Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Water plays a different role on activation thermodynamic parameters of alcoholysis reaction catalyzed by lipase in gaseous and organic media

The effect of water on the alcoholysis of methyl propionate and n-propanol catalyzed by immobilized Candida antarctica lipase B (CALB) has been compared in a continuous solid-gas reactor and in an organic liquid medium. The enthalpic and entropic contributions of water to the Gibbs free energy of activation in the gas phase were different from the ones in the organic phase, the inverse trends being observed for the variation of both DeltaH* and DeltaS* with water activity.Different phenomena were identified for their influence on the thermodynamic parameters. When increasing a(w), the enhanced flexibility of the enzyme was predominant in the gas phase whereas substrate-solvent interactions due to an increased polarity of the solvent affected mainly the thermodynamic parameters in the organic phase. The observed variations of DeltaG* with water activity were in accordance with kinetics results previously obtained in both reaction media.     

Virulence factors of the human opportunistic pathogen Serratia marcescens identified by in vivo screening

The human opportunistic pathogen Serratia marcescens is a bacterium with a broad host range, and represents a growing problem for public health. Serratia marcescens kills Caenorhabditis elegans after colonizing the nematode's intestine. We used C.elegans to screen a bank of transposon-induced S.marcescens mutants and isolated 23 clones with an attenuated virulence. Nine of the selected bacterial clones also showed a reduced virulence in an insect model of infection. Of these, three exhibited a reduced cytotoxicity in vitro, and among them one was also markedly attenuated in its virulence in a murine lung infection model. For 21 of the 23 mutants, the transposon insertion site was identified. This revealed that among the genes necessary for full in vivo virulence are those that function in lipopolysaccharide (LPS) biosynthesis, iron uptake and hemolysin production. Using this system we also identified novel conserved virulence factors required for Pseudomonas aeruginosa pathogenicity. This study extends the utility of C.elegans as an in vivo model for the study of bacterial virulence and advances the molecular understanding of S.marcescens pathogenicity.     

Genetic and plastic responses of a northern mammal to climate change

Climate change is predicted to be most severe in northern regions and there has been much interest in to what extent organisms can cope with these changes through phenotypic plasticity or microevolutionary processes. A red squirrel population in the southwest Yukon, Canada, faced with increasing spring temperatures and food supply has advanced the timing of breeding by 18 days over the last 10 years (6 days per generation). Longitudinal analysis of females breeding in multiple years suggests that much of this change in parturition date can be explained by a plastic response to increased food abundance (3.7 days per generation). Significant changes in breeding values (0.8 days per generation), were in concordance with predictions from the breeder's equation (0.6 days per generation), and indicated that an evolutionary response to strong selection favouring earlier breeders also contributed to the observed advancement of this heritable trait. The timing of breeding in this population of squirrels, therefore, has advanced as a result of both phenotypic changes within generations, and genetic changes among generations in response to a rapidly changing environment.     

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.     

Qualitative simulation of the carbon starvation response in Escherichia coli

In case of nutritional stress, like carbon starvation, Escherichia coli cells abandon their exponential-growth state to enter a more resistant, non-growth state called stationary phase. This growth-phase transition is controlled by a genetic regulatory network integrating various environmental signals. Although E. coli is a paradigm of the bacterial world, it is little understood how its response to carbon starvation conditions emerges from the interactions between the different components of the regulatory network. Using a qualitative method that is able to overcome the current lack of quantitative data on kinetic parameters and molecular concentrations, we model the carbon starvation response network and simulate the response of E. coli cells to carbon deprivation. This allows us to identify essential features of the transition between exponential and stationary phase and to make new predictions on the qualitative system behavior following a carbon upshift.     

Effect of imazaquin on absorption,translocation, and pattern of distribution of chlormequat chloride in winter wheat

The role of imazaquin in the absorption, translocation, and distribution of chlormequat chloride in CYCOCEL^* CL has been studied in winter wheat. Three treatments were applied to the 5th leaf of the main stem at growth stage 5 (Feekes Large scale): (1)¹⁴C-chlormequat chloride, (2) CYCOCEL^* CL containing¹⁴C-chlormequat chloride, and (3) CYCOCEL^* CL containing¹⁴C-imazaquin. Tracing of the radioactivity was followed in the treated leaf, main stem, tillers, and roots. Results showed that more than 85% of the radioactivity absorbed remained in the treated leaf. Ten days after the application of chlormequat chloride alone, 94.4% of the¹⁴C-chlormequat was found in the treated leaf, 2.9% in the main stem, 1.2% in the tillers, and 1.4% in the root system versus 88.2, 8.2, 2.1, and 1.4%, respectively, for the chlormequat chloride plus imazaquin treatment. It was concluded that imazaquin increases the mobility and the pattern of distribution of chlormequat chloride in the plant.