Evolution of the amino acid fingerprint in the unsterilized rhizosphere of a legume in relation to plant maturity

The exudation of low-molecular-weight compounds, such as amino acids, contributes to the structure of rhizospheric microbial communities and plays a crucial role in nutrient mobility throughout the vegetative cycle of the plant. Due to their low concentration and their constant removal by microorganisms, the study of amino acids is difficult in unsterilized rhizospheric soil. Our aim was to investigate the dynamics of amino acids in unsterilized soil. Since there was currently no available method to estimate accurately amino acid exudation in soil, a methodology was developed to assess amino-acid fingerprints in the rhizosphere. This proved a viable approach for understanding the dynamics of amino-acid release into the rhizosphere. The method was tested on Medicago truncatula, with the hypothesis that the fingerprint of amino acids in the rhizosphere should reflects amino acid exudation from leguminous plants, subject to the influence of microbiological components of the soil, and should be correlated with the stage of plant growth. M. truncatula plants were grown in either unsterilized or sterilized substrate. Amino acids in the substrate samples were identified and quantified by gas chromatography-mass spectrometry (GC-MS) and microbial biomass in the soil samples was also assessed. The total content of amino acids in the sterilized rhizospheric substrate increased substantially during plant growth with the highest content observed at the end of the vegetative stage. Despite the probable continual removal of amino acids by microorganisms, we also observed a significant quantitative and qualitative plant-dependent effect on the amino acid fingerprint in unsterilized soil. A clear chronological differentiation of this fingerprint in the rhizosphere of unsterilized and sterilized substrate was revealed by principal component analysis. We observed predominantly serine and glycine in the sterilized rhizosphere, whereas asparagine became prominent before flowering in the unsterilized rhizosphere soil. Root amino acid metabolic pathways and exudation appear to differ in unsterilized soil compared to in sterilized substrate, indicating an adaption to the rhizospheric environment. The established method could be used in other applications, for example, for future ecophysiological studies aiming at following root exudation of amino acids in response to changing environmental conditions