Plasma membrane-anchored fluorescent tracker based on boron-dipyrromethene

The construction of a stable-membrane tracker has significant implications for the visualization of the membrane in live cells.However,most current plasma trackers are not suitable for tracking plasma membranes for a long time due to their limited retention time.Herein,Mem580-F-Sulfo is designed to target and anchor cell membranes and therefore track cell membranes for a longer time.This tracker is composed of a lipophilic boron-dipyrromethene(BODIPY)derivative and a hydrophilic zwitterion to form an amphiphilic structure,which enables its targeting ability toward cell membranes.Moreover,a reactive ester group is included to bind with proteins through covalent bonds in cell membranes nonspecifically,which extends retention time in cell membranes.Mem580-F-Sulfo shows intense brightness(94600),with a high molar absorption coefficient of up to about 100000 L·mol^(-1)·cm^(-1)and a fluorescence quantum yield of up to 0.97.It shows fast cell membrane targeting ability and long retention up to 90 min.In brief,this work has not only developed a tracker with good cell membrane targetability but also provided a new strategy for improving the targeting stability of cell membranes.

Identification of P-type plasma membrane H^(+)-ATPases in common wheat and characterization of TaHA7 associated with seed dormancy and germination

The P-type plasma membrane(PM)H^(+)-ATPases(HAs)are crucial for plant development,growth,and defense.The HAs have been thoroughly characterized in many different plants.However,despite their importance,the functions of HAs in germination and seed dormancy(SD)have not been validated in wheat.Here,we identified 28 TaHA genes(TaHA1-28)in common wheat,which were divided into five subfamilies.An examination of gene expression in strong-and weak-SD wheat varieties led to the discovery of six candidate genes(TaHA7/-12/-14/-16/-18/-20).Based on a single nucleotide polymorphism(SNP)mutation(C/T)in the TaHA7 coding region,a CAPS marker(HA7)was developed and validated in 168 wheat varieties and 171 Chinese mini-core collections that exhibit diverse germination and SD phenotypes.We further verified the roles of the two allelic variations of TaHA7 in germination and SD using wheat mutants mutagenized with ethyl methane sulphonate(EMS)in‘Jimai 22’and‘Jing 411’backgrounds,and in transgenic Arabidopsis lines.TaHA7 appears to regulate germination and SD by mediating gibberellic acid(GA)and abscisic acid(ABA)signaling,metabolism,and biosynthesis.The results presented here will enable future research regarding the TaHAs in wheat.

Functional analysis of MdSUT2.1,a plasma membrane sucrose transporter from apple

Sugar content is a determinant of apple(Malus×domestica Borkh.)sweetness.However,the molecular mechanism underlying sucrose accumulation in apple fruit remains elusive.Herein,this study reported the role of the sucrose transporter MdSUT2.1 in the regulation of sucrose accumulation in apples.The MdSUT2.1 gene encoded a protein with 612 amino acid residues that could be localized at the plasma membrane when expressed in tobacco leaf protoplasts.MdSUT2.1 was highly expressed in fruit and was positively correlated with sucrose accumulation during apple fruit development.Moreover,complementary growth assays in a yeast mutant validated the sucrose transport activity of MdSUT2.1.MdSUT2.1 overexpression in apples and tomatoes resulted in significant increases in sucrose,fructose,and glucose contents compared to the wild type(WT).Further analysis revealed that the expression levels of sugar metabolism-and transport-related genes SUSYs,NINVs,FRKs,HXKs,and TSTs increased in apples and tomatoes with MdSUT2.1 overexpression compared to WT.Finally,unlike the tonoplast sugar transporters MdTST1 and MdTST2,the promoter of MdSUT2.1 was not induced by exogenous sugars.These findings provide valuable insights into the molecular mechanism underlying sugar accumulation in apples.

Effects of Salinity Stress on the Levels of Covalently and Noncovalently Conjugated Polyamines in Plasma Membrane and Tonoplast Isolated from Barley Seedlings

When the 7_d old barley ( Hordeum vulgare L.) seedlings were treated with different concentrations of NaCl for 3 d, the levels of the noncovalently conjugated polyamines (PAs) in the plasma membrane and tonoplast vesicles and the covalently conjugated PAs in the membrane proteins were promoted by NaCl of low concentrations and suppressed by NaCl of high concentrations. Among the noncovalently conjugated PAs in the vesicles, spermidine (Spd) level was the most abundant, while putrescine (Put) content was predominant among the covalently conjugated PAs, accounted for 40%-70%, 35%-60%, respectively. In addition, the TLC (thin_layer chromatography) profiles of the benzoylated PAs presented an unknown polyamine with Rf =0.92 (X 0.92 ), which conjugated covalently and noncovalently in root tonoplast and its content changed as well as Spd with NaCl treatment. The total PA contents in the roots were higher than that in the leaves, and the types and contents of covalently and noncovalently conjugated PAs in the tonoplast were higher than those in the plasma membrane. The results showed that the above two PAs associated with the membrane might be essential in salt adaption of cells and the maintenance of membrane function.

Effect of Sound Stimulation on the Lipid Physical States and Metabolism of Plasma Membrane from Chrysanthemum Callus

Plasma membrane vesicles of chrysanthemum ( Dendranthema morifolium (Ramat.) Tzvel.) callus was purified by sucrose gradient centrifugation to investigate the influence of sound stimulation on the lipid physical states and metabolism of plasma membrane. The results showed that sound stimulation decreased the content of phosphodiesters and the fluorescent intensity of DPH, but increased the light scattering value of the membrane, the fluorescent intensity of MC540 and the content of phosphomonoesters, indicating that the vesicles got looser, the charge density and hydrophobicity of membrane surface decreased under sound stimulation of some strength and frequency. However, the membrane fluidity increased under the condition. Meanwhile, the anabolism of membrane lipid increased and the catabolism decreased. It can be seen that the physical state and metabolism of membrane lipid is sensitive to sound stimulation.

Activation of Plasma Membrane NADPH Oxidase and Generation of H_2O_2 Mediate the Induction of PAL Activity and Saponin Synthesis byEndogenous Elicitor in Suspension-Cultured Cells of Panax ginseng

Endogenous elicitor, termed cellulase-degraded cell wall (CDW), was prepared from the cell wall of suspension-cultured ginseng (Panax ginseng C.A. Meyer) cells via cellulase degradation. CDW activated the NADPH oxidase activity of isolated plasma membranes and stimulated in vivo H2O2 generation in ginseng cell suspensions. CDW also increased the activity of phenylalanine ammonia lyase (PAL), expression of a P. ginseng squalene epoxidase (sqe) gene and saponin synthesis. NADPH oxidase inhibitors inhibited both in vitro NADPH oxidase activity and in vivo H2O2 generation. Induction of PAL activity, saponin synthesis and sqe gene expression were all inhibited by such inhibitor treatments and reduced by incubation with catalase and HA scavengers. These data indicate that activation of NADPH oxidase and generation of H2O2 are essential signalling events mediating defence responses induced by the endogenous elicitor(s) present in CDW.

Influence of K + on the Coupling Between ATP Hydrolysis and Proton Transport by the Plasma Membrane H +_ATPase from Soybean Hypocotyls

The plasma membrane vesicles were purified from soybean (Glycine max L.) hypocotyls by two_phase partitioning methods. The stimulatory effects of K + on the coupling between ATP hydrolysis and proton transport by the plasma membrane H +_ATPase were studied. The results showed that the proton transport activity was increased by 850% in the presence of 100 mmol/L KCl, while ATP hydrolytic activity was only increased by 28.2%. Kinetic studies showed that K m of ATP hydrolysis decreased from 1.14 to 0.7 mmol/L, while V max of ATP hydrolysis increased from 285.7 to 344.8 nmol Pi·mg -1 protein·min -1 in the presence of KCl. Experiments showed that the optimum pH was 6.5 and 6.0 in the presence and absence of KCl, respectively. Further studies revealed that K + could promote the inhibitory effects of hydroxylamines and vanadates on the ATP hydrolytic activity. The above results suggested that K + could regulate the coupling between ATP hydrolysis and proton transport of the plasma membrane H +_ATPase through modulating the structure and function of the kinase and phosphatase domains of the plasma membrane H +_ATPase.

Effect of Lysophosphatidylcholine on ATP and ρ-Nitrophenyl Phosphate Hydrolysis by the Plasma Membrane H^+-ATPase from Soybean Hypocotyls

The stimulatory effect of lysophosphatidylcholine (lyso_PC) on ATP and ρ_nitrophenyl phosphate (PNPP) hydrolysis by the plasma membrane H +_ATPase from soybean (Glycine max (L.) Merr.) hypocotyls was studied. Results showed that lyso_PC stimulated the hydrolysis of ATP; ATP hydrolysis was enhanced dramatically when lyso_PC was within 0-0.03%, and increased slightly when lyso_PC was higher than 0.03%. At the concentration of 0.03%, lyso_PC stimulated ATP hydrolysis by 80.5%. Kinetics analysis showed that V max increased from 0.46 μmol P i·mg -1 protein·min -1 to 0.87 μmol P i·mg -1 protein·min -1 while K m increased from 0.88 mmol/L to 1.15 mmol/L under lyso_PC treatment. The optimum pH of ATP hydrolysis was shifted from 6.5 to 7.0 . Moreover, it was found lyso_PC enhanced the inhibition of ATP hydrolysis by hydroxylamine. In the presence of 200 mmol/L hydroxylamine, ATP hydrolysis was inhibited by 74.4%, while it was inhibited by 84.4% when treated with lyso_PC. However, PNPP hydrolysis and the inhibitory effect of vanadate were not affected by lyso_PC. The above results indicated that the kinase domain might be an action site or regulatory region of the C_terminal autoinhibitory domain in the plant plasma membrane H +_ATPase.

Water relations and an expression analysis of plasma membrane intrinsic proteins in sensitive and tolerant rice during chilling and recovery

A symptom of chilling injury is development of water deficit in shoots, resulting from an imbalance of water transport and transpiration. In this work, two rice varieties （Oryza sativa L. var. Wasetoitsu and Somewake） seedlings were chilled at 7 ℃, followed by recovery at 28 ℃. Based on the growth phenotype and electrolyte leakage tests, Somewake was shown to be a chilling-tolerant variety, and Wasetoitsu a chilling-sensitive one. The chilling stress reduced markedly the relative water content （RWC） of leaves, accumulative transpiration and osmotic root hydraulic conductivity （Lp） in both varieties. But when retumed to 28 ℃, the water relation balance of Somewake recovered better. The mRNA expression profile of all the 11 plasma membrane intrinsic proteins （PIPs）, a subgroup of aquaporins, was subsequently determined by real-time reverse transcription （RT）-PCR with TaqMan-minor grove binder （MGB） probes derived from rice var. Nipponbare during chilling treatment and recovery. Most of the PIP genes was down-regulated at the low temperature, and recovered at the warm temperature. The relative expression of some PIPs in both Somewake and Wasetoitsu decreased in parallel during the chilling. However during the recovery, the relative expression of OsPIP1;1, OsPIP2;1, OsPIP2;7 in shoots and OsPIP1：1, OsPIP2：1 in roots were significantly higher in Somewake than Wasetoitsu. This supports the role of PIPs in re-establishing water balance after chilling conditions. We discuss the diversified roles played by members of the aquaporin PIP subfamily in plant chilling tolerance depending on aquaporin isoforms, plant tissue and the stage of chilling duration.

Effects of salinity on activities of H^+-ATPase, H^+-PPase and membrane lipid composition in plasma membrane and tonoplast vesicles isolated from soybean(Glycine max L.) seedlings

The effects of NaCl stress on the H +-ATPase, H +-PPase activity and lipid composition of plasma membrane(PM) and tonoplast(TP) vesicles isolated from roots and leaves of two soybean cultivars(Glycine max L.) differing in salt tolerance(Wenfeng7, salt-tolerant; Union, salt-sensitive) were investigated. When Wenfeng7 was treated with 0.3%(W/V) NaCl for 3 d, the H +-ATPase activities in PM and TP from roots and leaves exhibited a reduction and an enhancement, respectively. The H +-PPase activity in TP from roots also increased. Similar effects were not observed in roots of Union. In addition, the increases of phospholipid content and ratios of phospholipid to galactolipid in PM and TP from roots and leaves of Wenfeng7 may also change membrane permeability and hence affect salt tolerance.

Changes of plasma membrane ATPase activity, membrane potential and transmembrane proton gradient in Kandelia candel and Avicennia marina seedlings with various salinities

The salt-secreting mangrove, Avicennia marina, and non-salt-secreting mangrove, Kandelia candel were cultivated in sand with various salinities(0‰, 10‰, 20‰, 30‰, 40‰) for 60 d. Plasma membrane vesicles of high-purity in leaves and roots of A.marina and K. candel seedlings were obtained by two-phase partitioning. The function of the plasma membranes, the activity of ATPase, membrane potential and transmembrane proton gradient, at various salinities were investigated. The results showed that within a certain range of salinity(A. marina and roots of K. candel: 0—30‰; leaves of K.candel: 0—20‰), the activity of ATPase increased with increasing salinity, while high salinity(above 30‰ or 20‰) inhibited ATPase activity. In comparison with A. marina, K. candel appeared to be more sensitive to salinity. The dynamics of membrane potential and transmembrane proton gradient in leaves and roots of A. marina and K. candel seedlings were similar to that of ATPase. When treated directly by NaCl all the indexes were inhibited markedly: there was a little increase within 0—10‰(K. candel) or 0—20‰(A. marina) followed by sharp declining. It indicated that the structure and function of plasma membrane was damaged severely.

Involvement of Plasma Membrane Ca^(2+)/H^+ Antiporter in Cd^(2+) Tolerance

Cation exchangers （CAXs） belong to the cation/Ca2＋exchanger superfamily which have been extensively investigated in plant tonoplasts over the last decade. Recently, the roles of CAXs involved in heavy metal accumulation and tolerance in plants have been studied for phytoremediation and food security. In this mini review, we summarize the roles of the Ca2＋/H＋ antiporter in Ca2＋ signal transduction, maintaining ion homeostasis and sequestering heavy metals into the vacuole. Moreover, we present a possible role of the plasma membrane Ca2＋/H＋ antiporter in heavy metal detoxification.

Involvement of Plasma Membrane H+-ATPase in Adaption of Rice to Ammonium Nutrient

The preference of paddy rice for NH4+ rather than NO3ˉ is associated with its tolerance to low pH since a rhizosphere acidification occurs during NH4+ absorption.However,the adaptation of rice root to low pH has not been fully elucidated.The plasma membrane H+-ATPase is a universal electronic H+ pump,which uses ATP as energy source to pump H+ across the plasma membranes into the apoplast.The key function of this enzyme is to keep pH homeostasis of plant cells and generate a H+ electrochemical gradient,thereby providing the driving force for the active influx and efflux of ions and metabolites across the plasma membrane.This study investigated the acclimation of plasma membrane H+-ATPase of rice root to low pH.This mechanism might be partly responsible for the preference of rice plants to NH4+ nutrition.

Changes of Plasma Membrane H^+-ATPase Activities of Glycine max Seeds by PEG Treatment

The soybean （Glycine max） Heihe No. 23 is sensitive to imbibitional chilling injury. Polyethylene glycol （PEG） treatment can improve chilling tolerance of soybean seeds to a certain extent. The changes of hydrolytic ATPase in plasma membranes and H^＋-pumping responses in soybean seeds were investigated during PEG treatments. Effects of exogenous calcium and exogenous ABA on the hydrolytic ATPase were also examined in order to understand the mechanism of chilling resistance. Highly purified plasma membranes were isolated by 6.0% aqueous two-phase partitioning from soybean seeds, as judged by the sensitivity of hydrolytic ATPase to sodium vanadate. PEG treatment resulted in a slight increase of the hydrolytic ATPase activity in 12 h. Then the activity decreased gradually, but still higher than the control. The H^＋-pumping activity increased steadily during PEG treatment. Exogenous calcium had both activating and inhibiting effects on the hydrolytic ATPase, but the activity was inhibited in soybean seeds treated with exogenous ABA. Results suggested that PEG treatment, not the exogenous calcium and ABA, up-regulated H^＋-ATPase activities in soybean seeds.

Generation and Identification of Monoclonal Antibody Against Porcine Adipocyte Plasma Membrane Proteins

Production of monoclonal antibody against porcine adipocyte plasma membrane proteins to explore a new way of controlling body fat deposition and improving carcass quality is discussed in this article. Membrane proteins of pig adipocyte plasma membrane proteins were extracted with the help of sucrose density gradient centrifugation, and two kinds of proteins were obtained. The monoclonal antibody （designated 3B2 and 3F3） of IgG1 and IgG2b subclass against adipocyte membrane proteins were produced by immunization, with adipocyte membrane proteins as an antigen, and its titer was 1：105 detected by enzyme-linked immunoadsorbent assay （ELISA）. The cell strains were identified by analyzing the number of chromosomes, the heat stability, the acid and alkali, the types and subtypes of immnoglobulin, and its peculiarities and affinities. Through identification, the chromosome number of hybridoma cell strains was from 80 to 100 and the strains formed good hybridomas colonies. The strains＇ affinity constants were 4.63 × 10^9 and 3.75 × 10^9 （mol L^-1）-1, respectively. At the same time, the McAb secreted was stable to environmental factors, such as, temperature, acid, alkali and so on. The monoclonal antibodies had been obtained and their specificity to porcine adipocyte plasma membrane proteins had been identified.

Effects of Calcium on ATPase Activity and Lipid omposition of Plasma Membranes from Wheat Roots Under Aluminum Stress

Effects of calcium on ATPase activities, lipid contents, and fatty acid compositions of plasma membrane from wheat roots were assayed under aluminum stress. The results showed that the increase of calcium concentration in the nutrient solution increased the activity of H + ATPase and the phospholipid content, decreased the activity of Ca 2+ ATPase and the galactolipid of plasma membrane. Owing to the decrease of linolenic acid content, the index of unsaturated fatty acid (IUFA) and index of double bond (DBI) decreased in Altas66. The IUFA and DBI of plasma membrane from Scout66 roots increased because its linolenic acid content increased obviously and its palmitic acid content decreased apparently.

Asparagine Synthetase Is Partially Localized to the Plasma Membrane and Upregulated by L-asparaginase in U937 Cells

This study investigated the intracellular localization of asparagine synthetase （ASNS） in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

Preliminary study on plasma membrane fluidity of Psychrophilic Yeast Rhodotorula sp.NJ298 in low temperature

The ability of cell to modulate the fluidity of plasma membrane was crucial to the survival of microorganism at low temperature.Plasma membrane proteins,fatty acids and carotenoids profiles of Antarctic psychrophilc yeast Rhodotorula sp.NJ298 were investigated at-3 ℃,0 ℃ and 8 ℃.The results showed that plasma membrane protein content was greater at-3 ℃ than that at 8 ℃,and a unique membrane polypeptide composition with an apparent molecular mass of 94.7 kDa was newly synthesized with SDS-PAGE analysis;GC analysis showed that the main changes of fatty acids were the percentage of unsaturated fatty acids(C18:1 and C18:2) and shorter chain saturated fatty acid(C10:0) increased along with the decrease of the culture temperature from 8 ℃ to-3 ℃;HPLC analysis indicated that astaxanthin was the major functional carotenoids of the plasma membrane,percentage of which increased from 54.6±1.5% at 8 ℃ to 81.9±2.1% at-3 ℃.However the fluidity of plasma membrane which was determined by measuring fluorescence anisotropy was similar at-3 ℃,0 ℃ and 8 ℃.Hence these changes in plasma membrane’s characteristics were involved in the cellular cold-adaptation by which NJ298 could maintain normal plasma membrane fluidity at near-freezing temperature.

Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance plasma membrane H^(+)-ATPase activity and alkali stress tolerance by inhibiting PP2C-mediated dephosphorylation of TaHA2 in wheat

Alkali stress is a major constraint for crop production in many regions of saline-alkali land.However,little is known about the mechanisms through which wheat responds to alkali stress.In this study,we identified a calcium ion-binding protein from wheat,TaCCD1,which is critical for regulating the plasma membrane(PM)H^(+)-ATPase-mediated alkali stress response.PM H+-ATPase activity is closely related to alkali tolerance in the wheat variety Shanrong 4(SR4).We found that two D-clade type 2C protein phosphatases,TaPP2C.D1 and TaPP2C.D8(TaPP2C.D1/8),negatively modulate alkali stress tolerance by dephosphorylating the penultimate threonine residue(Thr926)of TaHA2 and thereby inhibiting PM H+-ATPase activity.Alkali stress induces the expression of TaCCD1 in SR4,and TaCCD1 interacts with TaSAUR215,an early auxin-responsive protein.These responses are both dependent on calcium signaling triggered by alkali stress.TaCCD1 enhances the inhibitory effect of TaSAUR215 on TaPP2C.D1/8 activity,thereby promoting the activity of the PM H^(+)-ATPase TaHA2 and alkali stress tolerance in wheat.Functional and genetic analyses verified the effects of these genes in response to alkali stress,indicating that TaPP2C.D1/8 function downstream of TaSAUR215 and TaCCD1.Collectively,this study uncovers a new signaling pathway that regulates wheat responses to alkali stress,in which Ca^(2+)-dependent TaCCD1 cooperates with TaSAUR215 to enhance PM H+-ATPase activity and alkali stress tolerance by inhibiting TaPP2C.D1/8-mediated dephosphorylation of PM H+-ATPase TaHA2 in wheat.

An AIE probe for long-term plasma membrane imaging and membrane-targeted photodynamic therapy

Two red-emissive luminogens(TPTH and TPTB) with typical aggregation-induced emission characteristics were developed. By introducing the heavy atom of Br at the end of alkyl chain, TPTB exhibited higher reactive oxygen species generation efficiency through both types I and II pathways. Due to its excellent biocompatibility and proper lipophilicity, TPTB could be used for long-term cell membrane staining and this staining ability was independent of the change of plasma membrane potential. Furthermore, TPTB could ablate the cancer cells through cell membrane-targeted photodynamic therapy.