Leguminous plants form a symbiotic relationship with nitrogen-fixing Rhizobium bacteria. The relationship is distinguished by the formation of specialised root structures called nodules. These nodules provide the niche where N2 gas is converted to ammonia for plant assimilation and use. Legumes developed a control system to regulate nodule numbers known as autoregulation of nodulation (AON). During the early stages of nodule development, two (and possibly more) CLE peptide elicitor signals (RIC1 and RIC2 in soybean) are produced by initiated nodule primordia. This or a second messenger is likely to be transported to the leaf vascular parenchyma, where it is detected by Nodulation Autoregulation Receptor Kinase (NARK) shown to be critical for AON through mutation studies. This perception leads to the production of reciprocal shoot-to-root signal, termed SDI (shoot-derived inhibitor), which is transported to the root where it arrests further nodule development.
A petiole-feeding bioassay was developed for the characterisation and determination of SDI in soybean (Glycine max) plants. This assay enabled the feeding of aqueous solutions to test plants, including dyes and radiolabelled tracers to determine the movement of the fed constituents such as hormones or leaf extracts to identify their suppressive effect on nodule numbers. Feeding leaf extracts from Bradyrhizobium japonicum-inoculated wild type (Glycine max cv. Bragg) or GmNARK mutant plants to GmNARK hypernodulating mutant (nts1116) test plants revealed that suppression activity was only present in wild type leaf extracts of Bradyrhizobium japonicum-inoculated plants. We demonstrated that SDI is a small, GmNARK-dependent, heat- and storage-stable molecule, and likely not of RNA or protein nature. Column separation methods coupled with analytical techniques, including mass spectrometry and NMR were used to identify potential SDI candidate molecules from the wild type leaf extracts of Bradyrhizobium japonicum-inoculated soybean plants. Structural elucidation of these SDI candidates is required to determine the chemical nature of SDI.