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Uptake of Humic Substances by Plants: a Study Using Tritium Autoradiography and FTICR MS Analysis
Natalia Kulikova1, Gennady Badun2, Erast Kunenkov2, Viktor Korobkov2, Zinaida Tyasto2, Maria Chernysheva2, Eugenia Tsvetkova3, Irina Perminova
1

2

Department of Soil Science, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 Department of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 N.D. Zelinski Institute of Organic Chemistry of RAS, Leninskii Ave. 47, 117913 Moscow, Russia

Moscow, Russia, knat@darvodgeo.ru
2

Moscow, Russia
3

Keywords: humic, uptake, autoradiography, FTICR mass spectrometry

1. INTRODUCTION

Bioavailability of humic substances (HS) to plants is still questionable due to difficulties in HS determination in plant tissues. The most convenient way is a usage of labeled compounds, and the availability of isotope-labeled HS is an important prerequisite to elucidate the fate of the heterogeneous organic matter in complex environments. On the other hand, presence of the isotope does not allow identifying specific compounds of HS that entered a target object. To solve this problem, isotope-labeled HS together with Fourier Transformed Ion Cyclotron Resonance mass spectrometry (FTICR MS) approach could be applied. This study was aimed (1) to find out uptake and distribution of HS in wheat seedlings and (2) to identify humic-derived compounds that can found in plants.
2. MATERIALS AND METHODS

For this study humic acids (HA) derived from leonardite was used. To synthesize isotope-labeled HA, an approach developed in (1) for the preparation of tritium-labeled HS was used. The obtained 3H-HA sample was dissolved in a phosphate buffer (0.028 M, pH 6.8) and purified by dialysis during a month at 4єC. It allowed eliminating exchangeable tritium of OH­, COOH­, and NHn groups of HA. Plants of wheat Triticum aestivum L. were used for the experiments. W heat seeds were germinated at 24°C in the dark for 72 h followed by transferring seedlings into the 0.5 l tanks containing Knopp nutrition solution. After another 72 h plants were transferred into the vials containing HA at concentration 50 mgl­1 with specific radioactivity 0.02 Curiel­1. After 24 h plants were harvested and subjected to autoradiography. To perform FTICR MS analysis, shoots' tips were colleted and subjected to methanol extraction followed by FTICR MS. FTICR was conducted at
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Apex Qe (Bruker Daltonics, Bremen, Germany) equipped with Apollo II electrospray ionization (ESI) source and 12 Tesla actively shielded superconductive magnet. Negative ionization mode was used with spray shield set to 3 kV and capillary voltage 3.5 kV. Flow rate was set to 120 mkl/hour. 100 scans in mass range 150...2000 Da were collected for each sample. To find tritium traces, FIRAN software (Kunenkov, 2006-2008) was used under the following considerations: a) there is only one tritium atom in the labeled ion; b) for each labeled ion there is ion without the label which consists only of
14 13 12

C, 1H,

16

O and

N; c) for each labeled ion there is ion without the label but with one of its

12

C replaced by

C (this consideration were used to find ion charge by mass difference between two

unlabeled ions); d) labeled ion present in mass spectrum of extract from shoots subjected to 3H-HA must be absent in mass spectrum of extract from shoots subjected to nonmodified HA.
3. RESULTS AND DISCUSSION

The obtained results revealed that coal HA were intensively adsorbed by wheat plants. The roots of the plants subjected to 3H-HA were characterized with homogeneous distribution of signal intensity. Distribution of HA within the shoots was also relatively homogeneous except for the tips of the leaves where local increase of signal density was observed. That finding was evident for the fact that HA could be taken up through plant roots and moved al least in the xylem with transpiration stream to areas of new growth. FTICR MS analysis of extract demonstrated presence of tritium-labelled fatty acids in the shoots of wheat plants treated with 3H-HA. As fatty acids are important moieties for plant lipids biosynthesis, the obtained data demonstrated clearly that HA-derived compounds can be used for membrane formation.
4. CONCLUSIONS

Distribution of HA in plants was demonstrated to be relatively homogeneous except for the tips of both roots and leaves where increase in 3H-HA concentration was observed. FTICR analysis of extract from tips leaves revealed presence of tritium-labelled fatty acids what was evident that HA-derived compounds could be used for membrane formation.
ACKNOWLEDGEMENTS

This work was supported by RFBR (#06-04-49017a).
REFERENCES
1. Badun, G. A. et al.: // Bulgarian J. of Ecol. Sci. 2(3-4), 26-27 (2003).

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