WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression.

Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.

Multivalent Proteins Rapidly and Reversibly Phase-Separate upon Osmotic Cell Volume Change.

Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration.

Excess microtubule and F-actin formation mediates shortening and loss of primary cilia in response to a hyperosmotic milieu.

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shock-induced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin.

Remarkable improvement in detection of readthrough downstream-of-gene transcripts by semi-extractable RNA-sequencing.

The mammalian cell nucleus contains dozens of membrane-less nuclear bodies that play significant roles in various aspects of gene expression. Several nuclear bodies are nucleated by specific architectural noncoding RNAs (arcRNAs) acting as structural scaffolds. We have reported that a minor population of cellular RNAs exhibits an unusual semi-extractable feature upon using the conventional procedure of RNA preparation and that needle shearing or heating of cell lysates remarkably improves extraction of dozens of RNAs. Because semi-extractable RNAs, including known arcRNAs, commonly localize in nuclear bodies, this feature may be a hallmark of arcRNAs. Using the semi-extractability of RNA, we performed genome-wide screening of semi-extractable long noncoding RNAs to identify new candidate arcRNAs for arcRNA under hyperosmotic and heat stress conditions. After screening stress-inducible and semi-extractable RNAs, hundreds of readthrough downstream-of-gene (DoG) transcripts over several hundreds of kilobases, many of which were not detected among RNAs prepared by the conventional extraction procedure, were found to be stress-inducible and semi-extractable. We further characterized some of the abundant DoGs and found that stress-inducible transient extension of the 3'-UTR made DoGs semi-extractable. Furthermore, they were localized in distinct nuclear foci that were sensitive to 1,6-hexanediol. These data suggest that semi-extractable DoGs exhibit arcRNA-like features and our semi-extractable RNA-seq is a powerful tool to extensively monitor DoGs that are induced under specific physiological conditions.

Hyperosmotic stress induces 2-cell-like cells through ROS and ATR signaling.

Mouse embryonic stem cells (ESCs) display pluripotency features characteristic of the inner cell mass of the blastocyst. Mouse embryonic stem cell cultures are highly heterogeneous and include a rare population of cells, which recapitulate characteristics of the 2-cell embryo, referred to as 2-cell-like cells (2CLCs). Whether and how ESC and 2CLC respond to environmental cues has not been fully elucidated. Here, we investigate the impact of mechanical stress on the reprogramming of ESC to 2CLC. We show that hyperosmotic stress induces 2CLC and that this induction can occur even after a recovery time from hyperosmotic stress, suggesting a memory response. Hyperosmotic stress in ESCs leads to accumulation of reactive-oxygen species (ROS) and ATR checkpoint activation. Importantly, preventing either elevated ROS levels or ATR activation impairs hyperosmotic-mediated 2CLC induction. We further show that ROS generation and the ATR checkpoint act within the same molecular pathway in response to hyperosmotic stress to induce 2CLCs. Altogether, these results shed light on the response of ESC to mechanical stress and on our understanding of 2CLC reprogramming.

Med3-mediated NADPH generation to help Saccharomyces cerevisiae tolerate hyperosmotic stress.

Hyperosmotic stress tolerance is crucial for Saccharomyces cerevisiae in producing value-added products from renewable feedstock. The limited understanding of its tolerance mechanism has impeded the application of these microbial cell factories. Previous studies have shown that Med3 plays a role in hyperosmotic stress in S. cerevisiae. However, the specific function of Med3 in hyperosmotic stress tolerance remains unclear. In this study, we showed that the deletion of the mediator Med3 impairs S. cerevisiae growth under hyperosmotic stress. Phenotypic analyses and yeast two-hybrid assays revealed that Med3 interacts with the transcription factor Stb5 to regulate the expression of the genes gnd1 and ald6, which are involved in NADPH production under hyperosmotic stress conditions. The deletion of med3 resulted in a decrease in intracellular NADPH content, leading to increased oxidative stress and elevated levels of intracellular reactive oxygen species under hyperosmotic stress, thereby impacting bud formation. These findings highlight the significant role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.IMPORTANCEHyperosmotic stress tolerance in the host strain is a significant challenge for fermentation performance in industrial production. In this study, we showed that the S. cerevisiae mediator Med3 is essential for yeast growth under hyperosmotic conditions. Med3 interacts with the transcription factor Stb5 to regulate the expression of genes involved in the NADPH-generation system during hyperosmotic stress. Adequate NADPH ensures the timely removal of excess reactive oxygen species and supports bud formation under these conditions. This work highlights the crucial role of Med3 as a regulator in maintaining NADPH generation and redox homeostasis in S. cerevisiae during hyperosmotic stress.

sRNA molecules participate in hyperosmotic stress response regulation in Sphingomonas melonis TY.

Drought and salinity are ubiquitous environmental factors that pose hyperosmotic threats to microorganisms and impair their efficiency in performing environmental functions. However, bacteria have developed various responses and regulatory systems to cope with these abiotic challenges. Posttranscriptional regulation plays vital roles in regulating gene expression and cellular homeostasis, as hyperosmotic stress conditions can lead to the induction of specific small RNA molecules (sRNAs) that participate in stress response regulation. Here, we report a candidate functional sRNA landscape of Sphingomonas melonis TY under hyperosmotic stress, and 18 sRNAs were found with a clear response to hyperosmotic stress. These findings will help in the comprehensive analysis of sRNA regulation in Sphingomonas species. Weighted correlation network analysis revealed a 263 nucleotide sRNA, SNC251, which was transcribed from its own promoter and showed the most significant correlation with hyperosmotic response factors. Deletion of snc251 affected biofilm formation and multiple cellular processes, including ribosome-related pathways, aromatic compound degradation, and the nicotine degradation capacity of S. melonis TY, while overexpression of SNC251 facilitated biofilm formation by TY under hyperosmotic stress. Two genes involved in the TonB system were further verified to be activated by SNC251, which also indicated that SNC251 is a trans-acting sRNA. Briefly, this research reports a landscape of sRNAs participating in the hyperosmotic stress response in S. melonis and reveals a novel sRNA, SNC251, which contributes to the S. melonis TY biofilm formation and thus enhances its hyperosmotic stress response ability.IMPORTANCESphingomonas species play a vital role in plant defense and pollutant degradation and survive extensively under drought or salinity. Previous studies have focused on the transcriptional and translational responses of Sphingomonas under hyperosmotic stress, but the posttranscriptional regulation of small RNA molecules (sRNAs) is also crucial for quickly modulating cellular processes to adapt dynamically to osmotic environments. In addition, the current knowledge of sRNAs in Sphingomonas is extremely scarce. This research revealed a novel sRNA landscape of Sphingomonas melonis and will greatly enhance our understanding of sRNAs' acting mechanisms in the hyperosmotic stress response.

Involvement of CCL2 in Salivary Gland Response to Hyperosmolar Stress Related to Sjögren's Syndrome.

In primary Sjögren's syndrome (pSS) patients, salivary gland (SG) epithelial cells (SGECs) could be exposed to chronic hyperosmotic stress (HOS), consecutive to their destruction and deregulation, that exacerbates an inflammatory response. The aims of this study were to assess the mechanism accounting for C-C motif chemokine ligand 2 (CCL2) expression in an immortalized human salivary gland epithelial acinar cell line (NS-SV-AC) subjected to HOS, as well as the involvement of CCL2 in pSS. CCL2 mRNA and protein levels were determined via RT-qPCR and ELISA. Reporter plasmids and a promoter pull-down assay were used to identify transcription factors associated with CCL2 mRNA increase. Our data showed that HOS-induced CCL2 mRNA increase was independent of the nuclear factor of activated T-cells 5 (NFAT5) and nuclear factor-kappa B (NFkB) but involved Kruppel-like factor 5 (KLF5). CCL2 protein levels, quantified by enzyme-linked immunosorbent assay (ELISA) in sera samples from pSS patients, correlated with the European Alliance of Associations for Rheumatology's Sjogren's syndrome disease activity index (ESSDAI) score for systemic activity. In addition, CCL2 protein levels were higher in patients with biological activity, cutaneous manifestations, and ESSDAI score superior or equal to five. Our data suggest that chronic HOS could exacerbate pSS disease by contributing to the inflammatory process induced by the expression and secretion of CCL2.

Actin cytoskeletal reorganization is involved in hyperosmotic stress-induced autophagy in tubular epithelial cells.

Tubular epithelial cells are routinely exposed to severe changes in osmolarity. Although the autophagic activity of cells is an indispensable process to maintain cellular homeostasis and respond to stressors, the effect of hyperosmotic stress on autophagic activity in tubular epithelial cells remains unknown. The aim of this study was to determine the effect of hyperosmotic stress on autophagy in rat kidney tubular epithelial cells focusing on the role of actin and microtubule cytoskeletons. Normal rat kidney (NRK)-52E cells exposed to mannitol-induced hyperosmotic stress. As a result, NRK-52E cells showed elevated protein levels of the autophagosome marker LC3-II, indicating enhancement of the autophagic flux. Hyperosmotic stress also transiently decreased cell volume and caused the reorganization of actin and microtubule cytoskeletal structures in NRK-52E cells. The inhibition of the actin cytoskeleton reorganization by cytochalasin D impaired the increase in the levels of LC3-II; however, disassembly of the microtubules following treatment with nocodazole did not affect the increase. These results indicate that hyperosmotic stress can induce autophagy mediated by the reorganization of the actin cytoskeleton in tubular epithelial cells.

A phosphatase-centric mechanism drives stress signaling response.

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase-driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho-proteomic response is the Endosulfine-mediated inhibition of protein phosphatase 2A-Cdc55 (PP2ACdc55 ), while we do not observe concurrent kinase activation. In fact, many of the stress-induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well-established kinase-centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re-evaluating the impact of phosphatases on shaping the phosphorylome.

Evidence for macromolecular crowding as a direct apoptotic stimulus.

Potassium loss and persistent shrinkage have both been implicated in apoptosis but their relationship and respective roles remain controversial. We approached this problem by clamping intracellular sodium and potassium in HeLa or MDCK cells using a combination of ionophores. Although ionophore treatment caused significant cell swelling, the initial volume could be restored and further reduced by application of sucrose. The swollen cells treated with ionophores remained viable for at least 8 h without any signs of apoptosis. Application of sucrose and the resulting shrinkage caused volume-dependent intrinsic apoptosis with all its classical features: inversion of phosphatidylserine, caspase activation and Bcl-2-dependent release of cytochrome c from mitochondria. In other experiments, apoptosis was induced by addition of the protein kinase inhibitor staurosporine at various degrees of swelling. Our results show that: (1) persistent shrinkage can cause apoptosis regardless of intracellular sodium or potassium composition or of the state of actin cytoskeleton; (2) strong potassium dependence of caspase activation is only observed in swollen cells with a reduced density of cytosolic proteins. We conclude that macromolecular crowding can be an important factor in determining the transition of cells to apoptosis.

Calcium influx through TRPV4 channels involve in hyperosmotic stress-induced epithelial-mesenchymal transition in tubular epithelial cells.

The epithelial-mesenchymal transition (EMT) is a biological process that occurs in the pathogenesis of kidney diseases in which injured tubular epithelial cells transform into myofibroblasts. We previously showed that mannitol-mediated hyperosmotic stress induces EMT of tubular epithelial cells. Although Ca2+ signaling is essential for the induction of EMT in tubular epithelial cells, the role of specific calcium channels is unknown. In this study, we assessed the transient receptor potential vanilloid 4 (TRPV4)-mediated Ca2+ influx in the hyperosmolarity-induced EMT. The Fluo-4 assay was used to examine the effect of hyperosmotic stress on the intracellular Ca2+ level of normal rat kidney (NRK)-52E cells. Expression of a mesenchymal marker α-smooth muscle actin (α-SMA) and an epithelial marker E-cadherin was also observed by fluorescence microscopy. The hyperosmotic stress caused a transient increase in intracellular Ca2+ concentration as well as a decrease in E-cadherin and an increase in α-SMA expressions in tubular epithelial cells, indicating the induction of EMT. A TRPV4 channel antagonist inhibited hyperosmotic stress-induced Ca2+ influx and the EMT, whereas, a TRPV4 channel agonist increased Ca2+ influx and EMT induction in tubular epithelial cells without the hyperosmotic stress. These findings suggest that Ca2+ influx through TRPV4 channels contributes to the hyperosmotic stress-induced EMT of tubular epithelial cells.

Nanoparticles Promote Bacterial Antibiotic Tolerance via Inducing Hyperosmotic Stress Response.

With the rapid development of nanotechnology, nanoparticles (NPs) are widely used in all fields of life. Nowadays, NPs have shown extraordinary antimicrobial activities and become one of the most popular strategies to combat antibiotic resistance. Whether they are equally effective in combating bacterial persistence, another important reason leading to antibiotic treatment failure, remains unknown. Persister cells are a small subgroup of phenotypic drug-tolerant cells in an isogenic bacterial population. Here, various types of NPs are used in combination with different antibiotics to destroy persisters. Strikingly, rather than eradicating persister cells, a wide range of NPs promote the formation of bacterial persistence. It is uncovered by PCR, thermogravimetric analysis, intracellular potassium ion staining, and molecular dynamics simulation that the persister promotion effect is achieved through exerting a hyperosmotic pressure around the cells. Moreover, protein mass spectrometry, fluorescence microscope images, and SDS-PAGE indicate NPs can further hijack cell osmotic regulatory circuits by inducing aggregation of outer membrane protein OmpA and OmpC. These findings question the efficacy of using NPs as antimicrobial agents and raise the possibility that widely used NPs may facilitate the global emergence of bacterial antibiotic tolerance.

Preclinical study of a new matrix to help the ocular surface in dry eye disease.

Dry eye disease (DED), a multifactorial disease of the tears and ocular system, causes loss of tear film homeostasis with damage to the ocular surface. This study aimed to assess whether a peculiar matrix based on sodium hyaluronate (HA), xanthan gum (XNT), glycine (GLY) and betaine (BET) as osmoprotectants, could be involved in biological responses. Wound healing assay on human corneal epithelial (HCE) cells in monolayer showed a synergistic effect of the combination of HA + XNT (**p ≤ 0.01) together with an efficient extracellular matrix remodeling of the formulation in SkinEthic™ HCE 3D-model sought by integrin beta-1 (ITGß1) expression and morphological analysis by hematoxylin and eosin (H&E), compared to a reference marketed product. The synergistic effect of HA + XNT + GLY + BET showed an antioxidant effect on HCE cells (***p ≤ 0.001). Real-time PCR analysis showed that the combination of GLY + BET seemed to ameliorate the effect exhibited by the single osmoprotectants in reducing tumor necrosis factor-alpha (TNFα, #p ≤ 0.05), interleukin-1 beta (IL1ß, ####p ≤ 0.0001) and cyclooxygenases-2 (COX2, ####p ≤ 0.0001) genes in SIRC cells under hyperosmotic stress. Furthermore, pretreatment with XNT, alone and in combination (##p ≤ 0.01), reduced COX2 expression in human non-small cell lung cancer cells (A549). Finally, the formulation was well-tolerated following q.i.d. ocular administration in rabbits during a 28-day study. Due to the synergistic effect of its components, the matrix proved able to repair the ocular surface restoring cell homeostasis and to protect the ocular surface from pro-inflammatory pathways activation and oxidative damage, thus behaving as a reactive oxygen species (ROS) scavenger.

Interleukin-20 is involved in dry eye disease and is a potential therapeutic target.

BACKGROUND: Dry eye disease (DED) is a common disease in ophthalmology, affecting millions of people worldwide. Recent studies have shown that inflammation is the core mechanism of DED. IL-20 is a proinflammatory cytokine involved in various inflammatory diseases. Therefore, we aimed to explore the role of this cytokine in the pathogenesis of DED and evaluate the therapeutic potential of the anti-IL-20 monoclonal antibody (mAb) 7E for DED treatment. METHODS: Clinical tear samples from patients with DED and non-DED controls were collected and their IL-20 protein levels were determined. We established three DED animal models to explore the role of IL-20 and the efficacy of IL-20 antibody in DED. Benzalkonium chloride (BAC)-induced over-evaporative DED, extra-orbital lacrimal gland excision (LGE)-induced aqueous tear-deficient DED, and desiccating stress (DS)-induced combined over-evaporative and aqueous tear-deficient DED animal models were established to investigate the role of IL-20. The anti-IL-20 antibody 7E was established to neutralize IL-20 activity. The effects of IL-20 or 7E on human corneal epithelial cells and macrophages under hyperosmotic stress were analyzed. 7E was topically applied to eyes to evaluate the therapeutic effects in the DED animal models. RESULTS: IL-20 was significantly upregulated in the tears of patients with DED and in the tears and corneas of DED animal models. Under hyperosmotic stress, IL-20 expression was induced via NFAT5 activation in corneal epithelial cells. 7E suppressed hyperosmotic stress-induced activation of macrophages. IL-20 induced cell death in corneal epithelial cells and 7E protected cells from hyperosmotic stress-induced cell death. Blocking IL-20 signaling with 7E protected mice from BAC-induced, LGE-induced, and DS-induced DED by reducing DED symptoms and inhibiting inflammatory responses, macrophage infiltration, apoptosis, and Th17 populations in the conjunctiva and draining lymph nodes. CONCLUSIONS: Our results demonstrated the functions of IL-20 in DED and presented a potential therapeutic option for this condition.

The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress.

The expression of flagellar proteins in Brucella species likely evolved through genetic transference from other microorganisms, and contributed to virulence, adaptability, and biofilm formation. Despite significant progress in defining the molecular mechanisms behind flagellar gene expression, the genetic program controlling biofilm formation remains unclear. The flagellar transcriptional factor (FtcR) is a master regulator of the flagellar system's expression, and is critical for B. melitensis 16M's flagellar biogenesis and virulence. Here, we demonstrate that FtcR mediates biofilm formation under hyperosmotic stress. Chromatin immunoprecipitation with next-generation sequencing for FtcR and RNA sequencing of ftcR-mutant and wild-type strains revealed a core set of FtcR target genes. We identified a novel FtcR-binding site in the promoter region of the osmotic-stress-response regulator gene betI, which is important for the survival of B. melitensis 16M under hyperosmotic stress. Strikingly, this site autoregulates its expression to benefit biofilm bacteria's survival under hyperosmotic stress. Moreover, biofilm reduction in ftcR mutants is independent of the flagellar target gene fliF. Collectively, our study provides new insights into the extent and functionality of flagellar-related transcriptional networks in biofilm formation, and presents phenotypic and evolutionary adaptations that alter the regulation of B. melitensis 16M to confer increased tolerance to hyperosmotic stress.

Alleviation of Endoplasmic Reticulum Stress Enhances Human Corneal Epithelial Cell Viability under Hyperosmotic Conditions.

Tear hyperosmolarity plays an essential role in the initiation and progression of dry-eye disease. Under a hyperosmotic environment, corneal epithelial cells experience perturbations in endoplasmic reticulum function that can lead to proinflammatory signaling and apoptosis. In this study, we investigated the effect of tauroursodeoxycholic acid (TUDCA), a chemical chaperone known to protect against endoplasmic reticulum stress, on corneal epithelial cells exposed to hyperosmotic conditions. We found that the expression of the genes involved in the activation of the unfolded protein response and the pro-apoptotic transcription factor DDIT3 were markedly upregulated in patients with Sjögren's dry-eye disease and in a human model of corneal epithelial differentiation following treatment with hyperosmotic saline. Experiments in vitro demonstrated that TUDCA prevented hyperosmotically induced cell death by reducing nuclear DNA fragmentation and caspase-3 activation. TUDCA supplementation also led to the transcriptional repression of CXCL8 and IL5, two inflammatory mediators associated with dry-eye pathogenesis. These studies highlight the role of hyperosmotic conditions in promoting endoplasmic reticulum stress in the cornea and identify TUDCA as a potential therapeutic agent for the treatment of dry-eye disease.

Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana.

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar 'Fuding Dabaicha'. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.

Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis.

As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.