Comparative Evaluation of Data Dependent and Data Independent Acquisition Workflows Implemented on an Orbitrap Fusion for Untargeted Metabolomics.

Constant improvements to the Orbitrap mass analyzer, such as acquisition speed, resolution, dynamic range and sensitivity have strengthened its value for the large-scale identification and quantification of metabolites in complex biological matrices. Here, we report the development and optimization of Data Dependent Acquisition (DDA) and Sequential Window Acquisition of all THeoretical fragment ions (SWATH-type) Data Independent Acquisition (DIA) workflows on a high-field Orbitrap FusionTM TribridTM instrument for the robust identification and quantification of metabolites in human plasma. By using a set of 47 exogenous and 72 endogenous molecules, we compared the efficiency and complementarity of both approaches. We exploited the versatility of this mass spectrometer to collect meaningful MS/MS spectra at both high- and low-mass resolution and various low-energy collision-induced dissociation conditions under optimized DDA conditions. We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.

Leaf status and environmental signals jointly regulate proline metabolism in winter oilseed rape.

Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).

A Genotypic Comparison Reveals That the Improvement in Nitrogen Remobilization Efficiency in Oilseed Rape Leaves Is Related to Specific Patterns of Senescence-Associated Protease Activities and Phytohormones.

Oilseed rape (Brassica napus L.) is an oleoproteaginous crop characterized by low N use efficiency (NUE) that is mainly related to a weak Nitrogen Remobilization Efficiency (NRE) during the sequential leaf senescence of the vegetative stages. Based on the hypothesis that proteolysis efficiency is crucial for the improvement of leafNRE, our objective was to characterize key senescence-associated proteolytic mechanisms of two genotypes (Ténor and Samouraï) previously identified with contrasting NREs. To reach this goal, biochemical changes, protease activities and phytohormone patterns were studied in mature leaves undergoing senescence in two genotypes with contrasting NRE cultivated in a greenhouse under limiting or ample nitrate supply. The genotype with the higher NRE (Ténor) possessed enhanced senescence processes in response to nitrate limitation, and this led to greater degradation of soluble proteins compared to the other genotype (Samouraï). This efficient proteolysis is associated with (i) an increase in serine and cysteine protease (CP) activities and (ii) the appearance of new CP activities (RD21-like, SAG12-like, RD19-like, cathepsin-B, XBCP3-like and aleurain-like proteases) during senescence induced by N limitation. Compared to Samouraï, Ténor has a higher hormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes senescence, particularly under low N conditions, and this is correlated with the stronger protein degradation and serine/CP activities observed during senescence. Short statement: The improvement in N recycling during leaf senescence in a genotype of Brassica napus L. characterized by a high nitrogen remobilization efficiency is related to a high phytohormonal ratio ([salicylic acid] + [abscisic acid])/([cytokinins]) that promotes leaf senescence and is correlated with an increase or the induction of specific serine and cysteine protease activities.

Amplifying recombination genome-wide and reshaping crossover landscapes in Brassicas.

Meiotic recombination by crossovers (COs) is tightly regulated, limiting its key role in producing genetic diversity. However, while COs are usually restricted in number and not homogenously distributed along chromosomes, we show here how to disrupt these rules in Brassica species by using allotriploid hybrids (AAC, 2n = 3x = 29), resulting from the cross between the allotetraploid rapeseed (B. napus, AACC, 2n = 4x = 38) and one of its diploid progenitors (B. rapa, AA, 2n = 2x = 20). We produced mapping populations from different genotypes of both diploid AA and triploid AAC hybrids, used as female and/or as male. Each population revealed nearly 3,000 COs that we studied with SNP markers well distributed along the A genome (on average 1 SNP per 1.25 Mbp). Compared to the case of diploids, allotriploid hybrids showed 1.7 to 3.4 times more overall COs depending on the sex of meiosis and the genetic background. Most surprisingly, we found that such a rise was always associated with (i) dramatic changes in the shape of recombination landscapes and (ii) a strong decrease of CO interference. Hybrids carrying an additional C genome exhibited COs all along the A chromosomes, even in the vicinity of centromeres that are deprived of COs in diploids as well as in most studied species. Moreover, in male allotriploid hybrids we found that Class I COs are mostly responsible for the changes of CO rates, landscapes and interference. These results offer the opportunity for geneticists and plant breeders to dramatically enhance the generation of diversity in Brassica species by disrupting the linkage drag coming from limits on number and distribution of COs.