Organic Nitrogen Uptake and Assimilation in Cucumis sativus Using Position-Specific Labeling and Compound-Specific Isotope Analysis

Organic nitrogen is now considered a significant source of N for plants. Although organic management practices increase soil organic C and N content, the importance of organic N as a source of crop N under organic farming management systems is still poorly understood. While dual-labeled (C-13 and N-15) molecule methods have been developed to study amino acid uptake by plants, multiple biases may arise from pre-uptake mineralization by microorganisms or post-uptake metabolism by the plant. We propose the combination of different isotopic analysis methods with molecule isotopologues as a novel approach to improve the accuracy of measured amino acid uptake rates in the total N budget of cucumber seedlings and provide a better characterization of post-uptake metabolism. Cucumber seedlings were exposed to solutions containing L-Ala-1-C-13, N-15 or U-L-Ala-C-13(3), N-15, in combination with ammonium nitrate, at total N concentrations ranging from 0 to 15 mM N and at inorganic/organic N ratios from 10: 1 to 500:1. Roots and shoots were then subjected to bulk stable isotope analysis (BSIA) by Isotope Ratio Mass Spectrometry (IRMS), and to compound-specific stable isotope analysis (CSIA) of the free amino acids by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS). Plants exposed to a lower inorganic: organic N ratio acquired up to 6.84% of their N from alanine, compared with 0.94% at higher ratio. No C-13 from L-Ala-1-C-13, N-15 was found in shoot tissues suggesting that post-uptake metabolism of Ala leads to the loss of the carboxyl-C as CO2. CSIA of the free amino acids in roots confirmed that intact Ala is indeed taken up by the roots, but that it is rapidly metabolized. C atoms other than from the carboxyl group and amino-N from Ala are assimilated in other amino acids, predominantly Glu, Gln, Asp, and Asn. Uptake rates reported by CSIA of the free amino acids are nevertheless much lower (16-64 times) than those reported by BSIA. Combining the use of isotopologues of amino acids with compound-specific isotope analysis helps reduce the bias in the assessment of organic N uptake and improves the understanding of organic N assimilation especially in the context of organic horticulture.