Ziegler J.,Institute of Plant Biochemistry |
Abel S.,Institute of Plant Biochemistry
Amino Acids | Year: 2014
A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC-ESI-MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/μl with a linear range of up to 125 pmol/μl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm). © 2014 Springer-Verlag Wien.
Handrick V.,Institute of Plant Biochemistry |
Vogt T.,Institute of Plant Biochemistry |
Frolov A.,Institute of Plant Biochemistry
Analytical and Bioanalytical Chemistry | Year: 2010
Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)-electrospray ionization-mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC-MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated. © 2010 Springer-Verlag.
Weichert H.,Leibniz University of Hanover |
Peschel S.,Leibniz University of Hanover |
Knoche M.,Leibniz University of Hanover |
Neumann D.,Institute of Plant Biochemistry
Journal of the American Society for Horticultural Science | Year: 2010
Recent studies established that some ferric salts, including FeCl3, decrease water permeability of the sweet cherry (Prunus avium L.) fruit exocarp and fruit cracking, presumably by a pH-dependent precipitation reaction that blocks high-flux pathways across the fruit surface. The objectives of our study were the following: to establish the effect of receiver pH on penetration of 55FeCl3 through excised exocarp segments (ES) and isolated cuticular membranes (CM) and to localize any Fe precipitates in the epidermal system of mature sweet cherry fruit. Penetration was studied using an infinite dose diffusion system where 55Fe penetrated from donor solutions of ferric salts (10 mM, pH 2.2-2.6) or EDTA-Na-Fe(III) (10 mM, pH 5.0) across an interfacing ES or CM into aqueous receiver solutions of pH values ranging from 2.0 to 6.0. For receiver pH 2.0, 55Fe penetration of the ES from a 10 mM FeCl3 donor (pH 2.6) was linear with time, but for receiver pH $ 3.0, penetration was low and insignificant. Increasing the pH of the water receiver from 2.0 to 6.0 in the course of an experiment resulted in an immediate halt of penetration regardless of whether 55Fe penetration occurred from FeCl3 (pH 2.6), Fe(NO3)3 (pH 2.6), or Fe2(SO4)3 (pH 2.4) as donor solutions (all at 10 mM). Only from EDTA-Na-Fe(III) (pH 5.0) 55Fe penetration continued to occur albeit at a decreased rate (-30%). At receiver pH 2.0, the 55FeCl3 flux through stomatous 'Sam' ES averaged 10.4 ± 2.3 pmol m-2 s-1 and was positively correlated to stomatal density. Conventional and analytical electron microscopy (energy dispersive X-ray analysis, electron spectroscopic imaging, and electron energy loss spectroscopy) identified ferric precipitates in periclinal and anticlinal cell walls of epidermal cells underlying the cuticle, but not within the cuticle. These data indicate that the lack of 55Fe penetration from donor solutions of ferric salts through the ES into a receiver solution at pH $ 3 and the previously reported decrease in water uptake and cracking as a response to immersing fruit in solutions of ferric salts are the result of a precipitation reaction at the cuticle/cell wall interface in the sweet cherry exocarp. Although spray application of ferric salts is prohibitive for ecotoxicological reasons, understanding their mechanism in decreasing water uptake and fruit cracking may be helpful in the search for alternate compounds that are effective and ecotoxicologically acceptable.
Nahar K.,Ghent University |
Kyndt T.,Ghent University |
Hause B.,Institute of Plant Biochemistry |
Hofte M.,Ghent University |
Gheysen G.,Ghent University
Molecular Plant-Microbe Interactions | Year: 2013
The importance of phytohormone balance is increasingly recognized as central to the outcome of plant-pathogen interactions. Next to their well-known developmental role, brassinosteroids (BR) were recently found to be involved in plant innate immunity. In this study, we examined the role of BR in rice (Oryza sativa) innate immunity during infection with the root-knot nematode Meloidogyne graminicola, and we studied the inter-relationship with the jasmonate (JA) pathway. Exogenous epibrassinolide (BL) supply at low concentrations induced susceptibility in the roots whereas high concentrations of BL enforced systemic defense against this nematode. Upon high exogenous BL supply on the shoot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) confirmed a strong feedback inhibitory effect, leading to reduced BR biosynthesis in the root. Moreover, we demonstrate that the immune suppressive effect of BR is at least partly due to negative cross-talk with the JA pathway. Mutants in the BR biosynthesis or signaling pathway accumulate slightly higher levels of the immediate JA-precursor 12-oxo-phytodienoic acid, and qRT-PCR data showed that the BR and JA pathway are mutually antagonistic in rice roots. Collectively, these results suggest that the balance between the BR and JA pathway is an effective regulator of the outcome of the rice-M. graminicola interaction. © 2013 The American Phytopathological Society.
Schmollinger S.,University of California at Los Angeles |
Muhlhaus T.,University of Kaiserslautern |
Muhlhaus T.,Max Planck Institute of Molecular Plant Physiology |
Boyle N.R.,University of California at Los Angeles |
And 19 more authors.
Plant Cell | Year: 2014
Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration. Transcripts and proteins of the Calvin-Benson cycle are reduced in N-deficient cells, resulting in the accumulation of cycle metabolic intermediates. Both cytosolic and chloroplast ribosomes are reduced, but via different mechanisms, reflected by rapid changes in abundance of RNAs encoding chloroplast ribosomal proteins but not cytosolic ones. RNAs encoding transporters and enzymes for metabolizing alternative N sources increase in abundance, as is appropriate for the soil environmental niche of C. reinhardtii. Comparison of the N-replete versus N-deplete proteome indicated that abundant proteins with a high N content are reduced in N-starved cells, while the proteins that are increased have lower than average N contents. This sparing mechanism contributes to a lower cellular N/C ratio and suggests an approach for engineering increased N-use efficiency. © American Society of Plant Biologists. All rights reserved.
PubMed | Institute of Plant Biochemistry
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2014
Plant glandular trichomes are specialized secretory structures located on the surface of the aerial parts of plants with large biosynthetic capacity, often with terpenoids as output molecules. The collection of plant trichomes requires a method to separate trichomes from leaf epidermal tissues. For metabolite profiling, trichome tissue needs to be rapidly quenched in order to maintain the indigenous state of intracellular intermediates. Appropriate extraction and chromatographic separation methods must be available, which address the wide-ranging polarity of metabolites. In this chapter, a protocol for trichome harvest using a frozen paint brush is presented. A work flow for broad-range metabolite profiling using LC-MS(2) analysis is described, which is applicable to assess very hydrophilic isoprenoid precursors as well as more hydrophobic metabolites from trichomes and other plant tissues.