Pollen tubes require a tightly regulated pectin secretion machinery to sustain the cell wall plasticity required for polar tip growth. Involved in this regulation at the apical plasma membrane are proteins and signaling molecules, including phosphoinositides and phosphatidic acid (PA). However, the contribution of diacylglycerol kinases (DGKs) is not clear. We transiently expressed tobacco DGKs in pollen tubes to identify a plasma membrane (PM)-localized isoform, and then to study its effect on pollen tube growth, pectin secretion and lipid signaling. In order to potentially downregulate DGK5 function, we overexpressed an inactive variant. Only one of eight DGKs displayed a confined localization at the apical PM. We could demonstrate its enzymatic activity and that a kinase-dead variant was inactive. Overexpression of either variant led to differential perturbations including misregulation of pectin secretion. One mode of regulation could be that DGK5-formed PA regulates phosphatidylinositol 4-phosphate 5-kinases, as overexpression of the inactive DGK5 variant not only led to a reduction of PA but also of phosphatidylinositol 4,5-bisphosphate levels and suppressed related growth phenotypes. We conclude that DGK5 is an additional player of polar tip growth that regulates pectin secretion probably in a common pathway with PI4P 5-kinases.

• Klíčová slova
• diacylglycerol kinase, lipid signaling, pectin, phosphatidic acid, pollen tube, secretion, tobacco (Nicotiana tabacum),
• MeSH
• buněčná membrána metabolismus   MeSH
• diacylglycerolkinasa genetika   metabolismus   MeSH
• fosfatidylinositoly metabolismus   MeSH
• pylová láčka * MeSH
• tabák * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diacylglycerolkinasa MeSH
• fosfatidylinositoly MeSH

Using Arabidopsis thaliana wild type (WT) plants and diacylglycerol kinase knockouts (single mutants - dgk3, dgk1, dgk6; double mutants - dgk3dgk7, dgk5dgk6, dgk1dgk2) we observed that the inhibitor of brassinosteroid (BR) biosynthesis, brassinazole (BRZ), drastically decreased germination of dgk mutants under salt stress, while BRZ co-administration with 24-epibrassinolide (EBL) partially improved germination rates. We also observed a statistically significant decrease in alternative and cytochrome respiratory pathways in response to BRZ treatment under salinity conditions. We showed that production of the lipid second messenger phosphatidic acid (PA) is impaired in dgk mutants in response to EBL treatment and inhibitor of diacylglycerol kinase (DGK) - R59022. This study demonstrates that dgk mutants possess lower germination rates, lower total respiration rates, an alternative respiratory pathway and PA content under optimal and high salinity conditions in response to EBL treatment comparing to WT plants.

• Klíčová slova
• Alternative oxidase, Arabidopsis thaliana, Brassinosteroids, Diacylglycerol kinase, Phosphatidic acid, Salinity,
• MeSH
• Arabidopsis chemie   metabolismus   MeSH
• brassinosteroidy farmakologie   MeSH
• diacylglycerolkinasa antagonisté a inhibitory   nedostatek   metabolismus   MeSH
• kyseliny fosfatidové chemie   metabolismus   MeSH
• salinita MeSH
• semena rostlinná účinky léků   růst a vývoj   metabolismus   MeSH
• triazoly farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• brassinazole MeSH Prohlížeč
• brassinosteroidy MeSH
• diacylglycerolkinasa MeSH
• kyseliny fosfatidové MeSH
• triazoly MeSH

Flagellin perception is a keystone of pattern-triggered immunity in plants. The recognition of this protein by a plasma membrane (PM) receptor complex is the beginning of a signaling cascade that includes protein phosphorylation and the production of reactive oxygen species (ROS). In both Arabidopsis (Arabidopsis thaliana) seedlings and suspension cells, we found that treatment with flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, caused a rapid and transient decrease in the level of phosphatidylinositol (PI) 4,5-bisphosphate along with a parallel increase in phosphatidic acid (PA). In suspension cells, inhibitors of either phosphoinositide-dependent phospholipases C (PLC) or diacylglycerol kinases (DGKs) inhibited flg22-triggered PA production and the oxidative burst. In response to flg22, receptor-like kinase-deficient fls2, bak1, and bik1 mutants (FLAGELLIN SENSITIVE 2, BRASSINOSTEROID INSENSITIVE 1-associated kinase 1, and BOTRYTIS-INDUCED KINASE 1, respectively) produced less PA than wild-type (WT) plants, whereas this response did not differ in NADPH oxidase-deficient rbohD (RESPIRATORY BURST OXIDASE HOMOLOG D) plants. Among the DGK-deficient lines tested, the dgk5.1 mutant produced less PA and less ROS after flg22 treatment compared with WT seedlings. In response to flg22, dgk5.1 plants showed lower callose accumulation and impaired resistance to Pseudomonas syringae pv. tomato DC3000 hrcC-. Transcriptomics revealed that the basal expression of defense-related genes was altered in dgk5.1 seedlings compared with the WT. A GFP-DGK5 fusion protein localized to the PM, where RBOHD and PLC2 (proteins involved in plant immunity) are also located. The role of DGK5 and its enzymatic activity in flagellin signaling and fine-tuning of early immune responses in plant-microbe interactions is discussed.

• MeSH
• Arabidopsis * metabolismus   MeSH
• diacylglycerolkinasa genetika   metabolismus   MeSH
• flagelin farmakologie   genetika   MeSH
• imunita rostlin MeSH
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• Pseudomonas syringae fyziologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BIK1 protein, Arabidopsis MeSH Prohlížeč
• diacylglycerolkinasa MeSH
• flagelin MeSH
• protein-serin-threoninkinasy MeSH
• proteiny huseníčku * MeSH
• reaktivní formy kyslíku MeSH

The distinction of lipid isomers is gaining more attention in lipidomics due to their different biochemical properties in the organism. Herein, we aimed to develop a method for the analysis of monoacylglycerol (MG) and diacylglycerol (DG) enantiomers in biological samples using chiral supercritical fluid chromatography and mass spectrometry (SFC-MS). Amylose-based chiral columns showed a certain degree of separation of MG and DG isomers, but low selectivity for the acylglycerol classes in total lipid extracts, which could not be improved by modifier composition or other chromatographic conditions. The coelution of MG and DG enantiomers with highly concentrated triacylglycerols (TGs) negatively affected their MS determination based on the peak area ratio, therefore the interclass selectivity of chiral SFC was adjusted by coupling with an achiral column. The connection of the amylose tris-(3,5-dimethylphenylcarbamate) chiral column with octadecyl achiral column using a methanol as a modifier provided an excellent interclass separation of acylglycerols with the resolution of 5.53 and 15.17 for oleic acid-based MG/DG and DG/TG classes, respectively. The developed method enabled the determination of MG and DG enantiomers in complex total lipid extracts of biological samples in a 15 min gradient without time-consuming sample prefractionation. Chiral SFC-MS analysis of egg yolk, human plasma, and porcine brain samples showed different ratios of enantiomers, suggesting their unique roles within each sample type.

• Klíčová slova
• Chiral separation, Column coupling, Diacylglycerol, Monoacylglycerol, Selectivity, Supercritical fluid chromatography,
• MeSH
• amylosa chemie   analogy a deriváty   MeSH
• diglyceridy * chemie   analýza   MeSH
• hmotnostní spektrometrie metody   MeSH
• lipidomika metody   MeSH
• monoglyceridy * chemie   analýza   MeSH
• stereoizomerie MeSH
• superkritická fluidní chromatografie * metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amylosa MeSH
• diglyceridy * MeSH
• monoglyceridy * MeSH

Diatom thylakoids contain much higher amounts of sulfoquinovosyl diacylglycerol (SQDG) than vascular plants and the hypothesis was brought forward that this relates to their special thylakoid structure. To test this hypothesis we created knock-down mutants in Thalassiosira pseudonana that exhibited a decreased SQDG content per cell. Surprisingly, the ratio between the different lipid classes did not change, pointing to strict regulation of thylakoid lipid composition. The antenna proteins, fucoxanthin-chlorophyll proteins (FCP), were reduced and photosystem (PS) I compared to PSII was increased as judged from absorbance spectra. CD spectroscopy indicated a tighter packing of chromophores. The reduction in FCP might help to avoid diametral changes in excitation energy transfer. In contrast, the increase in PSI in the mutants might counteract the diminishment of the usually huge PSI antenna. No changes in thylakoid structure were observed since the stoichiometry between different lipid classes seems to be carefully balanced.

• Klíčová slova
• algae, lipids, photosynthesis, sulfoquinovosyl diacylglycerol,
• Publikační typ
• časopisecké články MeSH

• Aluminium ions (Al) have been recognized as a major toxic factor for crop production in acidic soils. This study aimed to assess the impact of Al on the activity of phosphatidylcholine-hydrolysing phospholipase C (PC-PLC), a new member of the plant phospholipase family. • We labelled the tobacco cell line BY-2 and pollen tubes with a fluorescent derivative of phosphatidylcholine and assayed for patterns of fluorescently labelled products. Growth of pollen tubes was analysed. • We observed a significant decrease of labelled diacylglycerol (DAG) in cells treated with AlCl(3). Investigation of possible metabolic pathways that control DAG generation and consumption during the response to Al showed that DAG originated from the reaction catalysed by PC-PLC. The growth of pollen tubes was retarded in the presence of Al and this effect was accompanied by the decrease of labelled DAG similar to the case of the BY-2 cell line. The growth of pollen tubes arrested by Al was rescued by externally added DAG. • Our observation strongly supports the role of DAG generated by PC-PLC in the response of tobacco cells to Al.

• MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• časové faktory MeSH
• diglyceridy biosyntéza   MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosfolipasy typu C metabolismus   MeSH
• hliník toxicita   MeSH
• ionty MeSH
• porfobilinogen analogy a deriváty   metabolismus   MeSH
• pylová láčka růst a vývoj   metabolismus   MeSH
• tabák cytologie   účinky léků   enzymologie   MeSH
• viabilita buněk účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-diacylglycerol MeSH Prohlížeč
• 3-anilino difluoroboron dipyrromethene MeSH Prohlížeč
• diglyceridy MeSH
• fosfatidylcholiny MeSH
• fosfolipasy typu C MeSH
• hliník MeSH
• ionty MeSH
• phosphatidylcholine-specific phospholipase C MeSH Prohlížeč
• porfobilinogen MeSH

Regression coefficients and genetic variances for 40,890 single nucleotide polymorphisms (SNPs) for milk yield were calculated using mixed model equations, with deregressed proof (DRP) as the dependent variable. Bulls were genotyped using the Illumina BovineSNP50 v2 BeadChip and SNPs were edited according the minor allele frequency (MAF) and high incidence of missing genotype. Evaluation was conducted in two rounds. In the preliminary round, the direct genetic values (DGVs) of all genotyped bulls (2,904) were computed and the absolute difference between the DGV and the input DRP of each bull was investigated. Bulls with an absolute difference greater than the mean absolute difference plus two standard deviations were eliminated from the data set prior to the final analysis (2,766 bulls remaining). SNP regression coefficients from the final analysis had a mean absolute value of 0.506 kg and a standard deviation of 0.409 kg. The SNP with the highest regression coefficient and genetic variance was ARSBFGLNGS4939 on chromosome 14. This SNP is located within the gene DGAT1 (diacylglycerol O-acyltransferase 1). Other SNPs with high regression coefficients and genetic variance are localised in proximity to DGAT1. The mean genetic variance of an individual SNP was 0.170, with a standard deviation of 0.384 and a mean heterozygosity of 0.372. The sum of genetic variances of all SNPs was only 6,968.8, probably because of the existence of genetic covariances between loci. The largest sum of genetic variances was on chromosome 14 (498.4, 7.15 % of the total). After the final analysis, the correlation between the DGV and the input DRP was 0.951 for all bulls. The variance of the predicted DGV was 98.11 % of the variance of the input estimated breeding value (EBV) and 63.65 % of the variance of the DRP.

• MeSH
• chov MeSH
• diacylglycerol-O-acyltransferasa genetika   MeSH
• frekvence genu MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus * MeSH
• mlékárenství MeSH
• mléko * MeSH
• modely genetické MeSH
• skot genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot genetika   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diacylglycerol-O-acyltransferasa MeSH

Triacylglycerols (TGs) and diacylglycerols (DGs) in 16 plant oil samples (hazelnut, pistachio, poppy-seed, almond, palm, Brazil-nut, rapeseed, macadamia, soyabean, sunflower, linseed, Dracocephalum moldavica, evening primrose, corn, amaranth, Silybum arianum) were analyzed by HPLC-MS with atmospheric pressure chemical ionization (APCI) and UV detection at 205 nm on two Nova-Pak C18 chromatographic columns connected in series. A single chromatographic column and non-aqueous ethanol-acetonitrile gradient system was used as a compromise between the analysis time and the resolution for the characterization of TG composition of five plant oils. APCI mass spectra were applied for the identification of all TGs and other acylglycerols. The isobaric positional isomers can be distinguished on the basis of different relative abundances of the fragment ions formed by preferred losses of the fatty acid from sn-1(3) positions compared to the sn-2 position. Excellent chromatographic resolution and broad retention window together with APCI mass spectra enabled positive identification of TGs containing fatty acids with odd numbers of carbon atoms such as margaric (C17:0) and heptadecanoic (C17:1) acids. The general fragmentation patterns of TGs in both APCI and electrospray ionization mass spectra were proposed on the basis of MSn spectra measured with an ion trap analyzer. The relative concentrations of particular TGs in the analyzed plant oils were estimated on the basis of relative peak areas measured with UV detection at 205 nm.

• MeSH
• atmosférický tlak MeSH
• diglyceridy analýza   MeSH
• hmotnostní spektrometrie metody   MeSH
• oleje rostlin chemie   MeSH
• triglyceridy analýza   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diglyceridy MeSH
• oleje rostlin MeSH
• triglyceridy MeSH

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

• MeSH
• diglyceridy chemie   MeSH
• hmotnostní spektrometrie metody   MeSH
• hydrolýza MeSH
• katalýza MeSH
• lipasa * chemie   MeSH
• monoglyceridy MeSH
• prasata MeSH
• stereoizomerie MeSH
• superkritická fluidní chromatografie * MeSH
• triglyceridy analýza   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diglyceridy MeSH
• lipasa * MeSH
• monoglyceridy MeSH
• triglyceridy MeSH

Allelic and genotypic distribution of polymorphisms in diacylglycerol acyltransferase 1 (DGAT1), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor-γ coactivator-1α (PPARGC1A) genes were assessed in 679 Fleckvieh bulls. Single-locus genotype effects and the combined effect of the two polymorphisms within the FASN gene were evaluated for association with the intramuscular fat content and fatty acid profile determined in muscle and subcutaneous fat. The FASN (g.16024G>A) and FASN (g.17924A>G) polymorphisms were significantly associated mainly with C14:0, C16:0, and C18:1 n-9 concentrations as well as with the atherogenic index. The proportion of explained phenotypic variation markedly increased when the effect of a combination of the two polymorphisms within the FASN gene was tested, with the highest values of 8.6% and 14.8%, respectively, observed for C14:0 in muscle and subcutaneous fat. With a focus on improving the fatty acid composition of beef, the results of this study provide valuable information about the markers applicable in marker-assisted selection.

• Klíčová slova
• Adipose tissue, Cattle, Fatty acid, Gene polymorphism,
• MeSH
• alely MeSH
• ateroskleróza MeSH
• diacylglycerol-O-acyltransferasa genetika   metabolismus   MeSH
• druhová specificita MeSH
• fenotyp * MeSH
• genotyp * MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• maso analýza   MeSH
• mastné kyseliny metabolismus   MeSH
• podkožní tuk metabolismus   MeSH
• polymorfismus genetický * MeSH
• proteiny vázající mastné kyseliny genetika   metabolismus   MeSH
• skot MeSH
• svaly metabolismus   MeSH
• syntázy mastných kyselin genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diacylglycerol-O-acyltransferasa MeSH
• mastné kyseliny MeSH
• proteiny vázající mastné kyseliny MeSH
• syntázy mastných kyselin MeSH
• transkripční faktory MeSH