Document Type

Thesis - Open Access

Award Date


Degree Name

Master of Science (MS)

Department / School

Agronomy, Horticulture, and Plant Science

First Advisor

Senthil Subramanian


Nitrogen is one of the most limiting nutrients for plant growth and yield. Leguminous plants such as soybean (Glycine max) have developed the ability to form a symbiotic association with nitrogen fixing rhizobia. This symbiotic association results in the formation of unique structures called nodules that originate from root cortex via de novo cell differentiation. During soybean nodule development, two major nodule zones, the Nodule primordium (Npr) in the center and the nodule parenchyma (Npa) in the periphery, are clearly distinguishable. Npr gives rise to infection zone (IZ), and the Npa holds vascular bundles. However, it is not clear what early signaling pathways drive the conspicuous development of these two nodule zones. To bridge this knowledge gap, we adapted TRAP (Translating ribosome affinity purification) technique for use in soybean hairy root composite plants and evaluated the enrichment of selected mRNAs in translating ribosomes of Npa (using the ENOD2 promoter) and Npr/IZ (using the ENOD40 promoter) in soybean nodules. Confocal images confirmed the expected tissue-specific expression of ENOD2 promoter-driven TRAP gene cassettes in the Npa region and ENOD40 promoter-driven TRAP gene cassettes in the Npr/IZ. Absence of non-coding RNAs in TRAP samples confirmed that TRAP derived RNAs are free of contamination from total or nuclear RNAs. Enrichment of nodule zone-specific translated mRNAs was validated by RT-qPCR assays on three different marker genes: ENOD2, ENOD40, and CYP83B1 whose nodule zone-enriched expression patterns are previously demonstrated. The expected expression pattern of tissue-specific marker genes at 7 and 10 dpi validated the suitability of our system and methods to evaluate nodule zone-specific translated mRNA profiles. Relative enrichment of selected genes in each nodule zone was evaluated using RT-qPCR. Abundance of mRNAs encoding transcription factors such as MyB-related transcription factor (GLYMA03G42260.1), bZIP transcription factor (GLYMA19G43420.1), and bHLH transcription factor (GLYMA08G04661.1) were significantly higher in nodule infection zone vs. parenchyma at 10 dpi suggesting that these genes might be involved in nitrogen fixation. Similarly, the abundance of mRNA encoding GmARF5 (GLYMA17G37580, potential ortholog of Arabidopsis ARF5) was significantly enriched in nodule parenchyma at 7 and infection zone at 10 dpi suggesting the tissue-specific roles for auxin during nodule development and maturation. The abundance of nitrate transporter (GLYMA11G04500.2) mRNA was significantly enriched in infection zone at 7 dpi and 10 dpi suggesting a possible role for this gene in nitrogen fixation. The abundance of phosphate transporter 1 (GLYMA10G00720) mRNA was significantly higher in nodule parenchyma and might be involved in transporting Pi from roots to nitrogen fixing bacteroids. These results helped identify potential roles of specific genes in processes associated with distinct nodule zones. Global transcriptomic analysis yield broader insight on other key determinants and/or signaling components involved in nodule zone differentiation. Ultimately, this knowledge can be used to devise biotechnological strategies to enhance nitrogen fixation or even potentially transfer N-fixation trait to non-leguminous plants, and reduce environmental pollution caused by excessive use of chemical nitrogenous fertilizer.

Library of Congress Subject Headings

Soybean -- Genetics.
Gene expression.
Genetic transcription.
Nitrogen -- Fixation.



Number of Pages



South Dakota State University



Rights Statement

In Copyright