Document Type

Thesis - Open Access

Award Date

2017

Degree Name

Master of Science (MS)

Department / School

Agronomy, Horticulture, and Plant Science

First Advisor

Senthil Subramanian

Keywords

Auxin, microRNA, root nodule, soybean, transcription factor

Abstract

Soybean is an excellent candidate for sustainable agriculture due to its production of nutritious, versatile beans and the ability to form symbiotic organs called root nodules that perform nitrogen fixation. As demand for both yield and sustainable agriculture continue to increase, root nodules offer an attractive alternative to expensive and environmentally harmful nitrogen fertilizers. Understanding root nodule formation may open genetic engineering avenues for optimizing nitrogen fixation performance and transferring the nodule-formation ability to other plants. A major determinant of nodule numbers and quality in soybean is microRNA 160 (miR160), which dictates developmental stage-specific auxin sensitivity by targeting repressor auxin response factor (ARF) transcription factors during nodule development. However, the specific downstream targets of the miR160-ARF signaling module remain unknown. This project seeks to resolve these targets by evaluating the cellular, spatiotemporal, and DNAbinding activity of ARF16-2 (a key target of miR160 in soybean nodules), by confocal microscopy, DamID-Seq, and protein-DNA interaction studies. ARF16-2promoter:GUS or tdT transcriptional fusions revealed that ARF16-2 is transcriptionally active in dividing and differentiating tissues such as primary root tips, lateral root primordium, and emerging nodules. In addition, ARF16-2 promoter:GUS or tdT fusions show that this gene is active in the parenchyma and root stele of mature nodules. Interestingly, ARF16- 2 promoter:16-2 CDS:tdT translational fusions show a reduction of tdT signal in mature nodule tissues, underscoring post-transcriptional regulation of ARF16-2, likely by miR160 during nodule maturation. Compared to known activator ARFs, ARF16-2 does not appear to bind the conserved AuxRE TGTCTC motif with the same affinity as activator ARFs, suggesting alternative binding sites or functionality. In addition, ARF16- 2 appears to function as a monomer compared to the activator ARFs that appear to act as dimers. We hypothesize that ARF16-2 might form heterodimers with activator ARFs to reduce transactivation of target genes. When the genomic binding profile of ARF16-2 is identified and evaluated, we expect to identify key downstream genes affected by its activity during nodule development. A protein-DNA binding assay that can be performed using a simple UV trans-illuminator developed as part of this research could be accessible for teaching labs to demonstrate specificity of bio-molecular interactions.

Library of Congress Subject Headings

Soybean -- Genetic engineering.
Root-tubercles.
Auxin.
Transcription factors.

Description

Includes bibliographical references (91-99)

Format

application/pdf

Number of Pages

123

Publisher

South Dakota State University

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Rights Statement

In Copyright