Multiphase coalescence mediates Hippo pathway activation.

The function of biomolecular condensates is often restricted by condensate dissolution. Whether condensates can be suppressed without condensate dissolution is unclear. Here, we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and their coalescence into a common phase provides a mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inhibit STRIPAK function. These findings provide a paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a conceptual framework for understanding upstream regulation of the Hippo signaling pathway.

Macromolecular crowding sensing during osmotic stress in plants.

Osmotic stress conditions occur at multiple stages of plant life. Changes in water availability caused by osmotic stress induce alterations in the mechanical properties of the plasma membrane, its interaction with the cell wall, and the concentration of macromolecules in the cytoplasm. We summarize the reported players involved in the sensing mechanisms of osmotic stress in plants. We discuss how changes in macromolecular crowding are perceived intracellularly by intrinsically disordered regions (IDRs) in proteins. Finally, we review methods for dynamically monitoring macromolecular crowding in living cells and discuss why their implementation is required for the discovery of new plant osmosensors. Elucidating the osmosensing mechanisms will be essential for designing strategies to improve plant productivity in the face of climate change.

Ca2+-sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that low concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca2+ release from nearly all. Chelating cytoplasmic Ca2+ makes lysosomes more sensitive to GPN-induced rupture, suggesting a role for Ca2+ in lysosomal membrane resilience. GPN-elicited Ca2+ release causes the Ca2+-sensor Apoptosis Linked Gene-2 (ALG-2), along with Endosomal Sorting Complex Required for Transport (ESCRT) proteins it interacts with, to redistribute onto lysosomes. Functionally, ALG-2, but not its ESCRT binding-disabled ΔGF122 splice variant, increases lysosomal resilience to osmotic stress. Importantly, elevating juxta-lysosomal Ca2+ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca2+, through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.

Stretch-activated ion channel TMEM63B associates with developmental and epileptic encephalopathies and progressive neurodegeneration.

By converting physical forces into electrical signals or triggering intracellular cascades, stretch-activated ion channels allow the cell to respond to osmotic and mechanical stress. Knowledge of the pathophysiological mechanisms underlying associations of stretch-activated ion channels with human disease is limited. Here, we describe 17 unrelated individuals with severe early-onset developmental and epileptic encephalopathy (DEE), intellectual disability, and severe motor and cortical visual impairment associated with progressive neurodegenerative brain changes carrying ten distinct heterozygous variants of TMEM63B, encoding for a highly conserved stretch-activated ion channel. The variants occurred de novo in 16/17 individuals for whom parental DNA was available and either missense, including the recurrent p.Val44Met in 7/17 individuals, or in-frame, all affecting conserved residues located in transmembrane regions of the protein. In 12 individuals, hematological abnormalities co-occurred, such as macrocytosis and hemolysis, requiring blood transfusions in some. We modeled six variants (p.Val44Met, p.Arg433His, p.Thr481Asn, p.Gly580Ser, p.Arg660Thr, and p.Phe697Leu), each affecting a distinct transmembrane domain of the channel, in transfected Neuro2a cells and demonstrated inward leak cation currents across the mutated channel even in isotonic conditions, while the response to hypo-osmotic challenge was impaired, as were the Ca2+ transients generated under hypo-osmotic stimulation. Ectopic expression of the p.Val44Met and p.Gly580Cys variants in Drosophila resulted in early death. TMEM63B-associated DEE represents a recognizable clinicopathological entity in which altered cation conductivity results in a severe neurological phenotype with progressive brain damage and early-onset epilepsy associated with hematological abnormalities in most individuals.

DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here, in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput data-independent acquisition-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and SnRK2s, EGTA treatment also activates mitogen-activated protein kinase cascades, Calcium-dependent protein kinases, and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases and receptor-like protein kinases in the osmotic stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca2+ signaling, offering insights into the (exocellular) Ca2+ deprivation during early hyperosmolality sensing and signaling.

Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

BACKGROUND: Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. METHODS: Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. RESULTS: CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. CONCLUSIONS: Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

Hydration-mediated G-protein-coupled receptor activation.

The Rhodopsin family of G-protein­coupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (∼80­100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.

MAPK-dependent control of mitotic progression in S. pombe.

BACKGROUND: Mitogen-activated protein kinases (MAPKs) preserve cell homeostasis by transducing physicochemical fluctuations of the environment into multiple adaptive responses. These responses involve transcriptional rewiring and the regulation of cell cycle transitions, among others. However, how stress conditions impinge mitotic progression is largely unknown. The mitotic checkpoint is a surveillance mechanism that inhibits mitotic exit in situations of defective chromosome capture, thus preventing the generation of aneuploidies. In this study, we investigate the role of MAPK Pmk1 in the regulation of mitotic exit upon stress. RESULTS: We show that Schizosaccharomyces pombe cells lacking Pmk1, the MAP kinase effector of the cell integrity pathway (CIP), are hypersensitive to microtubule damage and defective in maintaining a metaphase arrest. Epistasis analysis suggests that Pmk1 is involved in maintaining spindle assembly checkpoint (SAC) signaling, and its deletion is additive to the lack of core SAC components such as Mad2 and Mad3. Strikingly, pmk1Δ cells show up to twofold increased levels of the anaphase-promoting complex (APC/C) activator Cdc20Slp1 during unperturbed growth. We demonstrate that Pmk1 physically interacts with Cdc20Slp1 N-terminus through a canonical MAPK docking site. Most important, the Cdc20Slp1 pool is rapidly degraded in stressed cells undergoing mitosis through a mechanism that requires MAPK activity, Mad3, and the proteasome, thus resulting in a delayed mitotic exit. CONCLUSIONS: Our data reveal a novel function of MAPK in preventing mitotic exit and activation of cytokinesis in response to stress. The regulation of Cdc20Slp1 turnover by MAPK Pmk1 provides a key mechanism by which the timing of mitotic exit can be adjusted relative to environmental conditions.

Transcriptional and post-transcriptional mechanisms modulate cyclopropane fatty acid synthase through small RNAs in Escherichia coli.

The small RNA (sRNA) RydC strongly activates cfa, which encodes the cyclopropane fatty acid synthase. Previous work demonstrated that RydC activation of cfa increases the conversion of unsaturated fatty acids to cyclopropanated fatty acids in membrane lipids and changes the biophysical properties of membranes, making cells more resistant to acid stress. The regulators that control RydC synthesis had not previously been identified. In this study, we identify a GntR-family transcription factor, YieP, that represses rydC transcription. YieP positively autoregulates its own transcription and indirectly regulates cfa through RydC. We further identify additional sRNA regulatory inputs that contribute to the control of RydC and cfa. The translation of yieP is repressed by the Fnr-dependent sRNA, FnrS, making FnrS an indirect activator of rydC and cfa. Conversely, RydC activity on cfa is antagonized by the OmpR-dependent sRNA OmrB. Altogether, this work illuminates a complex regulatory network involving transcriptional and post-transcriptional inputs that link the control of membrane biophysical properties to multiple environmental signals. IMPORTANCE: Bacteria experience many environmental stresses that challenge their membrane integrity. To withstand these challenges, bacteria sense what stress is occurring and mount a response that protects membranes. Previous work documented the important roles of small RNA (sRNA) regulators in membrane stress responses. One sRNA, RydC, helps cells cope with membrane-disrupting stresses by promoting changes in the types of lipids incorporated into membranes. In this study, we identified a regulator, YieP, that controls when RydC is produced and additional sRNA regulators that modulate YieP levels and RydC activity. These findings illuminate a complex regulatory network that helps bacteria sense and respond to membrane stress.

Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via regulation of pyruvate dehydrogenase phosphorylation.

A high-salt diet significantly impacts various diseases, ilncluding cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, we determined that the pyruvate dehydrogenase phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmosensing mechanism. Additionally, we demonstrate that Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Collectively, our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of pyruvate dehydrogenase phosphorylation by direct osmosensing within mitochondria.

Hydrogen sulfide alleviates osmotic stress-induced root growth inhibition by promoting auxin homeostasis.

Hydrogen sulfide (H2 S) promotes plant tolerance against various environmental cues, and d-cysteine desulfhydrase (DCD) is an enzymatic source of H2 S to enhance abiotic stress resistance. However, the role of DCD-mediated H2 S production in root growth under abiotic stress remains to be further elucidated. Here, we report that DCD-mediated H2 S production alleviates osmotic stress-mediated root growth inhibition by promoting auxin homeostasis. Osmotic stress up-regulated DCD gene transcript and DCD protein levels and thus H2 S production in roots. When subjected to osmotic stress, a dcd mutant showed more severe root growth inhibition, whereas the transgenic lines DCDox overexpressing DCD exhibited less sensitivity to osmotic stress in terms of longer root compared to the wild-type. Moreover, osmotic stress inhibited root growth through repressing auxin signaling, whereas H2 S treatment significantly alleviated osmotic stress-mediated inhibition of auxin. Under osmotic stress, auxin accumulation was increased in DCDox but decreased in dcd mutant. H2 S promoted auxin biosynthesis gene expression and auxin efflux carrier PIN-FORMED 1 (PIN1) protein level under osmotic stress. Taken together, our results reveal that mannitol-induced DCD and H2 S in roots promote auxin homeostasis, contributing to alleviating the inhibition of root growth under osmotic stress.

Pepper stress-associated protein 14 is a substrate of CaSnRK2.6 that positively modulates abscisic acid-dependent osmotic stress responses.

The phytohormone abscisic acid (ABA) plays a prominent role in various abiotic stress responses of plants. In the ABA-dependent osmotic stress response, SnRK2.6, one of the subclass III SnRK2 kinases, has been identified as playing a key role by phosphorylating and activating downstream genes. Although several modulatory proteins have been reported to be phosphorylated by SnRK2.6, the identities of the full spectrum of downstream targets have yet to be sufficiently established. In this study, we identified CaSAP14, a stress-associated protein in pepper (Capsicum annuum), as a downstream target of CaSnRK2.6. We elucidated the physical interaction between SnRK2.6 and CaSAP14, both in vitro and in vivo, and accordingly identified a C-terminal C2H2-type zinc finger domain of CaSAP14 as being important for their interaction. CaSAP14-silenced pepper plants showed dehydration- and high salt-sensitive phenotypes, whereas overexpression of CaSAP14 in Arabidopsis conferred tolerance to dehydration, high salinity, and mannitol treatment, with plants showing ABA-hypersensitive phenotypes. Furthermore, an in-gel kinase assay revealed that CaSnRK2.6 phosphorylates CaSAP14 in response to exogenous ABA, dehydration, and high-salinity stress. Collectively, these findings suggest that CaSAP14 is a direct substrate of CaSnRK2.6 and positively modulates dehydration- and high salinity-induced osmotic stress responses.

OsNF-YA3 regulates plant growth and osmotic stress tolerance by interacting with SLR1 and SAPK9 in rice.

The antagonism between gibberellin (GA) and abscisic acid (ABA) signaling pathways is vital to balance plant growth and stress response. Nevertheless, the mechanism by which plants determine the balance remains to be elucidated. Here, we report that rice NUCLEAR FACTOR-Y A3 (OsNF-YA3) modulates GA- and ABA-mediated balance between plant growth and osmotic stress tolerance. OsNF-YA3 loss-of-function mutants exhibit stunted growth, compromised GA biosynthetic gene expression, and decreased GA levels, while its overexpression lines have promoted growth and enhanced GA content. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis and transient transcriptional regulation assays demonstrate that OsNF-YA3 activates GA biosynthetic gene OsGA20ox1 expression. Furthermore, the DELLA protein SLENDER RICE1 (SLR1) physically interacts with OsNF-YA3 and thus inhibits its transcriptional activity. On the other side, OsNF-YA3 negatively regulates plant osmotic stress tolerance by repressing ABA response. OsNF-YA3 reduces ABA levels by transcriptionally regulating ABA catabolic genes OsABA8ox1 and OsABA8ox3 by binding to their promoters. Furthermore, OSMOTIC STRESS/ABA-ACTIVATED PROTEIN KINASE 9 (SAPK9), the positive component in ABA signaling, interacts with OsNF-YA3 and mediates OsNF-YA3 phosphorylation, resulting in its degradation in plants. Collectively, our findings establish OsNF-YA3 as an important transcription factor that positively modulates GA-regulated plant growth and negatively controls ABA-mediated water-deficit and salt tolerance. These findings shed light on the molecular mechanism underlying the balance between the growth and stress response of the plant.

Integrated physiological, metabolomic, and transcriptomic analyses elucidate the regulation mechanisms of lignin synthesis under osmotic stress in alfalfa leaf (Medicago sativa L.).

Alfalfa, an essential forage crop known for its high yield, nutritional value, and strong adaptability, has been widely cultivated worldwide. The yield and quality of alfalfa are frequently jeopardized due to environmental degradation. Lignin, a constituent of the cell wall, enhances plant resistance to abiotic stress, which often causes osmotic stress in plant cells. However, how lignin responds to osmotic stress in leaves remains unclear. This study explored the effects of osmotic stress on lignin accumulation and the contents of intermediate metabolites involved in lignin synthesis in alfalfa leaves. Osmotic stress caused an increase in lignin accumulation and the alteration of core enzyme activities and gene expression in the phenylpropanoid pathway. We identified five hub genes (CSE, CCR, CADa, CADb, and POD) and thirty edge genes (including WRKYs, MYBs, and UBPs) by integrating transcriptome and metabolome analyses. In addition, ABA and ethylene signaling induced by osmotic stress regulated lignin biosynthesis in a contradictory way. These findings contribute to a new theoretical foundation for the breeding of high-quality and resistant alfalfa varieties.

Arabidopsis thaliana ubiquitin-associated protein 2 (AtUAP2) functions as an E4 ubiquitin factor and negatively modulates dehydration stress response.

E4, a ubiquitin (Ub) chain assembly factor and post-translational modification protein, plays a key role in the regulation of multiple cellular functions in plants during biotic or abiotic stress. We have more recently reported that E4 factor AtUAP1 is a negative regulator of the osmotic stress response and enhances the multi-Ub chain assembly of E3 ligase Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1). To further investigate the function of other E4 Ub factors in osmotic stress, we isolated AtUAP2, an AtUAP1 homolog, which interacted with AtRZF1, using pull-down assay and bimolecular fluorescence complementation analysis. AtUAP2, a Ub-associated motif-containing protein, interacts with oligo-Ub5, -Ub6, and -Ub7 chains. The yeast functional complementation experiment revealed that AtUAP2 functions as an E4 Ub factor. In addition, AtUAP2 is localized in the cytoplasm, different from AtUAP1. The activity of AtUAP2 was relatively strongly induced in the leaf tissue of AtUAP2 promoter-ß-glucuronidase transgenic plants by abscisic acid, dehydration, and oxidative stress. atuap2 RNAi lines were more insensitive to osmotic stress condition than wild-type during the early growth of seedlings, whereas the AtUAP2-overexpressing line exhibited relatively more sensitive responses. Analyses of molecular and physiological experiments showed that AtUAP2 could negatively mediate the osmotic stress-induced signaling. Genetic studies showed that AtRZF1 mutation could suppress the dehydration-induced sensitive phenotype of the AtUAP2-overexpressing line, suggesting that AtRZF1 acts genetically downstream of AtUAP2 during osmotic stress. Taken together, our findings show that the AtRZF1-AtUAP2 complex may play important roles in the ubiquitination pathway, which controls the osmotic stress response in Arabidopsis.

CAD1 contributes to osmotic tolerance in Arabidopsis thaliana by suppressing immune responses under osmotic stress.

Acquired osmotolerance induced by initial exposure to mild salt stress is widespread across Arabidopsis thaliana ecotypes, but the mechanism underlying it remains poorly understood. To clarify it, we isolated acquired osmotolerance-deficient 1 (aod1), a mutant highly sensitive to osmotic stress, from ion-beam-irradiated seeds of Zu-0, an ecotype known for its remarkably high osmotolerance. Aod1 showed growth inhibition with spotted necrotic lesions on the rosette leaves under normal growth conditions on soil. However, its tolerance to salt and oxidative stresses was similar to that of the wild type (WT). Genetic and genome sequencing analyses suggested that the gene causing aod1 is identical to CONSTITUTIVELY ACTIVATED CELL DEATH 1 (CAD1). Complementation with the WT CAD1 gene restored the growth and osmotolerance of aod1, indicating that mutated CAD1 is responsible for the observed phenotypes in aod1. Although CAD1 is known to act as a negative regulator of immune response, transcript levels in the WT increased in response to osmotic stress. Aod1 displayed enhanced immune response and cell death under normal growth conditions, whereas the expression profiles of osmotic response genes were comparable to those of the WT. These findings suggest that autoimmunity in aod1 is detrimental to osmotolerance. Overall, our results suggest that CAD1 negatively regulates immune responses under osmotic stress, contributing to osmotolerance in Arabidopsis.

Function analysis of transcription factor OSR1 regulating osmotic stress resistance in maize.

BACKGROUND: Maize is a major cereal crop world widely, however, the yield of maize is frequently limited by dehydration and even death of plants, which resulted from osmotic stress such as drought and salinity. Dissection of molecular mechanisms controlling stress tolerance will enable plant scientists and breeders to increase crops yield by manipulating key regulatory components. METHODS: The candidate OSR1 gene was identified by map-based cloning. The expression level of OSR1 was verified by qRT-PCR and digital PCR in WT and osr1 mutant. Electrophoretic mobility shift assay, transactivation activity assay, subcellular localization, transcriptome analysis and physiological characters measurements were conducted to analyze the function of OSR1 in osmotic stress resistance in maize. RESULTS: The osr1 mutant was significantly less sensitive to osmotic stress than the WT plants and displayed stronger water-holding capacity, and the OSR1 homologous mutant in Arabidopsis showed a phenotype similar with maize osr1 mutant. Differentially expressed genes (DEGs) were identified between WT and osr1 under osmotic stress by transcriptome analysis, the expression levels of many genes, such as LEA, auxin-related factors, PPR family members, and TPR family members, changed notably, which may primarily involve in osmotic stress or promote root development. CONCLUSIONS: OSR1 may serve as a negative regulatory factor in response to osmotic stress in maize. The present study sheds new light on the molecular mechanisms of osmotic stress in maize.

Phosphoproteomic analysis reveals distinctive responses in Mangrovibacter phragmatis under high-salinity condition.

We conducted a thorough genome-wide investigation of protein phosphorylation in the halotolerant bacterium Mangrovibacter phragmitis (MPH) ASIOC01, using the Fe-IMAC enrichment method combined with tandem mass spectrometry under low- and high-salinity conditions. The phosphoproteome comprises 86 unique phosphorylated proteins, crucially involving pathways such as glycolysis/gluconeogenesis, the citrate cycle, chaperones, ribosomal proteins, and cell division. This study represents the first and most extensive investigation to-date comparing the bacterial phosphoproteome under different osmotic conditions using a gel-free approach. We identified 45 unique phosphoproteins in MPH cultured in media containing 1 % NaCl, and 33 exclusive phosphoproteins in MPH cultured in media containing 5 % NaCl. Eight phosphoproteins were detected in both growth conditions. Analysis of high-confidence phosphosites reveals that phosphorylation predominantly occurs on serine residues (52.3 %), followed by threonine (35.1 %) and tyrosine (12.6 %) residues. Interestingly, 34 % of the phosphopeptides display multiple phosphosites. Currently, prokaryotic phosphorylation site prediction platforms like MPSite and NetPhosBac 1.0 demonstrate an average prediction accuracy of only 21 % when applied to our dataset. Fourteen phosphoproteins did not yield matches when compared against dbPSP 2.0 (database of Phosphorylation Sites in Prokaryotes), indicating that these proteins may be novel phosphoproteins. These unique proteins undergoing phosphorylation under high salinity growth conditions potentially enhance their adaptive capabilities to environmental challenges.

Effect of carboxymethyl cellulose and gibberellic acid-enriched biochar on osmotic stress tolerance in cotton.

The deleterious impact of osmotic stress, induced by water deficit in arid and semi-arid regions, poses a formidable challenge to cotton production. To protect cotton farming in dry areas, it's crucial to create strong plans to increase soil water and reduce stress on plants. The carboxymethyl cellulose (CMC), gibberellic acid (GA3) and biochar (BC) are individually found effective in mitigating osmotic stress. However, combine effect of CMC and GA3 with biochar on drought mitigation is still not studied in depth. The present study was carried out using a combination of GA3 and CMC with BC as amendments on cotton plants subjected to osmotic stress levels of 70 (70 OS) and 40 (40 OS). There were five treatment groups, namely: control (0% CMC-BC and 0% GA3-BC), 0.4%CMC-BC, 0.4%GA3-BC, 0.8%CMC-BC, and 0.8%GA3-BC. Each treatment was replicated five times with a completely randomized design (CRD). The results revealed that 0.8 GA3-BC led to increase in cotton shoot fresh weight (99.95%), shoot dry weight (95.70%), root fresh weight (73.13%), and root dry weight (95.74%) compared to the control group under osmotic stress. There was a significant enhancement in cotton chlorophyll a (23.77%), chlorophyll b (70.44%), and total chlorophyll (35.44%), the photosynthetic rate (90.77%), transpiration rate (174.44%), and internal CO2 concentration (57.99%) compared to the control group under the 40 OS stress. Thus 0.8GA3-BC can be potential amendment for reducing osmotic stress in cotton cultivation, enhancing agricultural resilience and productivity.

Phototropin2 3'UTR overlaps with the AT5G58150 gene encoding an inactive RLK kinase.

BACKGROUND: This study examines the biological implications of an overlap between two sequences in the Arabidopsis genome, the 3'UTR of the PHOT2 gene and a putative AT5G58150 gene, encoded on the complementary strand. AT5G58150 is a probably inactive protein kinase that belongs to the transmembrane, leucine-rich repeat receptor-like kinase family. Phot2 is a membrane-bound UV/blue light photoreceptor kinase. Thus, both proteins share their cellular localization, on top of the proximity of their loci. RESULTS: The extent of the overlap between 3'UTR regions of AT5G58150 and PHOT2 was found to be 66 bp, using RACE PCR. Both the at5g58150 T-DNA SALK_093781C (with insertion in the promoter region) and 35S::AT5G58150-GFP lines overexpress the AT5G58150 gene. A detailed analysis did not reveal any substantial impact of PHOT2 or AT5G58150 on their mutual expression levels in different light and osmotic stress conditions. AT5G58150 is a plasma membrane protein, with no apparent kinase activity, as tested on several potential substrates. It appears not to form homodimers and it does not interact with PHOT2. Lines that overexpress AT5G58150 exhibit a greater reduction in lateral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may participate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identified proteins with ATPase activity, which are involved in proton transport and cell elongation, as putative interactors of AT5G58150. Membrane kinases, including other members of the LRR RLK family and BSK kinases (positive regulators of brassinosteroid signalling), can also act as partners for AT5G58150. CONCLUSIONS: AT5G58150 is a membrane protein that does not exhibit measurable kinase activity, but is involved in signalling through interactions with other proteins. Based on the interactome and root architecture analysis, AT5G58150 may be involved in plant response to salt and osmotic stress and the formation of roots in Arabidopsis.