scholarly journals SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity

2007 ◽  
Vol 27 (22) ◽  
pp. 7781-7790 ◽  
Author(s):  
Giorgia Batelli ◽  
Paul E. Verslues ◽  
Fernanda Agius ◽  
Quansheng Qiu ◽  
Hiroaki Fujii ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Atpase Activity ◽  
Ion Transport ◽  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Transport Activity ◽  
Sos Pathway

ABSTRACT The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo

PROTOPLASMA ◽  
2016 ◽  
Vol 254 (4) ◽  
pp. 1547-1562 ◽  
Author(s):  
Jana Majerská ◽  
Petra Procházková Schrumpfová ◽  
Ladislav Dokládal ◽  
Šárka Schořová ◽  
Karel Stejskal ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Tandem Affinity Purification ◽  
Interaction Partners

scholarly journals The signaling lipid PI(3,5)P2 stabilizes V1–Vo sector interactions and activates the V-ATPase

2014 ◽  
Vol 25 (8) ◽  
pp. 1251-1262 ◽  
Author(s):  
Sheena Claire Li ◽  
Theodore T. Diakov ◽  
Tao Xu ◽  
Maureen Tarsio ◽  
Wandi Zhu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Atpase Activity ◽  
Human Disease ◽  
Proton Pumps ◽  
Disease Phenotypes ◽  
Reversible Dissociation ◽  
Signaling Mechanisms ◽  
Vacuolar Acidification ◽  
Direct Binding

Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane Vo domain. Multiple stresses induce changes in V1-Vo assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-Vo assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified Vo complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of Vo-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-Vo interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-Vo assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1∆ and vac14∆ mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.

scholarly journals Evidence for High-Affinity Ca2+-ATPase Activity and ATP-Driven Ca2+-Transport in Membrane Preparations of the Gill Epithelium of the Cichlid Fish Oreochromis Mossambicus

1985 ◽  
Vol 119 (1) ◽  
pp. 335-347 ◽  
Author(s):  
GERT FLIK ◽  
JEANNE H. VAN RIJS ◽  
SJOERD E. WENDELAAR BONGA
Keyword(s):  
Atpase Activity ◽  
Plasma Membranes ◽  
Cichlid Fish ◽  
Transport Activity ◽  
High Affinity ◽  
Vesicle Preparation ◽  
Extrusion Mechanism ◽  
Branchial Epithelium ◽  
Inside Out

A high-affinity Ca2+-ATPase activity was demonstrated among the phosphatase activities in plasma membranes of tilapia branchial epithelium: its characteristics (K0.5 = 0.063 μmol l−1 Ca2+, Vmax = 6.02 -mol P1h−;1 mg−1 protein at 37°C) resemble those of Ca2+-translocating enzymes present in mammalian erythrocytes or enterocytes. The ratio of this Ca2+-ATPase activity to Na+/K+-ATPase activity was 1:20.4. Radioimmunoassayable calmodulin was demonstrated in the Ca2+-ATPase-containing membrane fraction. ATP-dependent Ca2+-transport was demonstrated in tight-vesicle preparations of the branchial cell membranes; 30 % of the vesicles in the preparation were inside-out, 44 % were right-side-out and 26 % were leaky. The characteristics of the active Ca2+-transport activity are consistent with a Ca2+-extrusion mechanism involving high-affinity Ca2+-ATPase activity. The branchial Ca2+-transport activity per fish, as calculated on the basis of the transport activity determined for the vesicle preparation, is of the order of the branchial Ca2+-influx rates observed in vivo. The data provide the first biochemical evidence for active Ca2+ -transport in plasma membranes of branchial epithelium. A model is presented for the mechanism of active transepithelial Ca2+-transport in fish gills.

scholarly journals Agronomical, Physiological and Biochemical Characterization of In Vitro Selected Eggplant Somaclonal Variants under NaCl Stress

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2544
Author(s):  
Sami Hannachi ◽  
Stefaan Werbrouck ◽  
Insaf Bahrini ◽  
Abdelmuhsin Abdelgadir ◽  
Hira Affan Siddiqui
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Normal Growth ◽  
Nacl Stress ◽  
Soluble Carbohydrates ◽  
Regenerated Plants ◽  
And Control ◽  
Physiological And Biochemical

Previously, an efficient regeneration protocol was established and applied to regenerate plants from calli lines that could grow on eggplant leaf explants after a stepwise in vitro selection for tolerance to salt stress. Plants were regenerated from calli lines that could tolerate up to 120 mM NaCl. For further in vitro and in vivo evaluation, four plants with a higher number of leaves and longer roots were selected from the 32 plants tested in vitro. The aim of this study was to confirm the stability of salt tolerance in the progeny of these four mutants (‘R18’, ‘R19’, ‘R23’ and ‘R30’). After three years of in vivo culture, we evaluated the impact of NaCl stress on agronomic, physiological and biochemical parameters compared to the parental control (‘P’). The regenerated and control plants were assessed under in vitro and in vivo conditions and were subjected to 0, 40, 80 and 160 mM of NaCl. Our results show significant variation in salinity tolerance among regenerated and control plants, indicating the superiority of four regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) when compared to the parental line (‘P’). In vitro germination kinetics and young seedling growth divided the lines into a sensitive and a tolerant group. ‘P’ tolerate only moderate salt stress, up to 40 mM NaCl, while the tolerance level of ‘R18’, ‘R19’, ‘R23’ and ‘R30’ was up to 80 mM NaCl. The quantum yield of PSII (ΦPSII) declined significantly in ‘P’ under salt stress. The photochemical quenching was reduced while nonphotochemical quenching rose in ‘P’ under salt stress. Interestingly, the regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) exhibited high apparent salt tolerance by maintaining quite stable Chl fluorescence parameters. Rising NaCl concentration led to a substantial increase in foliar proline, malondialdehyde and soluble carbohydrates accumulation in ‘P’. On the contrary, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ exhibited a decline in soluble carbohydrates and a significant enhancement in starch under salinity conditions. The water status reflected by midday leaf water potential (ψl) and leaf osmotic potential (ψπ) was significantly affected in ‘P’ and was maintained a stable level in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ under salt stress. The increase in foliar Na+ and Cl− content was more accentuated in parental plants than in regenerated plants. The leaf K+, Ca2+ and Mg2+ content reduction was more aggravated under salt stress in ‘P’. Under increased salt concentration, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ associate lower foliar Na+ content with a higher plant tolerance index (PTI), thus maintaining a normal growth, while foliar Na+ accumulation was more pronounced in ‘P’, revealing their failure in maintaining normal growth under salinity stress. ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an obvious salt tolerance by maintaining significantly high chlorophyll content. In ‘R18’, ‘R19’, ‘R23’ and ‘R30’, the enzyme scavenging machinery was more performant in the roots compared to the leaves. Salt stress led to a significant augmentation of catalase, ascorbate peroxidase and guaiacol peroxidase activities in the roots of ‘R18’, ‘R19’, ‘R23’ and ‘R30’. In contrast, enzyme activities were less enhanced in ‘P’, indicating lower efficiency to cope with oxidative stress than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. ACC deaminase activity was significantly higher in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. The present study suggests that regenerated plants ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an evident stability in tolerating salinity, which shows their potential to be adopted as interesting selected mutants, providing the desired salt tolerance trait in eggplant.

scholarly journals Responses of own-rooted grape cultivars to salt stress in vivo and in vitro

2020 ◽  
pp. 199-209
Author(s):  
Ирина Ильинична Рыфф ◽  
Светлана Петровна Березовская
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Leaf Area ◽  
Grape Juice ◽  
Testing System ◽  
Potted Plants ◽  
Shoot Number ◽  
Grape Cultivars

В связи с усиливающейся засоленностью почв в мировом масштабе возникает настоятельная потребность в методах тестирования солетолерантности у сельскохозяйственных культур. Цель настоящего исследования заключалась в оценке толерантности трех корнесобственных сортов винограда к хлориду натрия в условиях in vitro и в вегетационной культуре. Солевой стресс in vitro моделировали добавлением в среду NaCl в концентрациях 0, 50 и 100 mM. В качестве индикаторов солетолерантности использовали площадь листьев и общую длину корней. Солевой стресс in vivo обеспечивался орошением в течение 75 дней водой с содержанием NaCl в концентрациях 0, 80, 100 and 120 mM. Солетолерантность растений оценивали на основании агробиологических параметров (площадь листьев, длина побегов, нагрузка побегами, одревеснение побегов) и анализом урожая (характеристики гроздей, урожай с куста, массовая концентрация сахаров, титруемая кислотность и pH сока). Также определяли водные потенциалы листьев как показатели водного баланса растений и электропроводность почвы в качестве показателя засоленности. Реакции на солевой стресс растений, выращиваемых in vitro и в горшечной культуре, продемонстрировали полную корреляцию. Increasing soil salinity on a global level gives rise to an imperative need for methods to test salt tolerance in crops. This study aimed to evaluate the tolerance of three own-rooted grape cultivars to potassium chloride both in an in vitro testing system and potted culture. Salt stress in vitro was modeled by treating in vitro-grown plants with NaCl at 0, 50, and 100 mM. Leaf area and total root length were used as indicators of salt tolerance. Salt stress in potted culture was achieved by irrigation for 75 days with water containing NaCl at 0, 80, 100, and 120 mM. Salt tolerance of potted plants was evaluated by parameters of viticultural performance (leaf area, shoot length, shoot number, shoot lignification), yield (characteristics of bunches, yield per plant, sugars, titratable acids and pH of grape juice). Leaf water potentials as a measure of the water balance of the plants and electrical conductivity of the soil as another indicator of salinity were also determined. Responses of in vitro-grown and potted plants to salt stress correlated closely.

scholarly journals Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Dongjin Jeong ◽  
Hye Sung Kim ◽  
Hye Young Kim ◽  
Min Jueng Kang ◽  
Hyeryeon Jung ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Spectrometry Analysis ◽  
Soluble Fas ◽  
Soluble Fas Ligand ◽  
Deficient Mice

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Faslgld/gld)- and soluble FasL (FaslΔs/Δs)-deficient mice, but not in Fas (Faslpr/lpr and Fas–/–)- or membrane FasL (FaslΔm/Δm)-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL–Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-κB-dependent manner. Moreover, the sFasL–DR5 interaction-mediated CX3CL1–CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL–DR5 interaction promotes inflammation and is a potential therapeutic target.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance

2020 ◽  
Vol 40 (6) ◽  
pp. 731-745
Author(s):  
Huilong Zhang ◽  
Chen Deng ◽  
Xia Wu ◽  
Jun Yao ◽  
Yanli Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Salt Tolerance ◽  
Transgenic Plants ◽  
Recombinant Protein ◽  
Atpase Activity ◽  
Populus Euphratica ◽  
Hydrolytic Activity ◽  
In Vitro Assays

Abstract Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

scholarly journals Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses

2021 ◽  
Author(s):  
Andreas W Heumüller ◽  
Alisha Nicole Jones ◽  
André Mourão ◽  
Marius Klangwart ◽  
Chenyue Shi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Laminar Flow ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Circular Rnas ◽  
Cell Orientation ◽  
Spectrometry Analysis ◽  
Functional Conservation

Background : Circular RNAs (circRNAs) are generated by back-splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. Methods: Mechanistically, we identified the protein syndesmos (SDOS) to specifically interact with cZNF292 in endothelial cells by RNA affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganisation thereby recapitulating cZfp292 phenotypes. Results: Combining published endothelial RNA sequencing datasets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localisation and focal adhesion organisation. Conclusion: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

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