Saturday, 15 July 2006

New Analytical Techniques in Rhizodeposit Chemistry: Pyrolysis Field Ionization Mass Spectrometry, Liquid Injection Field Desorption Ionization – Mass Spectrometry and Synchrotron-based XANES.

Peter Leinweber, Univ of Rostock, Justus-von-Liebig-Weg, Rostock, Germany, André Schlichting, Steinbeis-Transferzentrum Soil Biotechgnology, Thuenenplatz 1, Gross Luesewitz, Germany, and Tom Regier, Canadian Light Source Inc., 101 Perimeter Road, Saskatoon, SK S7N 0X4, Canada.

We applied Pyrolysis Field Ionization Mass Spectrometry (Py-FIMS), Liquid Injection Field Desorption Ionization – (LIFDI) Mass Spectrometry (LIFDI-MS) and Synchrotron-based, soft X-ray Absorption Near-Edge Spectroscopy (XANES) to get new insights in the rhizosphere chemistry. Samples of rhizodeposits of non-transgenic (corn, wheat, potato) and transgenic (potato, Bt-corn) crops grown in experimental containers were collected by a leaching system and investigated by these techniques. Py-FIMS of the freeze-dried rhizodeposits enabled the detection of high-molecular compounds up to m/z 600, which can be either released by crops (e.g., phenols, sterols, suberin) or be products of the interaction of rhizodeposits with the surrounding humified soil organic matter (e.g., lignin dimers, alkylaromatics). Furthermore, photoperiod-resolved sampling and Py-FIMS proved differences in the rhizodeposit composition between day and night. For instance, corn released significantly larger proportions of amino acids, such as L-glutamic acid, during day-than during night-time (Melnitchouck et al., 2005). We applied this technique to investigate if genetic modification of potato crops (chimeric gene for T4-lysozyme) is likely to have any adverse effects of soil microbiota. Investigation of three independent sets of leachates showed that the rhizodeposits of three transgenic potato lines molecular-chemically differed from those of the non-transgenic potato crops (Melnitchouck et al., 2006). Despite this extreme sensitivity of Py-FIMS, potential undesired changes in the samples due to (1) freeze-drying and (2) the thermal energy impact during pyrolysis call for the application of complementary analytical techniques. Thus, we tested a newly developed LIFDI-MS system to analyze rhizosphere solution samples. In this system the liquid sample is directly placed at the FD emitter through a capillary. Direct (soft) field ionization and desorption from the emitter surface enables the detection of largely non-fragmented molecules. The initial experiments gave clearly resolved signals with constant high ion intensities for carbohydrates (m/z 96) and phenols and lignin monomers (m/z 150, 180, 210). Methodological improvements will be figured out to concentrate the target substances and lower the concentrations of undesired salts in the analyte. As organic N compounds were found to be important in rhizodeposits, and since there is an ongoing controversial debate on N heterocyclics in the literature, we recorded N K-edge XANES spectra at the Spherical Grating Monochromator (SGM) Beamline at the Canadian Light Source, Saskatoon. Spectra of rhizodeposit samples indicated the presence of some pyridinic and amide N compounds. Thus, the non-destructive N XANES supported the results of the mass spectrometric techniques. In ongoing experiments we will refine the analytical techniques to reduce the detection limits of compounds. Applications will be the risk assessment of genetically modified crops and reaction pathways for the formation of heterocyclic N in the myco-rhizosphere. References: (1) Melnitchouck, A., Leinweber, P., Eckhardt, K.-U., Beese, R. (2005) Qualitative differences between day- and nighttime rhizodeposition in maize (Zea mays L.) as investigated by pyrolysis-field ionization mass spectrometry. Soil Biology and Biochemistry 37: 155-162. (2) Melnitchouck, A., Leinweber, P., Eckhardt, K.-U., Beese, R. (2006) Pyrolysis-field ionization mass spectrometry of rhizodeposits – a new approach in the risk assessment of genetically modified plants. Environmental Biosafety Research (submitted).

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