Robust Method for Investigating Nitrogen Metabolism of N-15 Labeled Amino Acids Using AccQ center dot Tag Ultra Performance Liquid Chromatography-Photodiode Array-Electrospray Ionization-Mass Spectrometry: Application to a Parasitic Plant-Plant Interaction

International audienceAn AccQ center dot Tag ultra performance liquid chromatographyphotodiode array-electrospray ionization-mass spectrometry (AccQ center dot Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of N-15 isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing N-15 amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of N-15 isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as N-15-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to N-15(2)-glutamine. N-15(2)-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa

A high-throughput seed germination assay for root parasitic plants

BACKGROUND: Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope. RESULTS: We developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann’s protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose–response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves. CONCLUSION: The developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination

Robust Method for Investigating Nitrogen Metabolism of ¹⁵N Labeled Amino Acids Using AccQ•Tag Ultra Performance Liquid Chromatography-Photodiode Array-Electrospray Ionization-Mass Spectrometry: Application to a Parasitic Plant–Plant Interaction

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of ¹⁵N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie <i>Brassica napus</i> (rapeseed) and the parasitic weed <i>Phelipanche ramosa</i> (broomrape). This method was developed and validated using amino acid standard solutions containing ¹⁵N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of ¹⁵N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as ¹⁵N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to ¹⁵N₂-glutamine. ¹⁵N₂-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of <i>Phelipanche ramosa</i>

Response to Comment on “The Placental Mammal Ancestor and the Post–K-Pg Radiation of Placentals”

Tree-building with diverse data maximizes explanatory power. Application of molecular clock models to ancient speciation events risks a bias against detection of fast radiations subsequent to the Cretaceous-Paleogene (K-Pg) event. Contrary to Springer et al., post–K-Pg placental diversification does not require “virus-like” substitution rates. Even constraining clade ages to their model, the explosive model best explains placental evolution.Fil: O’Leary, Maureen A.. Stony Brook University; Estados UnidosFil: Bloch, Jonathan I.. University of Florida; Estados UnidosFil: Flynn, John J.. American Museum Of Natural History; Estados UnidosFil: Gaudin, Timothy J.. University of Tennessee; Estados UnidosFil: Giallombardo, Andres. American Museum Of Natural History; Estados UnidosFil: Giannini, Norberto Pedro. American Museum Of Natural History; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Goldberg, Suzann L.. American Museum Of Natural History; Estados UnidosFil: Kraatz, Brian P.. American Museum Of Natural History; Estados UnidosFil: Luo, Zhe-Xi. University of Chicago; Estados UnidosFil: Meng, Jin. American Museum Of Natural History; Estados UnidosFil: Ni, Xijun. American Museum Of Natural History; Estados UnidosFil: Novacek, Michael J.. American Museum Of Natural History; Estados UnidosFil: Perini, Fernando A.. Universidade Federal do Minas Gerais; BrasilFil: Randall, Zachary. University of Florida; Estados UnidosFil: Rougier, Guillermo Walter. The University Of Louisville; Estados UnidosFil: Sargis, Eric J.. University of Yale; Estados UnidosFil: Silcox, Mary T.. University of Toronto; CanadáFil: Simmons, Nancy B.. American Museum Of Natural History; Estados UnidosFil: Spaulding, Michelle. Carnegie Museum of Natural Histor; Estados UnidosFil: Velazco, Paúl M.. American Museum Of Natural History; Estados UnidosFil: Weksler, Marcelo. Universidade Federal do Rio de Janeiro; BrasilFil: Wible, John R.. American Museum Of Natural History; Estados UnidosFil: Cirranello, Andrea L.. American Museum Of Natural History; Estados Unido

The placental mammal ancestor and the post-KPg radiation of placentals

To discover interordinal relationships of living and fossil placental mammals and the time of origin of placentals relative to the Cretaceous-Paleogene (K-Pg) boundary, we scored 4541 phenomic characters de novo for 86 fossil and living species. Combining these data with molecular sequences, we obtained a phylogenetic tree that, when calibrated with fossils, shows that crown clade Placentalia and placental orders originated after the K-Pg boundary. Many nodes discovered using molecular data are upheld, but phenomic signals overturn molecular signals to show Sundatheria (Dermoptera + Scandentia) as the sister taxon of Primates, a close link between Proboscidea (elephants) and Sirenia (sea cows), and the monophyly of echolocating Chiroptera (bats). Our tree suggests that Placentalia first split into Xenarthra and Epitheria; extinct New World species are the oldest members of Afrotheria.Fil: O'Leary, Maureen A.. Stony Brook University; Estados Unidos. American Museum Of Natural History; Estados UnidosFil: Bloch, Jonathan I.. University Of Florida. Florida Museum Of History; Estados UnidosFil: Flynn, John J.. American Museum Of Natural History; Estados UnidosFil: Gaudin, Timothy J.. University Of Tennessee; Estados UnidosFil: Giallombardo, Andres. American Museum Of Natural History; Estados UnidosFil: Giannini, Norberto Pedro. American Museum Of Natural History; Estados UnidosFil: Goldberg, Suzann L.. American Museum Of Natural History; Estados UnidosFil: Kraatz, Brian P.. American Museum Of Natural History; Estados Unidos. Western University of Health Sciences. Department of Anatomy; Estados UnidosFil: Luo, Zhe Xi. Carnegie Museum of Natural History; Estados UnidosFil: Meng, Jin. American Museum Of Natural History; Estados UnidosFil: Ni, Xijun. American Museum Of Natural History; Estados UnidosFil: Novacek, Michael J.. American Museum Of Natural History; Estados UnidosFil: Perini, Fernando A.. American Museum Of Natural History; Estados UnidosFil: Randall, Zachary S.. University Of Florida. Florida Museum Of History; Estados UnidosFil: Rougier, Guillermo W.. The University Of Louisville; Estados UnidosFil: Sargis, Eric J.. University Of Yale; Estados UnidosFil: Silcox, Mary T.. University Of Toronto; CanadáFil: Simmons, Nancy B.. American Museum Of Natural History; Estados UnidosFil: Spaulding, Michelle. American Museum Of Natural History; Estados Unidos. Carnegie Museum of Natural History; Estados UnidosFil: Velazco, Paúl M.. American Museum Of Natural History; Estados UnidosFil: Weksler, Marcelo. American Museum Of Natural History; Estados UnidosFil: Wible, John R.. Carnegie Museum of Natural History; Estados UnidosFil: Cirranello, Andrea L.. Stony Brook University; Estados Unidos. American Museum Of Natural History; Estados Unido