Document Type

Dissertation - Open Access

Award Date

2018

Degree Name

Doctor of Philosophy (PhD)

Department / School

Agronomy, Horticulture, and Plant Science

First Advisor

Senthil Subramanian

Keywords

auxin, biosynthesis, CYP83B1, GH3, nodule, soybean

Abstract

Legume-rhizobia symbiosis leads to the development of secondary root organs called nodules. Rhizobia bacteria housed inside nodules assimilate atmospheric nitrogen and convert them into plant usable forms thereby reducing the need for fertilizer application in crop legumes like soybean. Nodule development is a coordinated process orchestrated by multiple plant hormones. In soybean, the auxin responsive gene expression was detected in nodule primordia and in the periphery of mature nodules, primarily in nodule vasculature. Auxin hypersensitivity reduces nodule formation in soybean and also polar auxin transport inhibition at the site of nodule development is not crucial for determinate nodule formation. Therefore, auxin distribution and sensitivity appear to be crucial for proper nodule development. However, the role of auxin metabolism in nodule development is unclear. Using global gene expression analysis, we have identified genes involved in the auxin metabolism that are specifically expressed in nodule tissues at early and mature stages. A cytochrome P450 oxidase gene, GmCYP83B1 was preferentially enriched in mature nodules and it was also identified to be a close ortholog of AtCYP83B1. Suppression of GmCYP83B1 expression through RNA interference (GmCYP83B1-RNAi) in soybean roots led to a significant reduction in nodule number and altered mature nodule morphology. Auxin accumulation was significantly higher in GmCYP83B1-RNAi nodules compared to vector control which suggested that suppression of GmCYP83B1 led to auxin accumulation which might have led to reduced nodule organogenesis and altered nodule development. Using the global gene expression data, we also identified three nodule-enriched genes encoding GRETCHEN HAGEN 3 (GH3) enzymes. Biochemical assays showed that the three GmGH3 enzymes can conjugate IAA with Asp for inactivation of free auxin levels. GmGH3-15 showed a broad substrate preference, especially with different forms of auxin. We hypothesized that these GH3s might maintain auxin homeostasis in soybean nodules. Promoter:GUS expression analysis indicated that GmGH3-14 acts primarily in the root epidermis and the nodule primordium where as GmGH3-15 might act in the vasculature. Silencing the expression of these GH3 genes in soybean composite plants led to altered nodule numbers, maturity, and size. Our results indicate that these GH3s are needed for proper nodule maturation in soybean, but the precise mechanism by which they regulate nodule development remains to be explained. Overall the results suggest that GmCYP83B1 and GmGH3 might act to regulate local auxin levels to direct proper soybean nodule development.

Library of Congress Subject Headings

Soybean -- Roots.
Auxin -- Metabolism.

Description

Includes bibliographical references

Format

application/pdf

Number of Pages

169

Publisher

South Dakota State University

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Rights Statement

In Copyright