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Presents results on patch clamp studies of Arabidopsis guard cells and other topics. Review of phosphorylation events regulating guard cell ion channels; Depolarization-activated background single channels in Arabidopsis guard cells; Hyperpolarization-activated background single channels; Instantaneous background currents in Arabidopsis guard cells.

Acidic stress is a formidable environmental factor that exerts adverse effects on plant growth and development, ultimately leading to a potential reduction in agricultural productivity. A low pH triggers Ca 2+ influx across the plasma membrane (PM), eliciting distinct responses under various acidic pH levels. However, the underlying mechanisms by which Arabidopsis plant cells generate stimulus-specific Ca 2+ signals in response to acidic stress remain largely unexplored. The experimentally induced stimulus may elicit spikes in cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) spikes or complex [Ca 2+ ] i oscillations that persist for 20 min over a long-term of 24 h or even several days within the plant cytosol and chloroplast. This study investigated the increase in [Ca 2+ ] i under a gradient of low pH stress ranging from pH 3.0 to 6.0. Notably, the peak of [Ca 2+ ] i elevation was lower at pH 4.0 than at pH 3.0 during the initial 8 h, while other pH levels did not significantly increase [Ca 2+ ] i compared to low acidic stress conditions. Lanthanum chloride (LaCl 3 ) can effectively suppress the influx of [Ca 2+ ] i from the apoplastic to the cytoplasm in plants under acid stress, with no discernible difference in intracellular calcium levels observed in Arabidopsis. Following 8 h of acid treatment in the darkness, the intracellular baseline Ca 2+ levels in Arabidopsis were significantly elevated when exposed to low pH stress. A moderately low pH, specifically 4.0, may function as a spatial-temporal input into the circadian clock system. These findings suggest that acid stimulation can exert a continuous influence on intracellular calcium levels, as well as plant growth and development.

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, are thought to function solely as ion channels. However, most 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 mutations along the transmembrane helix (TM) 4/6 interface allow non-scrambling TCS members to permeate phospholipids. In particular, 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., Competing Interests: AL, PL, MS, YW, ZP, HY, YZ No competing interests declared, (© 2024, Lowry, Liang et al.)

Background: Fried foods are favored for their unique crispiness, golden color and flavor, but they also face great challenge because of their high oil content, high calories and the existence of compounds such as acrylamide and polycyclic aromatic hydrocarbons. Long-term consumption of fried foods may adversely affect health. Therefore, it is necessary to explore fried foods with lower oil contents and a high quality to meet the demand., Results: A method of enzyme treatment was explored to investigate the effects of maltogenic amylase (MA), transglutaminase (TG) and bromelain (BRO) on the physicochemical properties of the batter and the quality of fried spring roll wrapper (FSRW). The results showed that the MA-, TG- or BRO-treated batters had a significant shear-thinning behavior, especially with an increase in viscosity upon increasing TG contents. FSRW enhanced its fracturability from 419.19 g (Control) to 616.50 g (MA-6 U g -1 ), 623.49 g (TG-0.75 U g -1 ) and 644.96 g (BRO-10 U g -1 ). Meanwhile, in comparison with BRO and MA, TG-0.5 U g -1 endowed batter with the highest density and thermal stability. MA-15 U g -1 and TG-0.5 U g -1 displayed FSRW with uniform and dense pores, and significantly reduced its oil content by 18.05% and 25.02%, respectively. Moreover, compared to MA and TG, BRO-50 U g -1 improved the flavor of FSRW., Conclusion: MA, TG or BRO played a key role in affecting the physicochemical properties of the batter and the quality of FSRW. TG-0.5 U g -1 remarkly reduced the oil content of FSRW with a great potential in practical application. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

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 Ca 2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration 1,2 , the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo 3-5 , 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 Ca 2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca 2+ 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 proteins 6 . 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 Ca 2+ -permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca 2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca 2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests D.W. holds equity in Computomics, which advises breeders. D.W. advises KWS SE, a plant breeder and seed producer., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Vitamin B6 is an essential water-soluble vitamin for humans. It is often used to prevent a variety of neuropathies, relieve vomiting, and relieve symptoms such as hand and foot neuritis., Aim: To evaluate whether vitamin B6 can alleviate the adverse reactions caused by the quadruple anti-Helicobacter pylori treatment regimen containing minocycline and metronidazole., Methods: In this randomized controlled trial, 280 patients with H. pylori infection were randomly placed into one of two treatment groups-the conventional treatment group and the vitamin B6 supplement treatment group-for 2 weeks. The primary endpoint was the total incidence of adverse reactions up to 2 weeks after treatment initiation. The study was designed according to CONSORT Medicinal Interventions. And it was registered with Chinese Clinical Trial Registry under the number ChiCTR2100053833., Results: In terms of efficacy, vitamin B6 does not affect the efficacy of conventional regimen. In the vitamin B6 supplement treatment group, the incidence of adverse reactions was 56.92%, which was significantly lower than the 74.62% observed in the conventional treatment group. In addition, the severity of adverse reactions was also significantly reduced. The proportion of moderate to severe central nervous system symptoms decreased from 58.7 to 14.63%. And, the proportion of moderate to severe gastrointestinal reactions decreased from 33.33 to 0%. We speculate that the mechanism of vitamin B6 of reducing adverse reaction may be related to the production of GABA in the brain., Conclusions: Vitamin B6 can alleviate adverse reactions of the quadruple anti-H. pylori regimen containing minocycline and metronidazole., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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.

Obstructive sleep apnea (OSA) is a highly prevalent type of sleep-disordered breathing, which is often comorbid with affective disorders such as anxiety. A 61-year-old woman who was diagnosed with OSA affected by anxiety disorder complained of poor sleep quality at night and anxiety symptoms, and showed chest tightness, dyspnea, snoring, and apnea events during sleep. The patient initially received treatment with positive airway pressure (PAP) combined with trazodone, and subsequently switched to auto-trilevel PAP (AtPAP) combined with trazodone therapy. The initial attempt to treat the patient's disease by auto-adjusting PAP combined with trazodone failed because of central sleep apnea (CSA), which frequently occurred at night. After switching to AtPAP combined with trazodone therapy, CSA was effectively eliminated. In addition, sleep quality, hypoxia, and anxiety disorders were improved. The first report of successful therapy of AtPAP combined with trazodone for OSA complicated by anxiety disorder provides a new therapeutic strategy for this patient population.

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 [Ca 2+ ] 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.

Genetic mutants defective in stimulus-induced Ca 2+ increases have been gradually isolated, allowing the identification of cell-surface sensors/receptors, such as the osmosensor OSCA1. However, determining the Ca 2+ -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 Ca 2+ increases, and demonstrated that osca1 is impaired primarily in Ca 2+ increases induced by the osmotic but not ionic stress. We recorded Ca 2+ increases induced by sorbitol (osmotic effect, OE) and NaCl/CaCl 2 (OE + ionic effect, IE) in Arabidopsis wild-type and osca1 seedlings. We assumed the NaCl/CaCl 2 total effect (TE) = OE + IE, then developed procedures for Ca 2+ 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 Ca 2+ -signaling mutants with distinct stimulus specificity but also the complex Ca 2+ signaling crosstalk among multiple concurrent stresses that occur naturally, and will enable us to specifically fine tune multiple signal pathways to improve crop yields., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Water is crucial to plant growth, development, and environmental adaptation. Water stress triggers cytosolic Ca 2+ ([Ca 2+ ] i ) increases, and the osmosensor OSCA1 (REDUCED-HYPEROSMOLALITY-INDUCED-[Ca 2+ ] 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., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Background: Nodular lymphoid hyperplasia (NLH) in the small intestine is a rare benign lesion characterized by multiple small nodules on the intestinal surface. Patients with terminal ileal NLH may experience long-term abdominal pain, diarrhea, and abdominal distension, among other symptoms. Supplementation with probiotics could mitigate these symptoms. NLH is linked to the immune system, and it may result from accumulation of plasma-cell precursors due to a maturational defect during the development of B lymphocytes. The intestinal microbiome plays an essential role in the immune system. Thus, we speculate that the gut flora plays a key role in terminal ileal NLH., Aim: To explore the correlation between intestinal flora and terminal ileal NLH., Methods: We collected mucosal biopsy samples that were obtained via colonoscopy from 15 patients with terminal ileal NLH (the test group) and 15 normal subjects (the control group). We subsequently performed 16S-rRNA gene amplicon sequencing of these samples, and the results were evaluated using alpha diversity, beta diversity and microbial composition analyses. The Phylogenetic Investigation of Communities by Reconstruction of Unobserved States was used to predict the metabolic pathways and orthologous groups according to the Kyoto Encyclopedia of Genes and Genomes database., Results: Compared with the control group, the terminal ileal NLH group showed an increased alpha diversity ( P < 0.05). The overall intestinal microbiota in the NLH group was significantly different from that of the control group ( P < 0.05), implying that there was the dysbiosis in the terminal ileal NLH patients. The relative abundance of phylum Bacteroidetes was significantly lower in the NLH group, while that of Patescibacteria and Campilobacterota was significantly higher. The genus Bacteroides was the dominant gut microbiota in both groups, but its abundance was significantly lower in the test group than it was in the control group. Conversely, the relative abundances of Haemophilus, Streptococcus, Pseudomonas, Actinomyces, TM7X, Fusobacterium nucleatum, Parvimonas, Granulicatella, Helicobacter , and the [ Eubacterium ] nodatum group were significantly higher in the test group than they were in the control group. In addition, several altered metabolic pathways, orthologous groups, and modules were found. For example, the Peptidoglycan biosynthesis and Aminoacyl tRNA biosynthesis were both increased in the test group., Conclusion: Maintaining the microbial balance and supplementing targeted protective bacteria could improve symptoms and potentially reduce the risk of lymphoma transformation in patients with terminal ileal NLH., Competing Interests: Conflict-of-interest statement: The authors declare that they have no competing interests., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

The flagellin epitope flg22, a pathogen-associated molecular pattern (PAMP), binds to the receptor-like kinase FLAGELLIN SENSING2 (FLS2), and triggers Ca 2+ influx across the plasma membrane (PM). The flg22-induced increases in cytosolic Ca 2+ concentration ([Ca 2+ ] 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 [Ca 2+ ] 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 Ca 2+ permeable channels. Taken together, we have carefully defined the FICA sensitivity adaptation, which will facilitate further molecular and genetic dissection of the Ca 2+ -mediated adaptive mechanisms in PAMP-triggered immunity., (© 2021 John Wiley & Sons Ltd.)

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 (Ca 2+ ) influx in plant and human cells. NRG1.1-dependent Ca 2+ influx and cell death were sensitive to Ca 2+ channel blockers and were suppressed by mutations affecting oligomerization or plasma membrane enrichment. Ca 2+ 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 Ca 2+ -permeable cation channels to directly regulate cytoplasmic Ca 2+ levels and consequent cell death. Thus, helper NLRs transduce cell death signals directly., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The circadian clock is a universal timing system that involved in plant physical responses to abiotic stresses. Moreover, OSCA1 is an osmosensor responsible for [Ca 2+ ] 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 Ca 2+ 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 Ca 2+ processes with other circadian signaling pathways.

Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues 1-4 . H 2 O 2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H 2 O 2 also exist on the cell surface remains unknown. In plant cells, H 2 O 2 triggers an influx of Ca 2+ ions, which is thought to be involved in H 2 O 2 sensing and signalling. Here, by using forward genetic screens based on Ca 2+ imaging, we isolated hydrogen-peroxide-induced Ca 2+ 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 H 2 O 2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H 2 O 2 -induced activation of Ca 2+ channels in guard cells and is required for stomatal closure. Our findings help to identify how the perception of extracellular H 2 O 2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.

Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in cytosolic Ca 2+ concentration, which activate Ca 2+ -binding proteins and upregulate the Na + /H + antiporter in order to remove Na + . Salt-induced increases in Ca 2+ 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 Ca 2+ -imaging-based forward genetic screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca 2+] 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, Ca 2+ spikes and waves, Na + /H + antiporter activation, and regulation of growth. Na + binds to GIPCs to gate Ca 2+ 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.

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.

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 Ca 2+ concentration ([Ca 2+ ] i ) via Ca 2+ influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS), such as H 2 O 2 . It is well established that short-term H 2 O 2 treatment also triggers a transient increase in [Ca 2+ ] i . However, whether and how long-term NaCl and H 2 O 2 treatments affect the basal levels of [Ca 2+ ] i as well as plant responses to additional NaCl and H 2 O 2 stresses remain poorly understood. Using an aequorin-based Ca 2+ imaging assay, we found that the long-term treatment of Arabidopsis seedlings with both moderate NaCl and H 2 O 2 in the growth media reduced the basal [Ca 2+ ] i levels. Interestingly, we found that the long-term treatment with NaCl, but not H 2 O 2, 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 H 2 O 2 might be an integrator of many stresses.

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&lt;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.

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.

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 Ca 2+ concentration ([Ca 2+ ] i ) via Ca 2+ influx across the plasma membrane, as well as bacterial flg22 and plant endogenous peptide Pep1. However, the interaction between abiotic stress-induced [Ca 2+ ] i increases and biotic stress-induced [Ca 2+ ] i increases is still not clear. Employing an aequorin-based Ca 2+ imaging assay, in this work, we investigated the [Ca 2+ ] i changes in response to flg22, Pep1, and NaCl treatments in Arabidopsis thaliana . We observed an additive effect on the [Ca 2+ ] i increase which induced by flg22, Pep1, and NaCl. Our results indicate that biotic and abiotic stresses may activate different Ca 2+ permeable channels. Further, calcium signal induced by biotic and abiotic stresses was independent in terms of spatial and temporal patterning.

Plant glutamate receptor homologs (GLRs) have long been proposed to function as ligand-gated Ca 2+ channels, but no in planta evidence has been provided. Here, we present genetic evidence that Arabidopsis GLR3.1 and GLR3.5 form Ca 2+ 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 Ca 2+ level, defective Ca 2+ -induced stomatal closure, and Ca 2+ -deficient growth disorder, all of which involved L-Met. Patch-clamp analyses of guard cells showed that GLR3.1/3.5 Ca 2+ channels are activated specifically by L-Met, with the activation abolished in glr3.1/3.5. Moreover, GLR3.1/3.5 Ca 2+ channels are distinct from previously characterized ROS-activated Ca 2+ channels and act upstream of ROS, providing Ca 2+ transients necessary for the activation of NADPH oxidases. Our data indicate that GLR3.1/3.5 constitute L-Met-activated Ca 2+ channels responsible for maintaining basal [Ca 2+ ] cyt , play a pivotal role in plant growth, and act upstream of ROS, thereby regulating stomatal aperture., (Copyright © 2016 Institute for Basic Science / DGIST. Published by Elsevier Inc. All rights reserved.)

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.

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca(2+)]i). Considering that in bacteria and animals osmosensing Ca(2+) channels serve as osmosensors, hyperosmolality-induced [Ca(2+)]i increases have been widely speculated to be involved in osmosensing in plants. However, the molecular nature of corresponding Ca(2+) channels remain unclear. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca(2+)]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca(2+)]i increase 1 (osca1), displays impaired osmotic Ca(2+) signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca(2+)]i increases induced by a stimulus in plants, opening up new avenues for studying Ca(2+) machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2',5')pA and A(3',5')pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Salinity is among the environmental factors that affect plant growth and development and constrain agricultural productivity. Salinity stress triggers increases in cytosolic free Ca(2+) concentration ([Ca(2+)]i) via Ca(2+) influx across the plasma membrane. Salinity stress, as well as other stresses, induces the production of reactive oxygen species (ROS). It is well established that ROS also triggers increases in [Ca(2+)]i. However, the relationship and interaction between salinity stress-induced [Ca(2+)]i increases and ROS-induced [Ca(2+)]i increases remain poorly understood. Using an aequorin-based Ca(2+) imaging assay we have analyzed [Ca(2+)]i changes in response to NaCl and H2O2 treatments in Arabidopsis thaliana. We found that NaCl and H2O2 together induced larger increases in [Ca(2+)]i in Arabidopsis seedlings than either NaCl or H2O2 alone, suggesting an additive effect on [Ca(2+)]i increases. Following a pre-treatment with either NaCl or H2O2, the subsequent elevation of [Ca(2+)]i in response to a second treatment with either NaCl or H2O2 was significantly reduced. Furthermore, the NaCl pre-treatment suppressed the elevation of [Ca(2+)]i seen with a second NaCl treatment more than that seen with a second treatment of H2O2. A similar response was seen when the initial treatment was with H2O2; subsequent addition of H2O2 led to less of an increase in [Ca(2+)]i than did addition of NaCl. These results imply that NaCl-gated Ca(2+) channels and H2O2-gated Ca(2+) channels may differ, and also suggest that NaCl- and H2O2-evoked [Ca(2+)]i may reduce the potency of both NaCl and H2O2 in triggering [Ca(2+)]i increases, highlighting a feedback mechanism. Alternatively, NaCl and H2O2 may activate the same Ca(2+) permeable channel, which is expressed in different types of cells and/or activated via different signaling pathways.

Background: Explicit comparisons based on the semantic similarity of Gene Ontology terms provide a quantitative way to measure the functional similarity between gene products and are widely applied in large-scale genomic research via integration with other models. Previously, we presented an edge-based method, Relative Specificity Similarity (RSS), which takes the global position of relevant terms into account. However, edge-based semantic similarity metrics are sensitive to the intrinsic structure of GO and simply consider terms at the same level in the ontology to be equally specific nodes, revealing the weaknesses that could be complemented using information content (IC)., Results and Conclusions: Here, we used the IC-based nodes to improve RSS and proposed a new method, Hybrid Relative Specificity Similarity (HRSS). HRSS outperformed other methods in distinguishing true protein-protein interactions from false. HRSS values were divided into four different levels of confidence for protein interactions. In addition, HRSS was statistically the best at obtaining the highest average functional similarity among human-mouse orthologs. Both HRSS and the groupwise measure, simGIC, are superior in correlation with sequence and Pfam similarities. Because different measures are best suited for different circumstances, we compared two pairwise strategies, the maximum and the best-match average, in the evaluation. The former was more effective at inferring physical protein-protein interactions, and the latter at estimating the functional conservation of orthologs and analyzing the CESSM datasets. In conclusion, HRSS can be applied to different biological problems by quantifying the functional similarity between gene products. The algorithm HRSS was implemented in the C programming language, which is freely available from http://cmb.bnu.edu.cn/hrss.

Maintaining nitric oxide (NO) homeostasis is essential for normal plant physiological processes. However, very little is known about the mechanisms of NO modulation in plants. Here, we report a unique mechanism for the catabolism of NO based on the reaction with the plant hormone cytokinin. We screened for NO-insensitive mutants in Arabidopsis and isolated two allelic lines, cnu1-1 and 1-2 (continuous NO-unstressed 1), that were identified as the previously reported altered meristem program 1 (amp1) and as having elevated levels of cytokinins. A double mutant of cnu1-2 and nitric oxide overexpression 1 (nox1) reduced the severity of the phenotypes ascribed to excess NO levels as did treating the nox1 line with trans-zeatin, the predominant form of cytokinin in Arabidopsis. We further showed that peroxinitrite, an active NO derivative, can react with zeatin in vitro, which together with the results in vivo suggests that cytokinins suppress the action of NO most likely through direct interaction between them, leading to the reduction of endogenous NO levels. These results provide insights into NO signaling and regulation of its bioactivity in plants.

As a second messenger, the free cytosolic calcium ion (Ca(2+)) plays important roles in many biochemical and physiological processes including photosynthesis in plants. In this study, we investigated morphological changes, chlorophyll accumulation and chloroplast development during early photomorphogenesis in etiolated seedlings of both Arabidopsis thaliana wild type (WT) and those with the antisense of CAS, a calcium sensor (CASas). Seedlings were grown at high, medium and low Ca(2+) concentrations to identify the roles of Ca(2+) and CAS in de-etiolation and chloroplast development. The results demonstrated that Ca(2+) and CAS are correlated with de-etiolation of A. thaliana after light exposure. High Ca(2+) significantly increased chlorophyll content and improved chloroplast development in both A. thaliana WT and CASas etiolated seedlings during de-etiolation. The analysis by western blot and real-time fluorescent quantitative polymerase chain reaction indicated that the expression levels of CAS mRNA and protein were upregulated by white light and external Ca(2+) significantly. Etiolated CASas plants showed much lower chlorophyll content and delay of chloroplast development as compared with WT plants, indicating that CAS functions in de-etiolation. All together, we concluded that the de-etiolation in A. thaliana was promoted by the high Ca(2+) concentration and CAS expression to a certain extent., (Copyright © Physiologia Plantarum 2011.)

The Arabidopsis calcium-sensing receptor CAS is a crucial regulator of extracellular calcium-induced stomatal closure. Free cytosolic Ca(2+) (Ca(2+)(i)) increases in response to a high extracellular calcium (Ca(2+)(o)) level through a CAS signalling pathway and finally leads to stomatal closure. Multidisciplinary approaches including histochemical, pharmacological, fluorescent, electrochemical, and molecular biological methods were used to discuss the relationship of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) signalling in the CAS signalling pathway in guard cells in response to Ca(2+)(o). Here it is shown that Ca(2+)(o) could induce H(2)O(2) and NO production from guard cells but only H(2)O(2) from chloroplasts, leading to stomatal closure. In addition, the CASas mutant, the atrbohD/F double mutant, and the Atnoa1 mutant were all insensitive to Ca(2+)(o)-stimulated stomatal closure, as well as H(2)O(2) and NO elevation in the case of CASas. Furthermore, it was found that the antioxidant system might function as a mediator in Ca(2+)(o) and H(2)O(2) signalling in guard cells. The results suggest a hypothetical model whereby Ca(2+)(o) induces H(2)O(2) and NO accumulation in guard cells through the CAS signalling pathway, which further triggers Ca(2+)(i) transients and finally stomatal closure. The possible cross-talk of Ca(2+)(o) and abscisic acid signalling as well as the antioxidant system are discussed.

To assess the potential contribution of nitric oxide (NO) emission from the plants grown under the increasing nitrogen (N) deposition to atmospheric NO budget, the effects of simulated N deposition on NO emission and various leaf traits (e.g., specific leaf area, leaf N concentration, net photosynthetic rate, etc.) were investigated in 79 plant species classified by 13 plant functional groups. Simulated N deposition induced the significant increase of NO emission from most functional groups, especially from conifer, gymnosperm and C(3) herb. Moreover, the change rate of NO emission was significantly correlated with the change rate of various leaf traits. We conclude that the plants grown under atmospheric N deposition, especially in conifer, gymnosperm and C(3) herb, should be taken into account as an important biological source of NO and potentially contribute to atmospheric NO budget., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study characterized the changes of nitric oxide (NO) production during the growth of Microcystis aerugrinosa, a cyanobacterium which usually cause cyanobacterial blooms. Results showed a drastic NO release accompanying with cell density and Chl-a content sharp rises when M. aerugrinosa grew from fifth day to sixth day. Moreover, high N:P ratio accelerated the cyanobacterial growth and NO burst. Sodium nitroprusside, an exogenous NO donor, promoted M. aerugrinosa growth with the optimal concentration of 0.1 mg/L. Experiments by supplementing with sodium nitrite and L-arginine demonstrated NO production in M. aerugrinosa cells was mainly through nitrate reductase (NR) pathway while minorly through NO synthase pathway. All these data suggested M. aerugrinosa produced increasing NO during its growth mainly by NR pathway, during which NO positively regulated the growth of M. aerugrinosa., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hydrogen sulphide (H(2)S) is emerging as a potential messenger molecule involved in modulation of physiological processes in animals and plants. In this report, the role of H(2)S in modulating photosynthesis of Spinacia oleracea seedlings was investigated. The main results are as follows. (i) NaHS, a donor of H(2)S, was found to increase the chlorophyll content in leaves. (ii) Seedlings treated with different concentrations of NaHS for 30 d exhibited a significant increase in seedling growth, soluble protein content, and photosynthesis in a dose-dependent manner, with 100 μM NaHS being the optimal concentration. (iii) The number of grana lamellae stacking into the functional chloroplasts was also markedly increased by treatment with the optimal NaHS concentration. (iv) The light saturation point (Lsp), maximum net photosynthetic rate (Pmax), carboxylation efficiency (CE), and maximal photochemical efficiency of photosystem II (F(v)/F(m)) reached their maximal values, whereas the light compensation point (Lcp) and dark respiration (Rd) decreased significantly under the optimal NaHS concentration. (v) The activity of ribulose-1,5-bisphosphate carboxylase (RuBISCO) and the protein expression of the RuBISCO large subunit (RuBISCO LSU) were also significantly enhanced by NaHS. (vi) The total thiol content, glutathione and cysteine levels, internal concentration of H(2)S, and O-acetylserine(thiol)lyase and L-cysteine desulphydrase activities were increased to some extent, suggesting that NaHS also induced the activity of thiol redox modification. (vii) Further studies using quantitative real-time PCR showed that the gene encoding the RuBISCO large subunit (RBCL), small subunit (RBCS), ferredoxin thioredoxin reductase (FTR), ferredoxin (FRX), thioredoxin m (TRX-m), thioredoxin f (TRX-f), NADP-malate dehydrogenase (NADP-MDH), and O-acetylserine(thiol)lyase (OAS) were up-regulated, but genes encoding serine acetyltransferase (SERAT), glycolate oxidase (GYX), and cytochrome oxidase (CCO) were down-regulated after exposure to the optimal concentration of H(2)S. These findings suggest that increases in RuBISCO activity and the function of thiol redox modification may underlie the amelioration of photosynthesis and that H(2)S plays an important role in plant photosynthesis regulation by modulating the expression of genes involved in photosynthesis and thiol redox modification., (© 2011 The Author(s).)

A proteomic study using 2-D gel electrophoresis and MALDI-TOF MS was performed to characterize the responses of Arabidopsis thaliana plants to simulated acid rain (SiAR) stress, which is a global environmental problem and has become a serious issue in China in recent years. The emphasis of the present study was to investigate the overall protein expression changes when exposed to SiAR. Out of over 1000 protein spots reproducibly resolved, 50 of them changed their abundance by at least 2-fold. Analysis of protein expression patterns revealed that a set of proteins associated with energy production, metabolism, cell rescue, cell defense and protein folding, etc., could play important roles in mediating plant response to SiAR. In addition to this, some proteins involved in stress responses and jasmonic acid pathway are also involved in plant response to SiAR. More interestingly, the expression of several ubiquitination-related proteins changed dramatically after 32-h SiAR treatment, suggesting that they may act as a molecular marker for the injury phenotype caused by SiAR. Based on our results, we proposed a schematic model to explain the mechanisms associated with the systematic response of Arabidopsis plants to SiAR.

We selected six tree species, Pinus massoniana Lamb., Cryptomeria fortunei Hooibr. ex Otto et Dietr., Cunninghamia lanceolata (Lamb.) Hook., Liquidambar formosana Hance, Pinus armandii Franch. and Castanopsis chinensis Hance, which are widely distributed as dominant species in the forest of southern China where acid deposition is becoming more and more serious in recent years. We investigated the effects and potential interactions between simulated acid rain (SiAR) and three calcium (Ca) levels on seed germination, radicle length, seedling growth, chlorophyll content, photosynthesis and Ca content in leaves of these six species. We found that the six species showed different responses to SiAR and different Ca levels. Pinus armandii and C. chinensis were very tolerant to SiAR, whereas the others were more sensitive. The results of significant SiAR × Ca interactions on different physiological parameters of the six species demonstrate that additional Ca had a dramatic rescue effect on the seed germination and seedling growth for the sensitive species under SiAR. Altogether, we conclude that the negative effects of SiAR on seed germination, seedling growth and photosynthesis of the four sensitive species could be ameliorated by Ca addition. In contrast, the physiological processes of the two tolerant species were much less affected by both SiAR and Ca treatments. This conclusion implies that the degree of forest decline caused by long-term acid deposition may be attributed not only to the sensitivity of tree species to acid deposition, but also to the Ca level in the soil.

To assess the suitability of dendrochemistry as an indicator of soil acidification, soil chemistry and tree ring information of Abies fabri were measured at two distinct sites (severe acid deposition site-Emei Mountain and clean site-Gongga Mountain) of the subalpine forest ecosystems of western Sichuan, southwest China. The actual soil acidity (pH) was significantly correlated with some of the recent xylem cation (Ca, Mg, Mn, Al, Sr and Ba) concentrations and their molar ratios. Xylem Ca/Mg and Ca/Mn of A. fabri were ultimately selected to reconstruct the historical changes of soil pH in Emei Mountain and Gongga Mountain, respectively. The validity of those rebuild was also verified to a certain extent. We conclude that xylem cation molar ratios of A. fabri were superior to the single cation concentrations in soil acidity rebuild at the study sites due to normalizing for concentration fluctuations., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plant's mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.

Objective: To study the preliminary antioxidant and antimicrobial activities of polysaccharide extracted from Lycoris aurea., Methods: The scavenging activities of the polysaccharide in vitro on superoxide radical (O2-*), hydroxyl radical (*OH), alkyl radical (R*) and hydrogen peroxide (H2O2) were investigated by modified chemical systems. Meanwhile, the antimicrobial activities were tested using paper-discagar diffusion method., Results: In general, the antioxidant activities of the polysaccharide were lower compared with Vc. However, the scavenging effects to *OH and H2O2 were parallel to Vc. Meanwhile, polysaccharide from Lycoris aurea had strong antimicrobial activities against Micrococcus luteus, Bacillus pumilus and Staphylococcus aureus., Conclusion: The polysaccharide extracted from L. aurea can scavenge *OH and H2O2 effectively and inhibit Gram-positive bacterias.

Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), which in turn directs release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.

It is well known that calcium acts as a vital intracellular second messenger that governs a large array of cellular processes. However, the molecular identification of a receptor for extracellular Ca2+, the extracellular calcium-sensing receptor, has opened up the possibility that Ca2+might also function as a messenger outside the cell. In animals, the Ca2+ sensor is the well-characterized extracellular-Ca2+- sensing receptor (CaR), a G- protein -coupled receptor originally isolated from the parathyroid gland. In addition, other receptors, channels and membrane proteins are all sensitive to external Ca2+ fluctuations. Recently, Han et al have cloned a receptor protein for extracellular calcium in Arabidopsis, which plays a key role in Ca2+-induced stomatal closing. Thus, the cloning of these receptors has prompted the consideration of Ca2+ also functioning as a 'first messenger' in animals and plants.

The correct timing of flowering is essential for plants to maximize reproductive success and is controlled by environmental and endogenous signals. We report that nitric oxide (NO) repressed the floral transition in Arabidopsis thaliana. Plants treated with NO, as well as a mutant overproducing NO (nox1), flowered late, whereas a mutant producing less NO (nos1) flowered early. NO suppressed CONSTANS and GIGANTEA gene expression and enhanced FLOWERING LOCUS C expression, which indicated that NO regulates the photoperiod and autonomous pathways. Because NO is induced by environmental stimuli and constitutively produced, it may integrate both external and internal cues into the floral decision.

Extracellular Ca(2+) (Ca(2+)(o)) is required for various physiological and developmental processes in animals and plants. In response to varied Ca(2+)(o) levels, plants maintain relatively constant internal Ca(2+) content, suggesting a precise regulatory mechanism for Ca(2+) homeostasis. However, little is known about how plants monitor Ca(2+)(o) status and whether Ca(2+)(o)-sensing receptors exist. The effects of Ca(2+)(o) on guard cells in promoting stomatal closure by inducing increases in the concentration of cytosolic Ca(2+) ([Ca(2+)](i)) provide a clue to Ca(2+)(o) sensing. Here we have used a functional screening assay in mammalian cells to isolate an Arabidopsis complementary DNA clone encoding a Ca(2+)-sensing receptor, CAS. CAS is localized to the plasma membrane, exhibits low-affinity/high-capacity Ca(2+) binding, and mediates Ca(2+)(o)-induced [Ca(2+)](i) increases. CAS is expressed predominantly in the shoot, including guard cells. Repression of CAS disrupts Ca(2+)(o) signalling in guard cells, and impairs bolting (swift upward growth at the transition to seed production) in response to Ca(2+) deficiency, so we conclude that CAS may be a primary transducer of Ca(2+)(o) in plants.

Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca(2+) increases and ABA- activation of plasma membrane Ca(2+)-permeable channels in guard cells. Exogenous H(2)O(2) rescues both Ca(2+) channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.

The hormone abscisic acid (ABA) regulates stress responses and developmental processes in plants. Calcium-permeable channels activated by reactive oxygen species (ROS) have been shown recently to function in the ABA signaling network in Arabidopsis guard cells. Here, we report that ABA activation of these I(Ca) Ca(2)+ channels requires the presence of NAD(P)H in the cytosol. The protein phosphatase 2C (PP2C) mutant abi1-1 disrupted ABA activation of I(Ca) channels. Moreover, in abi1-1, ABA did not induce ROS production. Consistent with these findings, in abi1-1, H(2)O(2) activation of I(Ca) channels and H(2)O(2)-induced stomatal closing were not disrupted, suggesting that abi1-1 impairs ABA signaling between ABA reception and ROS production. The abi2-1 mutation, which lies in a distinct PP2C gene, also disrupted ABA activation of I(Ca). However, in contrast to abi1-1, abi2-1 impaired both H(2)O(2) activation of I(Ca) and H(2)O(2)-induced stomatal closing. Furthermore, ABA elicited ROS production in abi2-1. These data suggest a model with the following sequence of events in early ABA signal transduction: ABA, abi1-1, NAD(P)H-dependent ROS production, abi2-1, I(Ca) Ca(2)+ channel activation followed by stomatal closing.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca(2+), calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg(2+) levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg(2+) inhibited fast vacuolar (FV) currents with a K(i) of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg(2+) at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg(2+), cytosolic Ca(2+) at less than 10 µM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg(2+) was present, submicromolar concentrations of cytosolic Ca(2+) activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg(2+)-Ca(2+) effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg(2+) concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca(2+) with an affinity in the submicromolar range and a cytosolic low-affinity Mg(2+)-Ca(2+) binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca(2+) and is inhibitory. In conclusion, cytosolic Mg(2+) sensitizes SV channels to physiological cytosolic Ca(2+) elevations. Furthermore, we propose that cytosolic and vacuolar Mg(2+) concentrations ensure that FV channels do not function as a continuous vacuolar K(+) leak, which would prohibit stomatal opening.

Desiccation of plants during drought can be detrimental to agricultural production. The phytohormone abscisic acid (ABA) reduces water loss by triggering stomatal pore closure in leaves, a process requiring ion-channel modulation by cytoplasmic proteins. Deletion of the Arabidopsis farnesyltransferase gene ERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing. ERA1 was expressed in guard cells. Double-mutant analyses of era1 with the ABA-insensitive mutants abi1 and abi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment.