Non-specific phospholipase C4 hydrolyzes phosphosphingolipids and phosphoglycerolipids and promotes rapeseed growth and yield

Phosphorus is a major nutrient vital for plant growth and development,with a substantial amount of cellular phosphorus being used for the biosynthesis of membrane phospholipids.Here,we report that NON-SPECIFIC PHOSPHOLIPASE C4(NPC4)in rapeseed(Brassica napus)releases phosphate from phospholipids to promote growth and seed yield,as plants with altered NPC4 levels showed significant changes in seed production under different phosphate conditions.Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated nuclease 9(Cas9)-mediated knockout of Bna NPC4 led to elevated accumulation of phospholipids and decreased growth,whereas overexpression(OE)of Bna NPC4resulted in lower phospholipid contents and increased plant growth and seed production.We demonstrate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in vitro,and plants with altered Bna NPC4 function displayed changes in their sphingolipid and glycerolipid contents in roots,with a greater change in glycerolipids than sphingolipids in leaves,particularly under phosphate deficiency conditions.In addition,Bna NPC4-OE plants led to the upregulation of genes involved in lipid metabolism,phosphate release,and phosphate transport and an increase in free inorganic phosphate in leaves.These results indicate that Bna NPC4 hydrolyzes phosphosphingolipids and phosphoglycerolipids in rapeseed to enhance phosphate release from membrane phospholipids and promote growth and seed production.

Plant phosphatidylinositol-specific phospholipase C at the center of plant innate immunity

Understanding plant resistance to pathogenic microbes requires detailed information on the molecular mechanisms controlling the execution of plant innate immune responses.A growing body of evidence places phosphoinositide-specific phospholipase C(PI-PLC) enzymes immediately downstream of activated immune receptors,well upstream of the initiation of early defense responses.An increase of the cytoplasmic levels of free Ca^(2+),lowering of the intercellular pH and the oxidative burst are a few examples of such responses and these are regulated by PI-PLCs.Consequently,PI-PLC activation represents an early primary signaling switch between elicitation and response involving the controlled hydrolysis of essential signaling phospholipids,thereby simultaneously generating lipid and non-lipid second messenger molecules required for a swift cellular defense response.Here,we elaborate on the signals generated by PI-PLCs and their respective downstream effects,while providing an inventory of different types of evidence describing the involvement of PI-PLCs in various aspects of plant immunity.We project the discussed information into a model describing the cellular events occurring after the activation of plant immune receptors.With this review we aim to provide new insights supporting future research on plant PI-PLCs and the development of plants with improved resistance.

Phospholipase C gamma(PLCγ)regulates soluble trehalase in the 20E-induced fecundity of Apolygus lucorum

Apolygus lucorum is the dominant pathogenic insect attacking Bacillus thuringiensis(Bt)cotton in China.Additionally,20-hydroxyecdysone(20E)has important functions in many biological processes,including insect reproduction.Phospholipase C(PLC),which is an essential enzyme for phosphoinositide metabolism,is involved in 20E signal transduction,but its function in 20E-mediated reproduction in A.lucorum remains unclear.In this study,20E increased A/PLCγ transcription as well as the abundance and activity of the encoded protein during molting and metamorphosis.The 20E treatment also induced the considerable accumulation of two second messengers,inositol triphosphate and diacylglycerol.The expression levels of genes encoding vitellogenin(AlVg)and soluble trehalase(AlTre-1)were similar to those of AlPLCy,and were upregulated in response to 20C.The silencing of AlPLCγ resulted in downregulated expression o f AITre-γ smdAlVg.However,the silencing of AlTre-1 and AlVg did not affect AlPLCγ expression.Moreover,the silencing of AlVg did not alter AlTre-1 expression.Furthermore,an examination of the insect specimens indicated that AlPLCy is required for female adult reproduction,and that downregulated expression of this gene is associated with decreases in fecundity,adult longevity,and egg hatching rate as well as delayed oocyte maturation.We propose that 20E regulates AlTre-1 expression via AlPLCy and affects Vg expression as well as ovary development to facilitate the reproductive activities of A.lucorum females.

Antigen unmasking does not improve the visualization of phospholipase C zeta in human spermatozoa

Phospholipase C zeta(PLCζ)is a sperm-specific protein that triggers oocyte activation.The analysis of PLCζexpression in human spermatozoa can be used as a diagnostic marker for oocyte activation deficiency.Our laboratory has previously optimized a standard"in-house"assay to determine PLCζexpression in human spermatozoa.However,one study has suggested that an antigen unmasking method(AUM)would be more efficient in visualizing PLCζin human sperm.This study aimed to compare our established assay and AUM(involving HCl,acidic Tyrode's solution[AT],and heat).The mean relative fluorescence(RF)intensity of PLCζin frozen-thawed spermatozoa from fourteen fertile donors stained with the in-house method was significantly higher than three other AUM groups(in-house[mean±standard error of mean]:18.87±2.39 arbitrary units[a.u.]vs non-AUM:11.44±1.61 a.u.,AT-AUM:12.38±1.89 a.u.,and HCl-AUM:12.51±2.16 a.u.,P<0.05,one-way analysis of variance).The mean RF intensity of PLCζin AT-and HCl-treated spermatozoa from 12 infertile males was not significantly different from that of the non-AUM group.However,the in-house method resulted in the highest RF intensity(12.11±1.36 a.u.,P<0.01).Furthermore,specificity testing of antibody-antigen binding indicated that the in-house method showed more specific binding than spermatozoa treated by the AUM.In conclusion,our in-house method showed superior visualization and reliability than the AUM,thus supporting the continued use of our in-house assay for clinical research screening.

磷脂酶C1基因rs2274223位点多态性与下咽癌临床病理特征及预后的相关性分析

目的 探讨磷脂酶C1(phospholipase C1,PLC1)基因rs2274223位点多态性与下咽癌临床病理特征及预后的相关性。方法 收集2016年1月~2022年12月间浙江大学医学院附属金华医院耳鼻咽喉头颈外科收治的42例下咽癌患者,所有患者均行根治手术,记录临床病理数据,并均取全血3 ml作DNA提取,Sanger法测序检测PLCE1基因rs2274223位点单核苷酸多态性,分析其与下咽癌临床病理特征及预后的相关性。结果 PLCE1基因rs2274223位点在42例下咽癌患者中有16例突变为AG,位点突变与肿瘤分化程度相关(χ^(2)=5.301,P=0.021),K-M生存分析提示rs2274223位点突变与未突变的下咽癌患者3年生存率分别为75.0%和83.1%,5年生存率分别为18.8%和31.2%,差异有统计学意义(χ^(2)=5.475,P=0.019)。结论PLCE1基因rs2274223位点突变与下咽癌低分化相关,该位点突变的下咽癌患者预后更差。

NYD-SP27,a novel intrinsic decapacitation factor in sperm

Prior to fertilization sperm has to undergo an activation process known as capaciation,leading to the acrosome reaction.Till now,little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved.In this study,we report that NYD-SP27,an isoform of phospholipase C Zeta 1(PLCZ1),is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody.Western blot and double staining analyses show NYD-SP27 becomes detached from sperm,as they undergo capacitation and acrosome reaction.The absence of HCO_(3)^(-),a key factor in activating capacitation,from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm.The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm,reduced the number of capacitated sperm,inhibited the acrosome reaction induced by ATP and progesterone,and inhibited agonist-induced PLC-coupled Ca^(2+)mobilization in sperm,which can be mimicked by the PLC inhibitor,U73122.These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.

PKCα signaling pathway involves in TNF-α-induced IP_3R1 expression in human mesangial cells

BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occurrence of hepatorenal syndrome(HRS).METHODS:HMCs were stimulated by tumor(TNF-a) with 100 ng/mL for different hours(2,4,8,and 24 hours).The expression changes of IP_3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting.Several inhibitors including D609,U73122,PP1,safingol,rottlerin and non-radioactive protein kinase C(PKC) were used to examine the mechanism of signal transduction ofTNF-a-regulated IP_3R1 in HMCs.RESULTS:The levels of IP_3R1 mRNA at 2 hours after TNF-a exposure were significantly enhanced and peaked at 8 hours in HMCs(P<0.01),then descended at 24 hours(P<0.01).The levels of IP_3R1 protein at 4 hours after TNF-a exposure were obviously increased and peaked at24 hours after TNF-a exposure(P<0.01).Compared to the control group,safingol(PKCa inhibitor)and D609(phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-ainduced expression of IP_3R1 mRNA(3.30±0.81 vs.1.95±0.13,P<0.05;2.10±0.49,P<0.01) and IP_3R1protein(3.09±0.13 vs.1.86+0.39,P<0.01;1.98±0.02,P<0.01).TNF-a promoted PKCa activation with maximal PKCa phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay,and the effect was markedly attenuated by pretreatment with D609 or safingol.CONCLUSION:TNF-a increased the expression of IP_3R1 and this was mediated,at least in part,through the PC-PLC/PKCa signaling pathways in HMCs.

The Difference in Calcium Levels in Aspergillus nidulans Grown on Glucose or Pectin

Understanding the growth regulatory mechanisms in filamentous fungi is very important for the production of medicines for antifungal therapies. It is well established that Ca2+ gradient is essential for hyphal growth and that one mechanism responsible for the Ca2+ cellular concentration starts with the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by receptor-regulated forms of phosphoinositide-specific phospholipase C (PI-PLC). In the present study the levels of calcium in Aspergillus nidulans wild type (A26) and plcA-deficient mutant (AP27) growing in a carbon source readily assimilated, as glucose or pectin a non-readily assimilated carbon source was investigated. Intracellular calcium levels in A26 were higher in the presence of glucose than in pectin, but lower in AP27 independently of the carbon source and in AP27 the vesicular calcium distribution occurred mainly at the apex of the hyphae. Delay in nuclear division was also observed if A26 and AP27 were grown in pectin presence when compared with growth in glucose. For the first time, it is demonstrated that the levels of intracellular Ca2+ were higher when A. nidulans was growing in glucose than in a non readily assimilated carbon source as pectin. Further, it also showed that the plcA gene, although not essential, may be responsible for high-molecular weight carbon source recongnation, for the intracellular Ca2+ levels maintenance and consequently by the nuclear division in A. nidulans.

An Extracellular Oligopeptide Permease May Be a Potential Virulence Factor of Vibrio harveyi

An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a(+)and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chro-matography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46×105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.

Preparation of Monoclonal Antibody Against Clostridium perfringensα-toxin and Screening and Identification of Phage Display Technology

Clostridium perfringens phospholipase C(plc),also calledα-toxin,is encoded by the plc gene Clostridium perfringers.The production ofα-toxin can lead to the occurrence of gas gangrene.Vaccination is considered as one of the best solutions against Clostridium infections.In this study,an anti-Cpα-toxin monoclonal antibody(mAbs)A10E5 was successfully prepared,which had better biological reactivity.Then,the phage random 12-peptide library was used to screen mAb A10E5 protein.After four rounds of screening,three peptides with high affinity to the anti-α-toxin mAbs were screened.Two 12-peptide peptide Q and peptide E with higher inhibition rate were obtained by indirect ELISA.Two polypeptides of 500μg·mL^(-1)synthesized in vitro were mixed with30μg·mL^(-1)α-toxin at a concentration to treat Hela cells.Cell viability was determined by MTT assay.The results showed that both of the peptides significantly increased the survival rate of Hela cells compared with theα-toxin group,and the effect of peptide Q was more obvious.The chickens were immunized with phages expressing two different affinity polypeptides and then challenged.The results of chicken weight change,intestinal lesion score,bacterial count,and antibody titer in peripheral blood showed that the two phages expressing the polypeptides had a certain protective effect on the chickens compared with the PBS group,and peptide Q had better protection effect.In conclusion,the high affinity peptide with mAb A10E5 was screened in this study,and the protective effect of the plc polypeptide vaccine was verified by in vivo and in vitro experiments,which was of great significance for the comprehensive prevention and treatment of the disease.

Advancing male age differentially alters levels and localization patterns of PLCzeta in sperm and testes from different mouse strains

Sperm-specific phospholipase C zeta(PLCζ)initiates intracellular calcium(Ca2+)transients which drive a series of concurrent events collectively termed oocyte activation.Numerous investigations have linked abrogation and absence/reduction of PLCζwith forms of male infertility in humans where oocyte activation fails.However,very few studies have examined potential relationships between PLCζand advancing male age,both of which are increasingly considered to be major effectors of male fertility.Initial efforts in humans may be hindered by inherent PLCζvariability within the human population,alongside a lack of sufficient controllable repeats.Herein,utilizing immunoblotting,immunofluorescence,and quantitative reverse transcription PCR(qRT-PCR)we examined for the first time PLCζprotein levels and localization patterns in sperm,and PLCζmRNA levels within testes,from mice at 8 weeks,12 weeks,24 weeks,and 36 weeks of age,from two separate strains of mice,C57BL/6(B6;inbred)and CD1(outbred).Collectively,advancing male age generally diminished levels and variability of PLCζprotein and mRNA in sperm and testes,respectively,when both strains were examined.Furthermore,advancing male age altered the predominant pattern of PLCζlocalization in mouse sperm,with younger mice exhibiting predominantly post-acrosomal,and older mice exhibiting both post-acrosomal and acrosomal populations of PLCζ.However,the specific pattern of such decline in levels of protein and mRNA was strain-specific.Collectively,our results demonstrate a negative relationship between advancing male age and PLCζlevels and localization patterns,indicating that aging male mice from different strains may serve as useful models to investigate PLCζin cases of male infertility and subfertility in humans.

磷脂酶C生物学特性及其在中耳胆脂瘤组织中异常表达

磷酸肌醇特异性磷脂酶C(phosphoinositide-specificphospholipaseC,PI-PLC)是磷酸肌醇信号转导途径中的一个关键酶。在哺乳动物中,已确认共有12种PI-PLC同工酶,根据其不同结构和调控机制分为5大类:β、γ、δ、ε和ζ。不同亚型的结构、调控和组织分布各有差异。PI-PLC在调控细胞信号转导途径中的特性使其在中耳胆脂瘤组织的形成中起到一定的作用。本文综述了PI-PLC家族5种亚型的结构、调控机制及其在中耳胆脂瘤组织异常表达的相关内容,并进一步探讨了PI-PLCγ1信号转导机制在胆脂瘤形成中可能的具体作用。