Document Type

Thesis - Open Access

Award Date

2025

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Senthil Subramanian

Abstract

Roots support water and nutrient uptake, anchorage, and soil interactions through continuous growth at the primary root tip (PRT). In common bean (Phaseolus vulgaris), new cells are produced in an open root apical meristem and subsequently elongate in the elongation zone, and together these processes drive root tip progression through the soil. Lateral roots (LRs) arise in the differentiation zone, where pairs of pericycle founder cells dedifferentiate, divide asymmetrically, and form lateral root primordia (LRP) that pass through defined developmental stages before emergence. Auxin and cytokinin (CK) are central regulators of both primary root growth and LR development, yet their simultaneous, cell-type–specific dynamics remain poorly characterized in common bean. In this study, we used composite common bean plants expressing the AuxCysensor (DR5rev::eGFP-NLS; TCSn::tdTomato-NLS) nuclear-localized auxin and CK reporters, which had been previously demonstrated in soybean to provide cell level auxin and cytokinin transcriptional outputs. Primary root tips were imaged using intravital 3D two-photon–induced fluorescence (TPIF) microscopy and subsequently, quantitative image analysis was used to quantify auxin and CK responses and calculate an Auxin–Cytokinin Relative Output (ACRO) ratios at single-nucleus resolution. The results demonstrated conserved tissue domain-level auxin-CK response patterns in common bean primary root tips and provided an analysis pipeline that could be used to quantify cell-level auxin–CK response patterns in other common bean root organs. We originally aimed to extend this approach to quantify ACRO ratios in both lateral root primordia and nodules; however, nodule analysis could not be completed because we were unable to consistently obtain nodulated common bean roots despite repeated inoculation attempts and troubleshooting. Therefore, we focused on mapping auxin and CK responses across six developmental stages of lateral root development and found that a strong auxin maximum is initially localized to pericycle founder cells at stage 1a, shifts toward inner cortical cells at the primordium tip by stages 3–4, and later relocates toward the primary root vasculature as tip cells transition from proliferation to differentiation. Thus, early LR development is characterized by high auxin and relatively low CK that favor primordium initiation and outgrowth, whereas later stages show more complex auxin–CK patterns associated with the onset of differentiation. Finally, we tested organ-specific promoter activity of Phaseolus vulgaris orthologs of TMO7 and LRP1 during LR development. Using transgenic hairy roots expressing pPvLRP1::GUS and pPvTMO7::GFP, we found that PvLRP1 is activated early in pericycle and inner cortical layers and remains confined to inner primordium cells, while PvTMO7 expression expands outward over time, becoming enriched in the primordium and epidermal/outer cortical layers and peaking at the primordium tip by stage 6. Together, these results show that PvLRP1 and PvTMO7 mark distinct but complementary domains within developing lateral roots. Since each nucleus is spatially and quantitatively resolved, these datasets can be integrated with single-cell RNA-seq being conducted by our collaborators to directly link hormone output patterns to underlying transcriptional programs in LR meristems. This integrated framework will help uncover conserved auxin–cytokinin regulatory pathways that coordinate lateral root development within and across legume species. This work generate knowledge that could be leveraged by the broader plant science and breeding communities to design legume root systems with improved water and nutrient uptake, ultimately supporting the development of stress-resilient, high-yielding crops for more sustainable agriculture.

Library of Congress Subject Headings

Beans -- Roots. 
Auxin. 
Cytokinins. 
Roots (Botany) -- Development.      

Publisher

South Dakota State University

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Rights Statement

In Copyright