Changes of phenolic secondary metabolite profiles in the reaction of narrow leaf lupin (Lupinus angustifolius) plants to infections with Colletotrichum lupini fungus or treatment with its toxin

Plant interactions with environmental factors cause changes in the metabolism and regulation of biochemical and physiological processes. Plant defense against pathogenic microorganisms depends on an innate immunity system that is activated as a result of infection. There are two mechanisms of triggering this system: basal immunity activated as a result of a perception of microbe-associated molecular patterns through pattern recognition receptors situated on the cell surface and effector-triggered immunity (ETI). An induced biosynthesis of bioactive secondary metabolites, in particular phytoalexins, is one of the mechanisms of plant defense to fungal infection. Results of the study on narrow leaf lupin (Lupinus angustifolius L.) plants infected with the anthracnose fungus Colletotrichum lupini and treated with fungal phytotoxic metabolites are described in the paper. The C. lupini phytotoxins were isolated from liquid cultures, purified and partially characterized with physicochemical methods. Accumulation of secondary metabolites on leaf surface and within the tissues of plants either infected, treated with the fungal phytotoxin or submitted to both treatments was studied using GC-MS and LC-MS, respectively. Substantial differences in isoflavone aglycones and glycoconjugate profiles occurred in response to different ways of plant treatment. Electronic supplementary material The online version of this article (doi:10.1007/s11306-012-0475-8) contains supplementary material, which is available to authorized users.

screw-cupped tubes. The solvent was removed in a vacuum concentrator at room temperature (Savant SPD 121P, Thermo Electron Corporation, Waltham, USA). Samples were dissolved in 300 µl of 80% methanol in water and centrifuged at 10,000 rpm for 10 min, transferred to autosampler vials and immediately subjected to LC-MS analyses.
For GC-MS analysis of compounds present on lupin leaf surface, green parts of lupin plants (five plants for each sample) were washed with 100 ml of CH 2 Cl 2 for 20 sec. The washing time was optimized to avoid damage of the cells, causing a leakage of cytosolic compounds that occurred with a prolonged action of the organic solvent. Collection of surface compounds was done at different time points after elicitation or infection. The obtained solutions were evaporated, then the sample was dissolved in 2 ml of CH 2 Cl 2 and the volume corresponding to 2 mg of the original dry weight from the each sample was transferred to the Teflon-lined screw-capped vials and taken for further GC-MS analysis. Rybitol (20 l of methanol solution at a concentration of 1 mg/ml) was added to each sample as an internal standard and a two-stage chemical derivatization procedure was performed.
Forty µl of O-methylhydroxylamine hydrochloride solution in pyridine (20 mg/mL) was added to the sample and heated at 40°C for 90 min followed by addition of 70 l MSTFA (N-acetyl-N-(trimethylsilyl)-trifluoroacetamide) and heating at 37°C for 30 min. The sample was centrifuged at 10,000 rpm for 10 min, transferred to autosampler glass vials and subjected to the GC-MS analyses.
Two biological samples were collected from each object and two independent extracts were prepared and analyzed for each sample.

Gas chromatography/mass spectrometry
GC-MS analyses of leaf surface compounds were performed with Agilent 6890 N gas chromatograph with a 7683 autosampler (Agilent Technologies, Stockport, UK) equipped with a DB-5 column (30 m × 0.25 mm i.d., film thickness 0.25 µm) from J&W Scientific Co. (USA) and coupled to the time-of-flight mass spectroscope (MS-ToF) analyzer from Waters. Helium was used as the carrier gas at a flow rate of 1 ml/min. The GC oven temperature program was as follows: 2 min at 70°C, raised by 10°C min −1 to 300°C, and held for 15 min at 300°C. The total analysis time was 45 minutes. The injector temperature was 250°C and 50% of the recovered vapor was passed into the chromatography column (split 50). The interface temperature was 230°C and source temperature was 250°C. Insource fragmentation was performed with 70 eV energy. Mass spectra were recorded in the 50-650 m/z range and data were analyzed using the Waters MassLynx ver. 4.1.

Liquid chromatography/mass spectrometry
LC-MS analyses of plant extracts were performed with an Agilent (Waldbronn, Germany) RR 1200 system (binary pump SL, diode array detector G1315C Starlight and a G1367C SL automatic injector) connected to a micrOToF-Q mass spectrometer from Bruker Daltonics (Bremen, Germany) and/or the TriVersa NanoMate system (Advion, USA).
Analyses were carried out using Zorbax Eclipse XDB C18 or Poroshell 120 EC-C18 columns (2.1×100 mm, granulation of 1.8 µm, Agilent). Chromatographic separation was performed at a 0.6 ml/min flow rate using mixtures of two solvents: A (99.5% H 2 O/0.5% formic acid v/v) and B (99.5% acetonitrile/0.5% formic acid v/v) with a 3:2 split of the column effluent, so 0.2 ml/min was delivered to the ESI ion source. The elution steps were as follows: 0-5 min linear gradient from 10 to 30% of B, 5-12 min isocratic at 30% of B, 12-13 min linear gradient from 30-95% of B, and 13-15 min isocratic at 95% of B. After returning back to the initial conditions, the equilibration was achieved after 4 min.
The ESI source of the micrOToF-Q mass spectrometer operated at a voltage of ±4.5 kV, ion transfer energy of 7 eV during the MS or MS 2 experiments. Nitrogen nebulization was performed at 1.2 bar and a dry gas flow of 8.0 l/min at temperature of 220°C. The instrument was operated using the micrOTOF control program ver. 2.3 and data were analyzed using the Bruker Data Analysis ver. 4 package. The system was calibrated externally using the calibration mixture containing sodium formate clusters. Additional internal calibration was performed for every run by injection of the calibration mixture using the diverter valve during the LC separation. All calculations of m/z values were done with the HPC quadratic algorithm with the accuracy of at least 5 ppm.        Table 1S. Quinolizidine alkaloids identified on the leaf surface of narrow leaf lupin (L. angustifolius) plants.

M + Rt Compound
PubChem ID