Quantitative Imaging of Cell-Level Auxin Cytokinin Outputs in Soybean and Model Plants

Auxin and cytokinin are major plant hormones that orchestrate plant development. These hormones are involved in cell division and differentiation and their spatio‐temporal balance controls organ initiation and maturation. Even though independent transcriptional responses to these hormones have been characterized, their concomitant outputs have not been described. We developed methodology to determine auxin-cytokinin relative output (ACRO) ratios at cellular resolution. Hormone-specific fluorescent markers that localize to the nucleus were used to report transcriptional outputs of auxin and cytokinin. Multiphoton microscopy and image data processing were used to quantify their relative ratio at cellular level in three-dimensional tissue volumes. This strategy was successfully applied to soybean (Glycine max) and two model plant species, Arabidopsis thaliana and Medicago truncatula to evaluate ACRO ratios in different organs of interest. The ACRO ratios estimated in soybean and Medicago root tips and lateral roots in this study agreed with previously reported outputs for each fluorescent reporter individually, suggesting that the method is reliable and accurate. Soybean and Medicago are legumes that can produce root nodules through a symbiotic partnership with nitrogen-fixing bacteria. Six soybean nodule developmental stages were studied and presented exclusive ACRO ratios patterns indicating a fine cell-specific regulation. We hypothesize that auxin bursts promote pattern initiation and cytokinin bursts promote cell differentiation. Based on ACRO ratios in nodules of soybean roots with reduced sensitivity to auxin, we speculated that lower auxincytokinin balance in nodule initials allows development of more primordia into nodules but delay their maturation. Similar ACRO ratios in infection zones and vascular bundles of soybean and Medicago nodules suggested a conserved role for auxin and cytokinin in these species that produce two different types of nodules. Evaluation of ACRO ratios in Arabidopsis roots treated with different concentrations of auxin or cytokinin revealed that the sensor responds to auxin in a dose-dependent manner and to cytokinin in a cell typespecific manner. Understanding the role of auxin and cytokinin balance in organ development can help improve plant productivity. For example, creating plants with more nodules that fix for a for a longer period could increase the soybean production in an ecofriendly manner