Identification of amino compounds synthesized and translocated in symbiotic Parasponia

We investigated the synthesis and translocation of amino compounds in Parasponia, a genus of the Ulmaceae that represents the only non-legumes known to form a root nodule symbiosis with rhizobia. In the xylem sap of P. andersonii we identified asparagine, aspartate, glutamine, glutamate and significant quantities of a non-protein amino acid, 4-methylglutamate (2-amino-4-methylpentanedioic acid). This identification was confirmed by two methods, capillary gas chromatography (GC) electron ionization (EI) mass spectrometry (MS) and reverse phase high pressure liquid chromatography (HPLC) analysis of derivatized compounds. In leaf, root and nodule samples from P. andersonii and P. parviflora we also identified the related compounds 4-methyleneglutamate and 4-methyleneglutamine. Using N-15(2) labelling and GC-MS analysis of root nodule extracts we followed N-2 fixation and ammonia assimilation in P. andersonii root nodules and observed label initially in glutamine and subsequently in glutamate, suggesting operation of the glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) pathway. Importantly, we observed the incorporation of significant quantities of N-15 into 4-methylglutamate in nodules, demonstrating the de novo synthesis of this non-protein amino acid and suggesting a role in the translocation of N in symbiotic Parasponia.