Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

TMEM16 and TMEM63/OSCA proteins share a conserved potential to permeate ions and phospholipids.

The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, likely function solely as ion channels. However, the remaining TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here we show that single lysine mutations in transmembrane helix (TM) 4 allow non-scrambling TCS members to permeate phospholipids. This study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.

Small proteins modulate ion-channel-like ACD6 to regulate immunity in Arabidopsis thaliana.

The multi-pass transmembrane protein ACCELERATED CELL DEATH 6 (ACD6) is an immune regulator in Arabidopsis thaliana with an unclear biochemical mode of action. We have identified two loci, MODULATOR OF HYPERACTIVE ACD6 1 (MHA1) and its paralog MHA1-LIKE (MHA1L), that code for ∼7 kDa proteins, which differentially interact with specific ACD6 variants. MHA1L enhances the accumulation of an ACD6 complex, thereby increasing the activity of the ACD6 standard allele for regulating plant growth and defenses. The intracellular ankyrin repeats of ACD6 are structurally similar to those found in mammalian ion channels. Several lines of evidence link increased ACD6 activity to enhanced calcium influx, with MHA1L as a direct regulator of ACD6, indicating that peptide-regulated ion channels are not restricted to animals.

Reactive Oxygen Species in Drought-Induced Stomatal Closure: The Potential Roles of NPR1.

Stomatal closure is a vital, adaptive mechanism that plants utilize to minimize water loss and withstand drought conditions. We will briefly review the pathway triggered by drought that governs stomatal closure, with specific focuses on salicylic acid (SA) and reactive oxygen species (ROS). We propose that the non-expressor of PR Gene 1 (NPR1), a protein that protects plants during pathogen infections, also responds to SA during drought to sustain ROS levels and prevent ROS-induced cell death. We will examine the evidence underpinning this hypothesis and discuss potential strategies for its practical implementation.

Systematic Characterization of the OSCA Family Members in Soybean and Validation of Their Functions in Osmotic Stress.

Since we discovered OSCA1, a hyperosmolarity-gated calcium-permeable channel that acted as an osmosensor in Arabidopsis, the OSCA family has been identified genome-wide in several crops, but only a few OSCA members' functions have been experimentally demonstrated. Osmotic stress seriously restricts the yield and quality of soybean. Therefore, it is essential to decipher the molecular mechanism of how soybean responds to osmotic stress. Here, we first systematically studied and experimentally demonstrated the role of OSCA family members in the osmotic sensing of soybean. Phylogenetic relationships, gene structures, protein domains and structures analysis revealed that 20 GmOSCA members were divided into four clades, of which members in the same cluster may have more similar functions. In addition, GmOSCA members in clusters III and IV may be functionally redundant and diverged from those in clusters I and II. Based on the spatiotemporal expression patterns, GmOSCA1.6, GmOSCA2.1, GmOSCA2.6, and GmOSCA4.1 were extremely low expressed or possible pseudogenes. The remaining 16 GmOSCA genes were heterologously overexpressed in an Arabidopsis osca1 mutant, to explore their functions. Subcellular localization showed that most GmOSCA members could localize to the plasma membrane (PM). Among 16 GmOSCA genes, only overexpressing GmOSCA1.1, GmOSCA1.2, GmOSCA1.3, GmOSCA1.4, and GmOSCA1.5 in cluster I could fully complement the reduced hyperosmolality-induced [Ca2+]i increase (OICI) in osca1. The expression profiles of GmOSCA genes against osmotic stress demonstrated that most GmOSCA genes, especially GmOSCA1.1, GmOSCA1.2, GmOSCA1.3, GmOSCA1.4, GmOSCA1.5, GmOSCA3.1, and GmOSCA3.2, strongly responded to osmotic stress. Moreover, overexpression of GmOSCA1.1, GmOSCA1.2, GmOSCA1.3, GmOSCA1.4, GmOSCA1.5, GmOSCA3.1, and GmOSCA3.2 rescued the drought-hypersensitive phenotype of osca1. Our findings provide important clues for further studies of GmOSCA-mediated calcium signaling in the osmotic sensing of soybean and contribute to improving soybean drought tolerance through genetic engineering and molecular breeding.

OSCA1 is an osmotic specific sensor: a method to distinguish Ca2+ -mediated osmotic and ionic perception.

Genetic mutants defective in stimulus-induced Ca2+ increases have been gradually isolated, allowing the identification of cell-surface sensors/receptors, such as the osmosensor OSCA1. However, determining the Ca2+ -signaling specificity to various stimuli in these mutants remains a challenge. For instance, less is known about the exact selectivity between osmotic and ionic stresses in the osca1 mutant. Here, we have developed a method to distinguish the osmotic and ionic effects by analyzing Ca2+ increases, and demonstrated that osca1 is impaired primarily in Ca2+ increases induced by the osmotic but not ionic stress. We recorded Ca2+ increases induced by sorbitol (osmotic effect, OE) and NaCl/CaCl2 (OE + ionic effect, IE) in Arabidopsis wild-type and osca1 seedlings. We assumed the NaCl/CaCl2 total effect (TE) = OE + IE, then developed procedures for Ca2+ imaging, image analysis and mathematic fitting/modeling, and found osca1 defects mainly in OE. The osmotic specificity of osca1 suggests that osmotic and ionic perceptions are independent. The precise estimation of these two stress effects is applicable not only to new Ca2+ -signaling mutants with distinct stimulus specificity but also the complex Ca2+ signaling crosstalk among multiple concurrent stresses that occur naturally, and will enable us to specifically fine tune multiple signal pathways to improve crop yields.

Evolution of osmosensing OSCA1 Ca2+ channel family coincident with plant transition from water to land.

Water is crucial to plant growth, development, and environmental adaptation. Water stress triggers cytosolic Ca2+ ([Ca2+ ]i ) increases, and the osmosensor OSCA1 (REDUCED-HYPEROSMOLALITY-INDUCED-[Ca2+ ]i -INCREASE 1), a member of the OSCA family, perceives the initial water stress and governs its downstream responses. OSCA homologs exist in eukaryotes and largely radiate in higher plants. However, it is enigmatic whether the OSCA family is crucial for plant evolution from aqueous to terrestrial environments and for the subsequent adaptation on land. Here, we carried out the first phylogenetic and molecular evolutionary analyses of the OSCA family. The family originated and diversified during the early evolution of protists, and three more lineages were established (a) in plants, (b) in fungi, and (c) in a complex clade of several major eukaryotic lineages. The chlorophyte algal cluster is directly basal to streptophyte-specific Clades 1-3, consistent with plant transition from water to land. The Clades 1-3 present different gene expansion pattern and together with previous functional analysis of OSCAs reveal that they probably have evolved diverse functions in respond to various mechanical stresses during the independent evolution of land plant clades. Moreover, variable selection pressures on different land plant lineages were explored. OSCAs in early land plants (mosses and lycophytes) were under decelerated evolution, whereas OSCAs in seed plants showed accelerated evolution. Together, we hypothesize OSCAs have evolved to sense water stress in the ancestor of euphyllophytes, which occupies typical leaves, typical roots, and phloem tissues, all of which require osmosensors to maintain water balance and food conduction through plant bodies.

Flg22-induced Ca2+ increases undergo desensitization and resensitization.

The flagellin epitope flg22, a pathogen-associated molecular pattern (PAMP), binds to the receptor-like kinase FLAGELLIN SENSING2 (FLS2), and triggers Ca2+ influx across the plasma membrane (PM). The flg22-induced increases in cytosolic Ca2+ concentration ([Ca2+ ]i ) (FICA) play a crucial role in plant innate immunity. It's well established that the receptor FLS2 and reactive oxygen species (ROS) burst undergo sensitivity adaptation after flg22 stimulation, referred to as desensitization and resensitization, to prevent over responses to pathogens. However, whether FICA also mount adaptation mechanisms to ensure appropriate and efficient responses against pathogens remains poorly understood. Here, we analysed systematically [Ca2+ ]i increases upon two successive flg22 treatments, recorded and characterized rapid desensitization but slow resensitization of FICA in Arabidopsis thaliana. Pharmacological analyses showed that the rapid desensitization might be synergistically regulated by ligand-induced FLS2 endocytosis as well as the PM depolarization. The resensitization of FICA might require de novo FLS2 protein synthesis. FICA resensitization appeared significantly slower than FLS2 protein recovery, suggesting additional regulatory mechanisms of other components, such as flg22-related Ca2+ permeable channels. Taken together, we have carefully defined the FICA sensitivity adaptation, which will facilitate further molecular and genetic dissection of the Ca2+ -mediated adaptive mechanisms in PAMP-triggered immunity.

Plant "helper" immune receptors are Ca2+-permeable nonselective cation channels.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) regulate immunity and cell death. In Arabidopsis, a subfamily of "helper" NLRs is required by many "sensor" NLRs. Active NRG1.1 oligomerized, was enriched in plasma membrane puncta, and conferred cytoplasmic calcium ion (Ca2+) influx in plant and human cells. NRG1.1-dependent Ca2+ influx and cell death were sensitive to Ca2+ channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca2+ influx and cell death mediated by NRG1.1 and ACTIVATED DISEASE RESISTANCE 1 (ADR1), another helper NLR, required conserved negatively charged N-terminal residues. Whole-cell voltage-clamp recordings demonstrated that Arabidopsis helper NLRs form Ca2+-permeable cation channels to directly regulate cytoplasmic Ca2+ levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly.

Osmotic stress alters circadian cytosolic Ca2+ oscillations and OSCA1 is required in circadian gated stress adaptation.

The circadian clock is a universal timing system that involved in plant physical responses to abiotic stresses. Moreover, OSCA1 is an osmosensor responsible for [Ca2+]i increases induced by osmotic stress in plants. However, there is little information on osmosensor involved osmotic stress-triggered circadian clock responses. Using an aequorin-based Ca2+ imaging assay, we found the gradient (0 mM, 200 mM, 500 mM) osmotic stress (induced by sorbitol) both altered the primary circadian parameter of WT and osca1 mutant. This means the plant switch to a fast day/night model to avoid energy consumption. In contrast, the period of WT and osca1 mutant became short since the sorbitol concentration increased from 0 mM to 500 mM. As the sorbitol concentration increased, the phase of the WT becomes more extensive compared with osca1 mutant, which means WT is more capable of coping with the environmental change. Moreover, the amplitude of WT also becomes broader than osca1 mutant, especially in high (500 mM) sorbitol concentration, indicate the WT shows more responses in high osmotic stress. In a word, the WT has much more flexibility to cope with the osmotic stress than osca1 mutant. It implies the OSCA1 might be involved in the circadian gated plant adaptation to the environmental osmotic stress, which opens an avenue to study Ca2+ processes with other circadian signaling pathways.

Hydrogen peroxide sensor HPCA1 is an LRR receptor kinase in Arabidopsis.

Hydrogen peroxide (H2O2) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues1-4. H2O2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H2O2 also exist on the cell surface remains unknown. In plant cells, H2O2 triggers an influx of Ca2+ ions, which is thought to be involved in H2O2 sensing and signalling. Here, by using forward genetic screens based on Ca2+ imaging, we isolated hydrogen-peroxide-induced Ca2+ increases (hpca) mutants in Arabidopsis, and identified HPCA1 as a leucine-rich-repeat receptor kinase belonging to a previously uncharacterized subfamily that features two extra pairs of cysteine residues in the extracellular domain. HPCA1 is localized to the plasma membrane and is activated by H2O2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H2O2-induced activation of Ca2+ channels in guard cells and is required for stomatal closure. Our findings help to identify how the perception of extracellular H2O2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.

Plant cell-surface GIPC sphingolipids sense salt to trigger Ca2+ influx.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca2+ concentration, which activate Ca2+-binding proteins and upregulate the Na+/H+ antiporter in order to remove Na+. Salt-induced increases in Ca2+ have long been thought to be involved in the detection of salt stress, but the molecular components of the sensing machinery remain unknown. Here, using Ca2+-imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca2+]i increases 1 (moca1), and identified MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca2+ spikes and waves, Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca2+ influx channels. This salt-sensing mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and could be used to improve salt resistance in crops.

Plasmonic Nanoprobes for in Vivo Multimodal Sensing and Bioimaging of MicroRNA within Plants.

Monitoring gene expression within whole plants is critical for many applications ranging from plant biology to agricultural biotechnology and biofuel development; however, no method currently exists for in vivo monitoring of genomic targets in plant systems without requiring sample extraction. Herein, we report a unique multimodal method based on plasmonic nanoprobes capable of in vivo imaging and biosensing of microRNA biotargets within whole plant leaves by integrating three different and complementary techniques: surface-enhanced Raman scattering (SERS), X-ray fluorescence (XRF), and plasmonics-enhanced two-photon luminescence (TPL). The method developed uses plasmonic nanostars, which not only provide large Raman signal enhancement but also allow for localization and quantification by XRF and plasmonics-enhanced TPL, owing to gold content and high two-photon luminescence cross sections. Our method uses inverse molecular sentinel nanoprobes for SERS bioimaging of microRNA within Arabidopsis thaliana leaves to provide a dynamic SERS map of detected microRNA targets while also quantifying nanoprobe concentrations using XRF and TPL. The nanoprobes were observed to occupy the intercellular spaces upon infiltration into the leaf tissues. This report lays the foundation for the use of plasmonic nanoprobes for in vivo functional imaging of nucleic acid biotargets in whole plants, a tool that will revolutionize bioengineering research by allowing the study of these biotargets with previously unmet spatial and temporal resolution, 200 µm and 30 min, respectively.

Both NaCl and H2O2 Long-Term Stresses Affect Basal Cytosolic Ca2+ Levels but Only NaCl Alters Cytosolic Ca2+ Signatures in Arabidopsis.

Salinity is one of the formidable environmental factors that affect plant growth and development and constrain agricultural productivity. Experimentally imposed short-term NaCl treatment triggers a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS), such as H2O2. It is well established that short-term H2O2 treatment also triggers a transient increase in [Ca2+]i. However, whether and how long-term NaCl and H2O2 treatments affect the basal levels of [Ca2+]i as well as plant responses to additional NaCl and H2O2 stresses remain poorly understood. Using an aequorin-based Ca2+ imaging assay, we found that the long-term treatment of Arabidopsis seedlings with both moderate NaCl and H2O2 in the growth media reduced the basal [Ca2+]i levels. Interestingly, we found that the long-term treatment with NaCl, but not H2O2, affected the responses of plants to additional NaCl stress, and remarkably the roots displayed enhanced responses while the leaves showed reduced responses. These findings suggest that plants adapt to the long-term NaCl stress, while H2O2 might be an integrator of many stresses.

GSK2193874 treatment at heatstroke onset reduced cell apoptosis in heatstroke mice.

Heatstroke is still a potentially fatal threat during summer heat waves, despite improved prevention and treatment. It is reported that the transient receptor potential vanilloid 4 (TRPV4) inhibitor may protect septicemia mice. Many aspects of heatstroke have been defined, from the sepsis-mimic inflammatory response to hyperthermia. Hence, TRPV4 may be a therapeutic target for heatstroke. The results in murine models of heatstroke verified that GSK2193874, as a selected TRPV4 inhibitor, was injected at heatstroke onset, and then reduced the reduction of core temperature, the death rate, wet/dry ratio of the lung, levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, coagulation indicators, the degree of organ injury, and caspase-3/7 activity (P<0.05). But GSK2193874 treatment before heat stress did not improve the symptoms of heatstroke mice. Therefore, TRPV4 should be involved in heatstroke-induced injury. Timely GSK2193874 administration may be useful to reduce heatstroke-induced injury. TRPV4 may be a potential new therapeutic target in fatal heatstroke.

Sustenance of endothelial cell stability in septic mice through appropriate activation of transient receptor potential vanilloid-4.

Therapeutic target transient receptor potential vanilloid-4 (TRPV-4) is frequently applied in endotoxemia research. It has been reported that HC067047, an inhibitor of TRPV-4, mitigated LPS-induced injury. However, the inhibition of TRPV-4 with HC06047 did not attenuate LPS-induced symptoms and exaggerated pathology. This study was carried with a view to unravelling the reason(s) behind these conflicting results. Different doses of the inhibitor were used in the same degree of sepsis, and their effects were determined through assays for sepsis-related physiological indicators such as endothelial injury markers, coagulation index, organ damage indicators, inflammatory factor levels, and cell apoptosis. The results showed that high or low inhibitor levels had no significant effect on sepsis-related physiological indicators. These findings suggest that proper activation of TRPV-4 in sepsis is important for maintaining normal physiological function. Thus, the degree of TRPV-4 activation should match the severity of sepsis.

Biotic and Abiotic Stresses Activate Different Ca2+ Permeable Channels in Arabidopsis.

To survive, plants must respond rapidly and effectively to various stress factors, including biotic and abiotic stresses. Salinity stress triggers the increase of cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane, as well as bacterial flg22 and plant endogenous peptide Pep1. However, the interaction between abiotic stress-induced [Ca2+]i increases and biotic stress-induced [Ca2+]i increases is still not clear. Employing an aequorin-based Ca2+ imaging assay, in this work, we investigated the [Ca2+]i changes in response to flg22, Pep1, and NaCl treatments in Arabidopsis thaliana. We observed an additive effect on the [Ca2+]i increase which induced by flg22, Pep1, and NaCl. Our results indicate that biotic and abiotic stresses may activate different Ca2+ permeable channels. Further, calcium signal induced by biotic and abiotic stresses was independent in terms of spatial and temporal patterning.

L-Met Activates Arabidopsis GLR Ca2+ Channels Upstream of ROS Production and Regulates Stomatal Movement.

Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca2+ channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca2+ channels activated by L-methionine (L-Met) at physiological concentrations and regulate stomatal apertures and plant growth. The glr3.1/3.5 mutations resulted in a lower cytosolic Ca2+ level, defective Ca2+-induced stomatal closure, and Ca2+-deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca2+ channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca2+ channels are distinct from previously characterized ROS-activated Ca2+ channels and act upstream of ROS, providing Ca2+ transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca2+ channels responsible for maintaining basal [Ca2+]cyt, play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture.

Genome-wide survey and expression analysis of the OSCA gene family in rice.

BACKGROUND: Reception of and response to exogenous and endogenous osmotic changes is important to sustain plant growth and development, as well as reproductive formation. Hyperosmolality-gated calcium-permeable channels (OSCA) were first characterised as an osmosensor in Arabidopsis and are involved in the perception of extracellular changes to trigger hyperosmolality-induced [Ca(2+)]i increases (OICI). To explore the potential biological functions of OSCAs in rice, we performed a bioinformatics and expression analysis of the OsOSCA gene family. RESULTS: A total of 11 OsOSCA genes were identified from the genome database of Oryza sativa L. Japonica. Based on their sequence composition and phylogenetic relationship, the OsOSCA family was classified into four clades. Gene and protein structure analysis indicated that the 11 OsOSCAs shared similar structures with their homologs in Oryza sativa L. ssp. Indica, Oryza glaberrima, and Oryza brachyantha. Multiple sequence alignment analysis revealed a conserved DUF221 domain in these members, in which the first three TMs were conserved, while the others were not. The expression profiles of OsOSCA genes were analysed at different stages of vegetative growth, reproductive development, and under osmotic-associated abiotic stresses. We found that four and six OsOSCA genes showed a clear correlation between the expression profile and osmotic changes during caryopsis development and seed imbibition, respectively. Orchestrated transcription of three OsOSCAs was strongly associated with the circadian clock. Moreover, osmotic-related abiotic stress differentially induced the expression of 10 genes. CONCLUSION: The entire OSCA family is characterised by the presence of a conserved DUF221 domain, which functions as an osmotic-sensing calcium channel. The phylogenetic tree of OSCA genes showed that two subspecies of cultivated rice, Oryza sativa L. ssp. Japonica and Oryza sativa L. ssp. Indica, are more closely related than wild rice Oryza glaberrima, while Oryza brachyantha was less closely related. OsOSCA expression is organ- and tissue-specific and regulated by different osmotic-related abiotic stresses in rice. These findings will facilitate further research in this gene family and provide potential target genes for generation of genetically modified osmotic-stress-resistant plants.