance. Our findings uncover a regulatory mechanism by which SnRK2.5-mediated phosphorylation of PIN2 dynamically adjusts auxin flux in response to water availability, representing a critical adaptive strategy that optimizes plant growth under osmotic stress.

Abstract Chloride (Cl − ) ions cause major damage to crops in saline soils. Understanding the key factors that influence Cl − uptake and translocation will aid the breeding of more salt-tolerant crops. Here, using genome-wide association study and transcriptomic analysis, we identified a NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER family (NPF) protein, GmNPF7.5, as the dominant gene locus influencing Cl − homeostasis in soybean (Glycine max). A natural SNP variation resulted in two haplotypes (GmNPF7.5 HapA and GmNPF7.5 HapB ), which was associated with Cl − content. GmNPF7.5 HapA mediated Cl − or nitrate (NO 3 − ) uptake in a pH-dependent manner and exhibited higher permeability for Cl − over NO 3 − . The suppression of GmNPF7.5 HapA expression decreased Cl − accumulation and salt damage in plants, whereas its overexpression showed the opposite effects. The elite haplotype GmNPF7.5 HapB diminished Cl − transport activity independently from NO 3 − permeability, thus enhancing soybean salt tolerance. Furthermore, the protein kinase GmPI4Kγ4 could phosphorylate GmNPF7.5, which repressed Cl − uptake without affecting NO 3 − permeability. Our findings define a regulatory mechanism for Cl − control under NaCl stress, providing a strategy for the improvement of salt tolerance in soybean plants.

Cellular membranes primarily consist of proteins and lipids. These proteins perform cellular functions such as metabolic regulation, environmental and hormonal signal sensing, and nutrient transport. There is increasing experimental evidence that certain lipids, particularly anionic phospholipids, can act as signaling molecules. Specific examples of functional regulation by anionic phospholipids in plant cells have been reported for transporters, channels, and even receptors. By regulating the structure and activity of membrane-integral proteins, these phospholipids mediate the transport of phytohormones and ions, and elicit physiological responses to developmental and environmental cues. Phospholipids also control membrane protein abundance and lipid composition and abundance by facilitating vesicular trafficking. In this review, we discuss recent research that elucidates the mechanisms by which membrane-integral transporters and channels are controlled via phospholipid signaling, as well as the regulation of membrane protein accumulation by phospholipids through coordinated removal, recycling, and degradation processes.

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.

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.

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.

Although there has been long-standing recognition that stimuli-induced cytosolic pH alterations coincide with changes in calcium ion (Ca 2+ ) levels, the interdependence between protons (H + ) and Ca 2+ remains poorly understood. We addressed this topic using the light-gated channelrhodopsin Hc KCR2 from the pseudofungus Hyphochytrium catenoides , which operates as a H + conductive, Ca 2+ impermeable ion channel on the plasma membrane of plant cells. Light activation of Hc KCR2 in Arabidopsis guard cells evokes a transient cytoplasmic acidification that sparks Ca 2+ release from the endoplasmic reticulum. A H + -induced cytosolic Ca 2+ signal results in membrane depolarization through the activation of Ca 2+ -dependent SLAC1/SLAH3 anion channels, which enabled us to remotely control stomatal movement. Our study suggests a H + -induced Ca 2+ release mechanism in plant cells and establishes Hc KCR2 as a tool to dissect the molecular basis of plant intracellular pH and Ca 2+ signaling.

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.

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.

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.

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 (NH 4 + ) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1 Hap 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.

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.

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.

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 b(5) (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 b(5) and highlighted the coordinated action of two proteins to perceive Na+ in response to salt stress.

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.

Anionic phospholipid phosphatidic acid (PA) behaves as an important second messenger involved in many cellular processes, such as development, cytoskeletal dynamics, vesicle trafficking, and stress response. Recently, it was reported that PA can directly bind with the rice Shaker K(+)channel OsAKT2 to inhibit its channel activity. Two adjacent arginine residues (R644 and R645) in ANK domain were identified as a PA-binding site essential to the PA-mediated inhibition of OsAKT2. However, there may be still other PA-binding sites unidentified in OsAKT2. Here, using a PA biosensor (PAleon), we found that the exogenous PA treatment significantly increased the PA level at the plasma membrane ofXenopusoocytes which were used to express OsAKT2 for electrophysiological assays. As reported previously, exogenous PA markedly inhibited OsAKT2 K(+)currents. Replacement of two adjacent basic residues (R190 and K191) in the S4 voltage sensor by glycine completely abolished the time-dependent K+ currents of OsAKT2, but this variant was insensitive to PA treatment. In addition, we also identified other two adjacent arginines (R755 and R756) located in the cytosolic domain as a PA-binding site, which were also essential to the PA-mediated inhibition of OsAKT2. These results provide a more comprehensive understanding of the PA-K+ channel interaction mechanism. Combining the findings here with the previous study, we propose that multiple basic residues (R190/K191, R644/R645, and R755/R756) in different domains of OsAKT2 contribute to PA-mediated regulation of OsAKT2.

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. 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 △gef1 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. 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.