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During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction. SnRK2 kinases activate abiotic stress responses in plants following ABA-dependent phosphatase inhibition or ABA-independent osmotic stress signalling. Here Takahashi et al. show that MAPKK-kinases phosphorylate and activate SnRK2s thus enabling robust ABA and osmotic stress signal transduction.

SNF1-related protein kinases 2 (SnRK2s) are key regulators governing the plant adaptive responses to osmotic stresses, such as drought and high salinity. Subclass III SnRK2s function as central regulators of abscisic acid (ABA) signalling and orchestrate ABA-regulated adaptive responses to osmotic stresses. Seed plants have acquired other types of osmotic stress-activated but ABA-unresponsive subclass I SnRK2s that regulate mRNA decay and promote plant growth under osmotic stresses. In contrast to subclass III SnRK2s, the regulatory mechanisms underlying the rapid activation of subclass I SnRK2s in response to osmotic stress remain elusive. Here, we report that three B4 Raf-like MAP kinase kinase kinases (MAPKKKs) phosphorylate and activate subclass I SnRK2s under osmotic stress. Transcriptome analyses reveal that genes downstream of these MAPKKKs largely overlap with subclass I SnRK2-regulated genes under osmotic stress, which indicates that these MAPKKKs are upstream factors of subclass I SnRK2 and are directly activated by osmotic stress. SnRK2 protein kinases play key roles in signaling during plant responses to abiotic stress. Here Soma et al . report three Arabidopsis Raf-like MAP kinase kinase kinases phosphorylate and activate a subclass of SnRK2s that rapidly respond to osmotic stress independently of ABA signaling.

Salinity is a growing problem affecting soils and agriculture in many parts of the world. The presence of salt in plant cells disrupts many basic metabolic processes, contributing to severe negative effects on plant development and growth. This review focuses on the effects of salinity on chloroplasts, including the structures and function of these organelles. Chloroplasts house various important biochemical reactions, including photosynthesis, most of which are considered essential for plant survival. Salinity can affect these reactions in a number of ways, for example, by changing the chloroplast size, number, lamellar organization, lipid and starch accumulation, and interfering with cross-membrane transportation. Research has shown that maintenance of the normal chloroplast physiology is necessary for the survival of the entire plant. Many plant species have evolved different mechanisms to withstand the harmful effects of salt-induced toxicity on their chloroplasts and its machinery. The differences depend on the plant species and growth stage and can be quite different between salt-sensitive (glycophyte) and salt-tolerant (halophyte) plants. Salt stress tolerance is a complex trait, and many aspects of salt tolerance in plants are not entirely clear yet. In this review, we discuss the different mechanisms of salt stress tolerance in plants with a special focus on chloroplast structure and its functions, including the underlying differences between glycophytes and halophytes.

Mixed lineage kinase domain-like protein （MLKL） was identified to function downstream of receptor interacting protein 3 （RIP3） in tumor necrosis factor-α （TNF）-induced necrosis （also called necroptosis）. However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain （HBD＊） triggers necroptosis. Notably, forcing together the N-terminal domain （ND） but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL （1-130 amino acids） is sufficient to trigger necroptosis. Both the HBD＊-mediated and TNF-induced complexes of MLKL（ND） or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plas- ma membrane location is located in the junction of the first and second a-helices of MLKL. The plasma membrane translocation of MLKL or MLKL（ND） leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.

Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of β-AMYLASE1 (BAM1) and α-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. 14C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.

Macromolecular crowding affects the physicochemistry of the cytoplasm and, in turn, microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range (a process that we refer to as 'homeocrowding') and discuss possible mechanisms for achieving this. Macromolecular crowding affects the mobility of biomolecules, protein folding and stability, and the association of macromolecules with each other. Local differences in crowding that arise as a result of subcellular components and supramolecular assemblies contribute to the structural organization of the cytoplasm. In this Opinion article we discuss how macromolecular crowding affects the physicochemistry of the cytoplasm and how this, in turn, affects microbial physiology. We propose that cells maintain the overall concentration of macromolecules within a narrow range and discuss possible mechanisms for achieving crowding homeostasis. In addition, we propose that the term 'homeocrowding' is used to describe the process by which cells maintain relatively constant levels of macromolecules.

The plant hormone abscisic acid (ABA) triggers cellular tolerance responses to osmotic stress caused by drought and salinity. ABA controls the turgor pressure of guard cells in the plant epidermis, leading to stomatal closure to minimize water loss. However, stomatal apertures open to uptake CO2 for photosynthesis even under stress conditions. ABA modulates its signaling pathway via negative feedback regulation to maintain plant homeostasis. In the nuclei of guard cells, the clade A type 2C protein phosphatases (PP2Cs) counteract SnRK2 kinases by physical interaction, and thereby inhibit activation of the transcription factors that mediate ABA-responsive gene expression. Under osmotic stress conditions, PP2Cs bind to soluble ABA receptors to capture ABA and release active SnRK2s. Thus, PP2Cs function as a switch at the center of the ABA signaling network. ABA induces the expression of genes encoding repressors or activators of PP2C gene transcription. These regulators mediate the conversion of PP2C chromatins from a repressive to an active state for gene transcription. The stress-induced chromatin remodeling states of ABA-responsive genes could be memorized and transmitted to plant progeny; i.e., transgenerational epigenetic inheritance. This review focuses on the mechanism by which PP2C gene transcription modulates ABA signaling.

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in plant biological processes and stress responses. Here, we characterized the functional roles of BpNAC012 in white birch (Betula platyphylla). We found that BpNAC012 serves as a transcriptional activator. Gain- and loss-of-function analyses revealed that the transcript level of BpNAC012 was positively associated with salt and osmotic stress tolerance. BpNAC012 activated the core sequence CGT[G/A] to induce the expression of abiotic stress-responsive downstream genes, including Δ-1-pyrroline-5-carboxylate synthetase, superoxide dismutase, and peroxidase, resulting in enhanced salt and osmotic stress tolerance in BpNAC012 overexpression transgenic birch lines. We also showed that BpNAC012 is expressed predominantly in mature stems and that RNA interference-induced suppression of BpNAC012 caused a drastic reduction in the secondary wall thickening of stem fibers. Overexpression of BpNAC012 activated the expression of secondary wall-associated downstream genes by directly binding to the secondary wall NAC-binding element sites, resulting in ectopic secondary wall deposition in the stem epidermis. Moreover, salt and osmotic stresses elicited higher expression levels of lignin biosynthetic genes and elevated lignin accumulation in BpNAC012 overexpression lines. These findings provide insight into the functions of NAC transcription factors.

Pollen grains undergo dramatic changes in cellular water potential as they deliver the male germ line to female gametes, and it has been proposed that mechanosensitive ion channels may sense the resulting mechanical stress. Here, we identify and characterize MscS-like 8 (MSL8), a pollen-specific, membrane tension–gated ion channel required for pollen to survive the hypoosmotic shock of rehydration and for full male fertility. MSL8 negatively regulates pollen germination but is required for cellular integrity during germination and tube growth. MSL8 thus senses and responds to changes in membrane tension associated with pollen hydration and germination. These data further suggest that homologs of bacterial MscS have been repurposed in eukaryotes to function as mechanosensors in multiple developmental and environmental contexts.