Quantitative 3D imaging of cell level auxin and cytokinin response ratios in soybean roots and nodules

Legume-Rhizobium symbiosis results in root nodules where rhizobia fix atmospheric nitrogen into plant usable forms in exchange for plant-derived carbohydrates. The development of these specialized root organs involves a set of carefully orchestrated plant hormone signalling. In particular, a spatio-temporal balance between auxin and cytokinin appears to be crucial for proper nodule development. We put together a construct that carried nuclear localized fluorescence sensors for auxin and cytokinin and used two photon induced fluorescence microscopy for concurrent quantitative 3-dimensional imaging to determine cellular level auxin and cytokinin outputs and ratios in root and nodule tissues of soybean. The use of nuclear localization signals on the markers and nuclei segmentation during image processing enabled accurate monitoring of outputs in 3D image volumes. The ratiometric method used here largely compensates for variations in individual outputs due to sample turbidity and scattering, an inherent issue when imaging thick root and nodule samples typical of many legumes. Overlays of determined auxin/cytokinin ratios on specific root zones and cell types accurately reflected those predicted based on previously reported outputs for each hormone individually. Importantly, distinct auxin/cytokinin ratios corresponded to distinct nodule cell types indicating a key role for these hormones in nodule cell type identity.