Rhizobacteria Bacillus spp. mitigate osmotic stress and improve seed germination in mustard by regulating osmolyte and plant hormone signaling.

Drought, a widespread abiotic stressor, exerts a profound impact on agriculture, impeding germination and plant growth, and reducing crop yields. In the present investigation, the osmotolerant rhizobacteria Bacillus casamancensis strain MKS-6 and Bacillus sp. strain MRD-17 were assessed for their effects on molecular processes involved in mustard germination under osmotic stress conditions. Enhancement in germination was evidenced by improved germination percentages, plumule and radicle lengths, and seedling vigor upon rhizobacterial inoculation under no stress and osmotic stress conditions. Under osmotic stress, rhizobacteria stimulated the production of gibberellins and reserve hydrolytic enzymes (lipases, isocitrate lyase, and malate synthase), bolstering germination. Furthermore, these rhizobacteria influenced the plant hormones such as gibberellins and abscisic acid (ABA), as well as signalling pathways, thereby promoting germination under osmotic stress. Reduced proline and glycine betaine accumulation, and down-regulation of transcription factors BjDREB1_2 and BjDREB2 (linked to ABA-independent signalling) in rhizobacteria-inoculated seedlings indicated that bacterial treatment mitigated water deficit stress during germination, independently of these pathways. Hence, the advantageous attributes exhibited by these rhizobacterial strains can be effectively harnessed to alleviate drought-induced stress in mustard crops, potentially through the development of targeted bio-formulations.

How plants sense and respond to osmotic stress.

Drought is one of the most serious abiotic stresses to land plants. Plants sense and respond to drought stress to survive under water deficiency. Scientists have studied how plants sense drought stress, or osmotic stress caused by drought, ever since Charles Darwin, and gradually obtained clues about osmotic stress sensing and signaling in plants. Osmotic stress is a physical stimulus that triggers many physiological changes at the cellular level, including changes in turgor, cell wall stiffness and integrity, membrane tension, and cell fluid volume, and plants may sense some of these stimuli and trigger downstream responses. In this review, we emphasized water potential and movements in organisms, compared putative signal inputs in cell wall-containing and cell wall-free organisms, prospected how plants sense changes in turgor, membrane tension, and cell fluid volume under osmotic stress according to advances in plants, animals, yeasts, and bacteria, summarized multilevel biochemical and physiological signal outputs, such as plasma membrane nanodomain formation, membrane water permeability, root hydrotropism, root halotropism, Casparian strip and suberin lamellae, and finally proposed a hypothesis that osmotic stress responses are likely to be a cocktail of signaling mediated by multiple osmosensors. We also discussed the core scientific questions, provided perspective about the future directions in this field, and highlighted the importance of robust and smart root systems and efficient source-sink allocations for generating future high-yield stress-resistant crops and plants.

Bp-miR408a participates in osmotic and salt stress responses by regulating BpBCP1 in Betula platyphylla.

The microRNAs, which are small RNAs of 18-25 nt in length, act as key regulatory factors in posttranscriptional gene expression during plant growth and development. However, little is known about their regulatory roles in response to stressful environments in birch (Betula platyphylla). Here, we characterized and further explored miRNAs from osmotic- and salt-stressed birch. Our analysis revealed a total of 190 microRNA (miRNA) sequences, which were classified into 180 conserved miRNAs and 10 predicted novel miRNAs based on sequence homology. Furthermore, we identified Bp-miR408a under osmotic and salt stress and elucidated its role in osmotic and salt stress responses in birch. Notably, under osmotic and salt stress, Bp-miR408a contributed to osmotic and salt tolerance sensitivity by mediating various physiological changes, such as increases in reactive oxygen species accumulation, osmoregulatory substance contents and Na+ accumulation. Additionally, molecular analysis provided evidence of the in vivo targeting of BpBCP1 (blue copper protein) transcripts by Bp-miR408a. The overexpression of BpBCP1 in birch enhanced osmotic and salt tolerance by increasing the antioxidant enzyme activity, maintaining cellular ion homeostasis and decreasing lipid peroxidation and cell death. Thus, we reveal a Bp-miR408a-BpBCP1 regulatory module that mediates osmotic and salt stress responses in birch.

The positive impact of the NtTAS14-like1 gene on osmotic stress response in Nicotiana tabacum.

KEY MESSAGE: NtTAS14-like1 enhances osmotic tolerance through coordinately activating the expression of osmotic- and ABA-related genes. Osmotic stress is one of the most important limiting factors for tobacco (Nicotiana tabacum) growth and development. Dehydrin proteins are widely involved in plant adaptation to osmotic stress, but few of these proteins have been functionally characterized in tobacco. Here, to identify genes required for osmotic stress response in tobacco, an encoding dehydrin protein gene NtTAS14-like1 was isolated based on RNA sequence data. The expression of NtTAS14-like1 was obviously induced by mannitol and abscisic acid (ABA) treatments. Knock down of NtTAS14-like1 expression reduced osmotic tolerance, while overexpression of NtTAS14-like1 conferred tolerance to osmotic stress in transgenic tobacco plants, as determined by physiological analysis of the relative electrolyte leakage and malonaldehyde accumulation. Further expression analysis by quantitative real-time PCR indicated that NtTAS14-like1 participates in osmotic stress response possibly through coordinately activating osmotic- and ABA-related genes expression, such as late embryogenesis abundant (NtLEA5), early responsive to dehydration 10C (NtERD10C), calcium-dependent protein kinase 2 (NtCDPK2), ABA-responsive element-binding protein (NtAREB), ABA-responsive element-binding factor 1 (NtABF1), dehydration-responsive element-binding genes (NtDREB2A), xanthoxin dehydrogenase/reductase (NtABA2), ABA-aldehyde oxidase 3 (NtAAO3), 9-cis-epoxycarotenoid dioxygenase (NtNCED3). Together, this study will facilitate to improve our understandings of molecular and functional properties of plant TAS14 proteins and to improve genetic evidence on the involvement of the NtTAS14-like1 in osmotic stress response of tobacco.

Genome-Wide Analysis of Stress-Responsive Genes and Alternative Splice Variants in Arabidopsis Roots under Osmotic Stresses.

Plant roots show distinct gene-expression profiles from those of shoots under abiotic stress conditions. In this study, we performed mRNA sequencing (mRNA-Seq) to analyze the transcriptional profiling of Arabidopsis roots under osmotic stress conditions-high salinity (NaCl) and drought (mannitol). The roots demonstrated significantly distinct gene-expression changes from those of the aerial parts under both the NaCl and the mannitol treatment. We identified 68 closely connected transcription-factor genes involved in osmotic stress-signal transduction in roots. Well-known abscisic acid (ABA)-dependent and/or ABA-independent osmotic stress-responsive genes were not considerably upregulated in the roots compared to those in the aerial parts, indicating that the osmotic stress response in the roots may be regulated by other uncharacterized stress pathways. Moreover, we identified 26 osmotic-stress-responsive genes with distinct expressions of alternative splice variants in the roots. The quantitative reverse-transcription polymerase chain reaction further confirmed that alternative splice variants, such as those for ANNAT4, MAGL6, TRM19, and CAD9, were differentially expressed in the roots, suggesting that alternative splicing is an important regulatory mechanism in the osmotic stress response in roots. Altogether, our results suggest that tightly connected transcription-factor families, as well as alternative splicing and the resulting splice variants, are involved in the osmotic stress response in roots.

The osmotic stress-activated receptor-like kinase DPY1 mediates SnRK2 kinase activation and drought tolerance in Setaria.

Plant genomes encode many receptor-like kinases (RLKs) that localize to the cell surface and perceive a wide variety of environmental cues to initiate downstream signaling cascades. Whether these RLKs participate in dehydration stress signaling in plants is largely unknown. DROOPY LEAF1 (DPY1), a leucine-rich repeat (LRR)-RLK, was recently shown to regulate plant architecture by orchestrating early brassinosteroid signaling in foxtail millet (Setaria italica). Here, we show that DPY1 is essential for the acclimation of foxtail millet to drought stress. DPY1 can be phosphorylated and activated in response to osmotic stress and is required for more than half of osmotic stress-induced global phosphorylation events, including the phosphorylation of sucrose nonfermenting kinase 2s (SnRK2s), the central kinases involved in osmotic stress. DPY1 acts upstream of STRESS-ACTIVATED PROTEIN KINASE 6 (SAPK6, a subclass I SnRK2) and is required for full SAPK6 activation, thereby allowing regulation of downstream genes to mount a response against drought stress. These signaling events are largely independent of DPY1-mediated brassinosteroid signaling. The DPY1-SAPK6 module is specific to seed plants and is absent in ancestral nonseed plants. Our findings reveal a dehydration stress-activated RLK that plays an indispensable role in osmotic stress signaling and mediates SnRK2 activation at the cell surface.

Osmotic stress-induced localisation switch of CBR1 from mitochondria to the endoplasmic reticulum triggers ATP production via ß-oxidation to respond to osmotic shock.

Drought and high salinity are major environmental factors that reduce plant growth and development, leading to loss of plant productivity in agriculture. Under these stress conditions, photosynthesis is greatly suppressed despite the high cellular energy cost of stress response processes. Currently, the process that allows plants to secure the energy required for osmotic stress responses remains elusive. Here, we provide evidence that cytochrome b5 reductase 1 (CBR1), a cytochrome b5 reductase, plays an important role in ATP production in response to NaCl and dehydration stresses. Overexpression and loss of function of CBR1 led to enhanced resistance and sensitivity, respectively, to osmotic stress. Upon exposure to osmotic stress, CBR1 was localised to the endoplasmic reticulum (ER) instead of to mitochondria, where it was localised under normal conditions. Transgenic plants overexpressing ER-targeted CBR1 showed enhanced resistance to osmotic stress. Moreover, CBR1-ER and CBR1-OX plants, had higher levels of ATP and unsaturated fatty acids under osmotic stress. However, these effects were abrogated by thioridazine and 2-deoxy glucose, inhibitors of ß-oxidation and glycolysis, respectively. Based on these results, we propose that ER-localised CBR1 triggers ATP production via the production and ß-oxidation of polyunsaturated fatty acids under osmotic stress.

Knockout of cyclase-associated protein CAP1 confers tolerance towards salt and osmotic stress in Arabidopsis.

As a regulator of actin filament turnover, Arabidopsis thaliana CAP1 plays an important role in plant growth and development. Here, we analyzed the phenotypes of two Arabidopsis cap1 mutants: cap1-1 (a T-DNA insertion mutant) and Cas9-CAP1 (generated by CRISPR-Cas9 gene editing). Phenotypic analysis demonstrated that loss of CAP1 results in defects in seed germination and seedling morphology, with some seedlings exhibiting one or three cotyledons. The cap1-1 mutant took longer than the wild type to complete its life cycle, but its flowering time was normal, indicating that loss of CAP1 prolongs reproductive but not vegetative growth. Moreover, loss of CAP1 severely reduces seed production in self-pollinated plants, due to disruption of pollen tube elongation. RNA-seq and qRT-PCR analyses demonstrated that CAP1 may be involved in osmotic stress responses. Indeed, the cap1-1 mutant showed increased tolerance of salt and mannitol treatment, indicating that CAP1 plays a negative role in osmotic stress tolerance in Arabidopsis. Taken together, our results demonstrate that CAP1 functions not only in plant growth and development, but also in Arabidopsis responses to osmotic stress.

PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions.

Polyglutamine binding protein 5 (PQBP5), also called nucleolar protein 10 (NOL10), binds to polyglutamine tract sequences and is expressed in the nucleolus. Using dynamic imaging of high-speed atomic force microscopy, we show that PQBP5/NOL10 is an intrinsically disordered protein. Super-resolution microscopy and correlative light and electron microscopy method show that PQBP5/NOL10 makes up the skeletal structure of the nucleolus, constituting the granule meshwork in the granular component area, which is distinct from other nucleolar substructures, such as the fibrillar center and dense fibrillar component. In contrast to other nucleolar proteins, which disperse to the nucleoplasm under osmotic stress conditions, PQBP5/NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins. Droplet and thermal shift assays show that the biophysical features of PQBP5/NOL10 remain stable under stress conditions, explaining the spatial role of this protein. PQBP5/NOL10 can be functionally depleted by sequestration with polyglutamine disease proteins in vitro and in vivo, leading to the pathological deformity or disappearance of the nucleolus. Taken together, these findings indicate that PQBP5/NOL10 is an essential protein needed to maintain the structure of the nucleolus.

Tomato (Solanum lycopersicum) WRKY23 enhances salt and osmotic stress tolerance by modulating the ethylene and auxin pathways in transgenic Arabidopsis.

Osmotic stress is one of the biggest problems in agriculture, which adversely affects crop productivity. Plants adopt several strategies to overcome osmotic stresses that include transcriptional reprogramming and activation of stress responses mediated by different transcription factors and phytohormones. We have identified a WRKY transcription factor from tomato, SlWRKY23, which is induced by mannitol and NaCl treatment. Over-expression of SlWRKY23 in transgenic Arabidopsis enhances osmotic stress tolerance to mannitol and NaCl and affects root growth and lateral root number. Transgenic Arabidopsis over-expressing SlWRKY23 showed reduced electrolyte leakage and higher relative water content than Col-0 plants upon mannitol and NaCl treatment. These lines also showed better membrane integrity with lower MDA content and higher proline content than Col-0. Responses to mannitol were governed by auxin as treatment with TIBA (auxin transport inhibitor) negatively affected the osmotic tolerance in transgenic lines by inhibiting lateral root growth. Similarly, responses to NaCl were controlled by ethylene as treatment with AgNO3 (ethylene perception inhibitor) inhibited the stress response to NaCl by suppressing primary and lateral root growth. The study shows that SlWRKY23, a osmotic stress inducible gene in tomato, imparts tolerance to mannitol and NaCl stress through interaction of the auxin and ethylene pathways.

Transcriptomic Analysis in Marine Medaka Gill Reveals That the Hypo-Osmotic Stress Could Alter the Immune Response via the IL17 Signaling Pathway.

Fish gills are the major osmoregulatory tissue that contact the external water environment and have developed an effective osmoregulatory mechanism to maintain cellular function. Marine medaka (Oryzias melastigma) has the ability to live in both seawater and fresh water environments. The present study performed a seawater (SW) to 50% seawater (SFW) transfer, and the gill samples were used for comparative transcriptomic analysis to study the alteration of hypo-osmotic stress on immune responsive genes in this model organism. The result identified 518 differentiated expressed genes (DEGs) after the SW to SFW transfer. Various pathways such as p53 signaling, forkhead box O signaling, and the cell cycle were enriched. Moreover, the immune system was highlighted as one of the top altered biological processes in the enrichment analysis. Various cytokines, chemokines, and inflammatory genes that participate in the IL-17 signaling pathway were suppressed after the SW to SFW transfer. On the other hand, some immunoglobulin-related genes were up-regulated. The results were further validated by real-time qPCR. Taken together, our study provides additional gill transcriptome information in marine medaka; it also supports the notion that osmotic stress could influence the immune responses in fish gills.

The Combined Effect of Heat and Osmotic Stress on Suberization of Arabidopsis Roots.

The simultaneous occurrence of heat stress and drought is becoming more regular as a consequence of climate change, causing extensive agricultural losses. The application of either heat or osmotic stress increase cell-wall suberization in different tissues, which may play a role in improving plant resilience. In this work, we studied how the suberization process is affected by the combination of drought and heat stress by following the expression of suberin biosynthesis genes, cell-wall suberization and the chemical composition in Arabidopsis roots. The Arabidopsis plants used in this study were at the onset of secondary root development. At this point, one can observe a developmental gradient in the main root, with primary development closer to the root tip and secondary development, confirmed by the suberized phellem, closer to the shoot. Remarkably, we found a differential response depending on the root zone. The combination of drought and heat stress increased cell wall suberization in main root segments undergoing secondary development and in lateral roots (LRs), while the main root zone, at primary development stage, was not particularly affected. We also found differences in the overall chemical composition of the cell walls in both root zones in response to combined stress. The data gathered showed that, under combined drought and heat stress, Arabidopsis roots undergo differential cell wall remodeling depending on developmental stage, with modifications in the biosynthesis and/or assembly of major cell wall components.

Genetic and cellular characterization of MscS-like putative channels in the filamentous fungus Aspergillus nidulans.

Mechanosensitive ion channels are integral membrane proteins ubiquitously present in bacteria, archaea, and eukarya. They act as molecular sensors of mechanical stress to serve vital functions such as touch, hearing, osmotic pressure, proprioception and balance, while their malfunction is often associated with pathologies. Amongst them, the structurally distinct MscL and MscS channels from bacteria are the most extensively studied. MscS-like channels have been found in plants and Schizosaccharomyces pombe, where they regulate intracellular Ca2+ and cell volume under hypo-osmotic conditions. Here we characterize two MscS-like putative channels, named MscA and MscB, from the model filamentous fungus Aspergillus nidulans. Orthologues of MscA and MscB are present in most fungi, including relative plant and animal pathogens. MscA/MscB and other fungal MscS-like proteins share the three transmembrane helices and the extended C-terminal cytosolic domain that form the structural fingerprint of MscS-like channels with at least three additional transmembrane segments than Escherichia coli MscS. We show that MscA and MscB localize in Endoplasmic Reticulum and the Plasma Membrane, respectively, whereas their overexpression leads to increased CaCl2 toxicity or/and reduction of asexual spore formation. Our findings contribute to understanding the role of MscS-like channels in filamentous fungi and relative pathogens.

Overexpression of VvASMT1 from grapevine enhanced salt and osmotic stress tolerance in Nicotiana benthamiana.

Salt and drought stresses are major environmental conditions that severely limit grape growth and productivity, while exogenous melatonin can alleviate the drought and salt damage to grapevines. N-acetylserotonin methyltransferase (ASMT) is the key enzyme in melatonin synthesis, which plays a critical role in regulating stress responses. However, the roles of ASMTs from grapevine under drought and salt stresses responses remain largely unclear. In this study, the VvASMT1 gene was isolated from grapevine, and its physiological functions in salt and mimic drought stress tolerance were investigated. Expression pattern analysis revealed that VvASMT1 was significantly induced by different salt and osmotic stresses. Ectopic expression of VvASMT1 in Nicotiana benthamiana significantly enhanced melatonin production in transgenic plants. Compared with wild-type plants, the transgenic lines exhibited a higher germination ratio, longer root length, lower degree of leaf wilting and relative water content (RWC) under salt and osmotic stresses. In addition, under salt and osmotic stresses, overexpression of VvASMT1 improved proline and malondialdehyde (MDA) contents, increased the activity of antioxidant enzymes and decreased the accumulation of reactive oxygen species (ROS). Taken together, our results demonstrate the explicit role of VvASMT1 in salt and osmotic stress responses, which provides a theoretical foundation for the genetic engineering of grapevine.

mTORC2 suppresses cell death induced by hypo-osmotic stress by promoting sphingomyelin transport.

Epithelial cells are constantly exposed to osmotic stress. The influx of water molecules into the cell in a hypo-osmotic environment increases plasma membrane tension as it rapidly expands. Therefore, the plasma membrane must be supplied with membrane lipids since expansion beyond its elastic limit will cause the cell to rupture. However, the molecular mechanism to maintain a constant plasma membrane tension is not known. In this study, we found that the apical membrane selectively expands when epithelial cells are exposed to hypo-osmotic stress. This requires the activation of mTORC2, which enhances the transport of secretory vesicles containing sphingomyelin, the major lipid of the apical membrane. We further show that the mTORC2-Rab35 axis plays an essential role in the defense against hypotonic stress by promoting the degradation of the actin cortex through the up-regulation of PI(4,5)P2 metabolism, which facilitates the apical tethering of sphingomyelin-loaded vesicles to relieve plasma membrane tension.

Expression profiling of TaARGOS homoeologous drought responsive genes in bread wheat.

Drought tolerant germplasm is needed to increase crop production, since water scarcity is a critical bottleneck in crop productivity worldwide. Auxin Regulated Gene involved in Organ Size (ARGOS) is a large protein family of transcription factors that plays a vital role in organ size, plant growth, development, and abiotic stress responses in plants. Although, the ARGOS gene family has been discovered and functionalized in a variety of crop plants, but a comprehensive and systematic investigation of ARGOS genes in locally used commercial wheat cultivars is still yet to be reported. The relative expression of three highly conserved TaARGOS homoeologous genes (TaARGOS-A, TaARGOS-B, TaARGOS-D) was studied in three drought-tolerant (Pakistan-2013, NARC-2009 and NR-499) and three sensitive (Borlaug-2016, NR-514 and NR-516) wheat genotypes under osmotic stress, induced by PEG-6000 at 0 (exogenous control), 2, 4, 6, and 12 h. The normalization of target genes was done using ß-actin as endogenous control, whereas DREB3, as a marker gene was also transcribed, reinforcing the prevalence of dehydration in all stress treatments. Real-time quantitative PCR revealed that osmotic stress induced expression of the three TaARGOS transcripts in different wheat seedlings at distinct timepoints. Overall, all genes exhibited significantly higher expression in the drought-tolerant genotypes as compared to the sensitive ones. For instance, the expression profile of TaARGOS-A and TaARGOS-D showed more than threefold increase at 2 h and six to sevenfold increase after 4 h of osmotic stress. However, after 6 h of osmotic stress these genes started to downregulate, and the lowest gene expression was noticed after 12 h of osmotic stress. Among all the homoeologous genes, TaARGOS-D, in particular, had a more significant influence on controlling plant growth and drought tolerance as it showed the highest expression. Altogether, TaARGOSs are involved in seedling establishment and overall plant growth. In addition, the tolerant group of genotypes had a much greater relative fold expression than the sensitive genotypes. Ultimately, Pakistan-2013 showed the highest relative expression of the studied genes than other genotypes which shows its proficiency to mitigate osmotic stress. Therefore, it could be cultivated in arid and semi-arid regions under moisture-deficient regimes. These findings advocated the molecular mechanism and regulatory roles of TaARGOS genes in plant growth and osmotic stress tolerance in contrasting groups of wheat genotypes, accompanied by the genetic nature of identified genotypes in terms of their potential for drought tolerance.

Root Membrane Ubiquitinome under Short-Term Osmotic Stress.

Osmotic stress can be detrimental to plants, whose survival relies heavily on proteomic plasticity. Protein ubiquitination is a central post-translational modification in osmotic-mediated stress. In this study, we used the K-Ɛ-GG antibody enrichment method integrated with high-resolution mass spectrometry to compile a list of 719 ubiquitinated lysine (K-Ub) residues from 450 Arabidopsis root membrane proteins (58% of which are transmembrane proteins), thereby adding to the database of ubiquitinated substrates in plants. Although no ubiquitin (Ub) motifs could be identified, the presence of acidic residues close to K-Ub was revealed. Our ubiquitinome analysis pointed to a broad role of ubiquitination in the internalization and sorting of cargo proteins. Moreover, the simultaneous proteome and ubiquitinome quantification showed that ubiquitination is mostly not involved in membrane protein degradation in response to short osmotic treatment but that it is putatively involved in protein internalization, as described for the aquaporin PIP2;1. Our in silico analysis of ubiquitinated proteins shows that two E2 Ub-conjugating enzymes, UBC32 and UBC34, putatively target membrane proteins under osmotic stress. Finally, we revealed a positive role for UBC32 and UBC34 in primary root growth under osmotic stress.

Wheat TaTIP4;1 Confers Enhanced Tolerance to Drought, Salt and Osmotic Stress in Arabidopsis and Rice.

Tonoplast aquaporins (intrinsic proteins, TIPs) have been indicated to play important roles in plant tolerance to water deficit and salinity. However, the functions of wheat TIPs in response to the stresses are largely unknown. In this study, we observed that transgenic plants overexpressing wheat TaTIP4;1 in Arabidopsis and rice displayed clearly enhanced seed germination and seedling growth under drought, salt and osmotic stress. Compared with wild type plants, Arabidopsis and rice overexpression lines had heightened water contents, reduced leaf water loss, lowered levels of Na+, Na+/K+, H2O2 and malondialdehyde, and improved activities of catalase and/or superoxide dismutase, and increased accumulation of proline under drought, salinity and/or osmotic stresses. Moreover, the expression levels of multiple drought responsive genes clearly elevated upon water dehydration, and the transcription of some salt responsive genes was markedly induced by NaCl treatment in the overexpression lines. Also, the yeast cells containing TaTIP4;1 showed increased tolerance to NaCl and mannitol, and mutation in one of three serines of TaTIP4;1 caused decreased tolerance to the two stresses. These results suggest that TaTIP4;1 serves as an essential positive regulator of seed germination and seedling growth under drought, salt and/or osmotic stress through impacting water relations, ROS balance, the accumulation of Na+ and proline, and stimulating the expression of dozens of stress responsive genes in Arabidopsis and rice. Phosphorylation may modulate the activity of TaTIP4;1.

Affinity Purification Followed by Liquid Chromatography-Tandem Mass Spectrometry to Identify Proteins Interacting with ABA Signaling Components.

Abscisic acid (ABA) is a key phytohormone involved in plant development, seed germination and responses to osmotic stresses, such as drought and high salinity. SNF1-related protein kinases (SnRK2s) play important roles in ABA-dependent and ABA-independent osmotic stress signaling. SnRK2s phosphorylate transcription factors and ion channels in response to ABA or osmotic stress to induce the expression of stress-responsive genes and stomatal closure, respectively, to confer osmotic stress tolerance. The activity of SnRK2s is directly or indirectly regulated by several protein factors. Identification of downstream substrates or upstream regulators of SnRK2s is very useful for elucidating protein components that regulate ABA and osmotic stress signaling. Here, we describe the use of affinity purification by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry to identify protein complexes involved in ABA and osmotic stress signaling in plants. We previously identified several protein factors that regulate ABA and osmotic stress signaling by using this method.

Quantification of pulsed electric field for the rupture of giant vesicles with various surface charges, cholesterols and osmotic pressures.

Electropermeabilization is a promising phenomenon that occurs when pulsed electric field with high frequency is applied to cells/vesicles. We quantify the required values of pulsed electric fields for the rupture of cell-sized giant unilamellar vesicles (GUVs) which are prepared under various surface charges, cholesterol contents and osmotic pressures. The probability of rupture and the average time of rupture are evaluated under these conditions. The electric field changes from 500 to 410 Vcm-1 by varying the anionic lipid mole fraction from 0 to 0.60 for getting the maximum probability of rupture (i.e., 1.0). In contrast, the same probability of rupture is obtained for changing the electric field from 410 to 630 Vcm-1 by varying the cholesterol mole fraction in the membranes from 0 to 0.40. These results suggest that the required electric field for the rupture decreases with the increase of surface charge density but increases with the increase of cholesterol. We also quantify the electric field for the rupture of GUVs containing anionic mole fraction of 0.40 under various osmotic pressures. In the absence of osmotic pressure, the electric field for the rupture is obtained 430 Vcm-1, whereas the field is 300 Vcm-1 in the presence of 17 mOsmL-1, indicating the instability of GUVs at higher osmotic pressures. These investigations open an avenue of possibilities for finding the electric field dependent rupture of cell-like vesicles along with the insight of biophysical and biochemical processes.