Time and substrate dependent exudation of carboxylates by Lupinus albus L. and Brassica napus L.

Root exudates influence significantly physical, chemical and biological characteristics of rhizosphere soil. Their qualitative and quantitative composition is affected by environmental factors such as pH, soil type, oxygen status, light intensity, soil temperature, plant growth, nutrient availability and microorganisms. The aim of the present study was to assess the influence of growth substrate and plant age on the release of carboxylates from Lupinus albus L. and Brassica napus L. Both plant species were studied in continuously percolated microcosms filled with either sand, soil or sand + soil (1:1) mixture. Soil solution was collected every week at 7, 14, 21, 28 and 35 days after planting (DAP). Carboxylate concentrations were determined by reversed-phase liquid chromatography - electrospray ionization - time of flight mass spectrometry (LC-ESI-TOFMS). Oxalate, citrate, succinate, malate and maleate were detected in soil solutions of both plant species. Their concentrations were correlated with the physiological status of the plant and the growth substrate. Oxalate was the predominant carboxylate detected within the soil solution of B. napus plants while oxalate and citrate were the predominant ones found in the soil solutions of L. albus plants. The sampling determination of carboxylates released by plant roots with continuous percolation systems seems to be promising as it is a non-destructive method and allows sampling and determination of soluble low molecular weight organic compounds derived from root exudation as well as the concentration of soluble nutrients, which both might reflect the nutritional status of plants.

LC-MS analysis of low molecular weight organic acids derived from root exudation.

A sensitive method for quantification of citric, fumaric, malic, malonic, oxalic, trans aconitic, and succinic acid in soil- and root-related samples is presented. The method is based on a novel, fast, and simple esterification procedure and subsequent analysis via liquid chromatography-mass spectrometry. Derivatization comprises in situ generation of HCl, which catalyzes the Fischer esterification with benzyl alcohol. As a key advance, the esterification with the aromate allows reversed-phase separation and improves electrospray ionization efficiency. The method provided procedural detection limits of 1 nM for citric, 47 nM for fumaric, 10 nM for malic, 10 nM for malonic, 16 nM for oxalic, 15 nM for succinic, and 2 nM for aconitic acid utilizing 500 µL of liquid sample. The working range was 3 nM to 10 µM for citric acid, 158 nM to 10 µM for fumaric acid, 34 nM to 10 µM for malic acid, 33 nM to 10 µM for malonic acid, 53 nM to 10 µM for oxalic acid, 48 nM to 10 µM for succinic acid, and 6 nM to 10 µM for aconitic acid. Quantification of the analytes in soil-related samples was performed via external calibration of the entire procedure utilizing (13)C-labeled oxalic and citric acid as internal standards. The robustness of the method was tested with soil extracts and samples from hydroponic experiments. The latter concerned the regulation of phosphorus solubilization via plant root exudation of citric, malic, and oxalic acid.

Hydrophilic interaction LC combined with electrospray MS for highly sensitive analysis of underivatized amino acids in rhizosphere research.

Analysis of underivatized amino acids is challenging regarding the separation as well as the detection of these small polar analytes. Hydrophilic interaction LC using a 2.1x150 mm ZIC (ZIC, zwitterionic)-hydrophilic interaction LC from SeQuant as stationary phase with 1% v/v formic acid in water and ACN as eluents was combined with MS/MS in multiple reaction monitoring mode for the separation and the detection of 16 underivatized amino acids. Regression coefficients of eight or seven point calibrations varied from 0.9454 to 0.9993. Absolute LODs and LOQ (on column) were in the fmol range (0.1-12 and 0.4-41 fmol, respectively). A fast screening method of 19 min total runtime has been developed offering applicability to samples from rhizosphere studies--characterized by low analyte concentrations and complex matrices. A successful application to the analysis of tyrosine in samples from soil adsorption experiments is presented as well as an evaluated enrichment procedure for amino acids derived from plant culture in nutrient solution.