Document Type

Thesis - Open Access

Award Date

2025

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Senthil Subramanian

Abstract

Lateral roots (LRs) and nodules are post-embryonic root organs in legumes formed through the activity of de novo meristem. LRs, which are present in all vascular plants, are developed primarily via intrinsic developmental cues, whereas root nodules, found only in a small group of plants known as legumes, are formed in response to legume–rhizobium symbiosis. LRs arise from the pericycle layer of the root, whereas nodule initiation can begin either in the pericycle (indeterminate nodules with persistent meristem, as in Medicago) or in the cortex (determinate nodules with transient meristem, as in soybean), pointing toward a conserved developmental origin, especially between LRs and indeterminate nodules. Auxin and cytokinin are plant hormones that play an important role in the initiation and maturation of root lateral organs. Although these hormones interact closely in regulating plant development, their individual responses, but not simultaneous, stage-specific quantification, have been studied in LR and nodule development in Medicago. In this study, we used the Medicago truncatula AuxCysensor line, which employs fluorescent reporters (eGFP and tdTomato) that localize to the nucleus (NLS), enabling quantification of hormonal response at the cell level. The auxin-responsive DR5 promoter fused with eGFP (DR5:eGFP-NLS) and the cytokinin-responsive TCSn promoter fused with tdTomato (TCSn:tdTomato-NLS), allowed us to visualize and quantify auxin and cytokinin responses within tissue at single-cell resolution. Using two-photon–induced fluorescence (TPIF) microscopy for in vivo 3D imaging, together with a robust biological image-analysis pipeline, Auxin–Cytokinin Relative Output (ACRO) ratios were calculated across developmental stages of lateral roots and nodules. Lateral root development was predominantly auxin-driven, with increasing auxin response at the tip of developing LR primodia at early stages and later followed by the establishment of an auxin-dominated region forming an ACRO peak at later stages, potentially marking the region that will establish the future lateral root meristem. We hypothesize that the cells that will eventually form the lateral root meristem acquire their identity at very early stages of primordium development alongside auxin maxima. Another notable observation is that, after emergence, the lateral root displays an ACRO distribution highly similar to that of the primary root tip. In contrast to LR, early nodule primordia show a strong cytokinin response in the cortex, with increasing cytokinin response during subsequent stages in parallel with extensive cortical cell proliferation. Tissue-specific quantification at a later stage of nodule shows a unique ACRO signature across the nodule vasculature, nodule periphery, central zone, and nodule meristem, despite these tissues are derived from cortical cell division. Our findings will complement single-cell RNA-seq analyses by correlating gene expression with observed auxin-cytokinin distribution in LR and nodules. This integrated approach will enable the discovery of new conserved hormonal pathways that coordinate LR and nodule development both within and across species. Ultimately, a deeper understanding of these hormonal and developmental programs may support efforts to engineer non-nodulating plants to form nodules and meet their own nitrogen demands, thereby making agriculture more sustainable.

Publisher

South Dakota State University

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Rights Statement

In Copyright