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Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)


Plant Science

First Advisor

Senthil Subramanian


Biological nitrogen fixation in symbiotic nodules of leguminous crops viz. soybean reduces the use of chemical fertilizers providing economic and environmental benefits. Though not completely understood, the molecular signaling events during nodule development are being identified. Class III homeodomain leucine zipper (HDZIPIII) proteins are a group of plant specific transcriptional factors that are known to regulate several developmental process in Arabidopsis and other plants. Transcriptional, post-transcriptional and post-translational regulation of two GmHD-ZIPIII genes (GmHD-ZIPIII-1 & GmHD-ZIPIII-2) was examined in soybean roots inoculated with its symbiont Bradyrhizobium japonicum, to understand their role in root and nodule development. The expression of these genes was monitored using promoter:GUS and promoter:tdTomato constructs in hairy root composite soybean plants, at different time points post B. japonicum inoculation. These genes were expressed in the root tips, root vasculature, emerging lateral root, emerging nodule and vasculature of mature nodules suggesting a possible role of GmHD-ZIPIII in root lateral organ development. Examination of GmHD-ZIPIII gene expression along a time course of nodule development suggested that GmHD-ZIPIII-2 is up-regulated by B. japonicum inoculation. HD-ZIPIIIs are regulated in a post-transcriptional manner by miR166 in many plant species. Cleavage of GmHD-ZIPIII transcripts by miR166 was experimentally validated using 5'-RLM-RACE assays. The expression levels of miR166 and the target GmHD-ZIPIIIs were identified using qRT-PCR and RNA sequencing respectively in emerging lateral roots, young lateral roots, emerging nodules and mature nodules. The results suggested that GmHD-ZIPIIIs are expressed at higher levels in emerging lateral organs compared to the respective mature organs. Examination of miR166 activity using a miR166 sensor suggested that miR166 spatially restricts GmHDZIPIII expression to the metaxylem and epidermis in the roots as observed in Arabidopsis. In symbiotic nodules, miR166 appears to spatially restrict GmHD-ZIPIII expression to the basal part of the nodule vasculature. The activity of HD-ZIPIII proteins are regulated in a post translational manner by small leucine zipper proteins (ZPRs). The interaction of GmHD-ZIPIII-1 & 2 proteins with all eight predicted ZPR proteins in soybean was examined using yeast-two-hybrid assay. Both GmHD-ZIPIII genes showed positive interaction with ZPR3b, ZPR3c and ZPR3d. Quantitative assays of interaction based on β-GAL activity (a marker gene in the assay) indicated that ZPR3d and GmHDZIPIII- 2 pair had the strongest interaction among the tested GmHD-ZIPIII and ZPR pairs. These observations suggested that GmHD-ZIPIII genes are indeed regulated at the transcriptional, post-transcriptional and post-translational levels in soybean. Subsequent experiments to examine significance of GmHD-ZIPIII regulation during nodule development would provide crucial insights in this crucial process.

Library of Congress Subject Headings

Soybean -- Genetics
Plant proteins
Roots (botany) -- Development


Includes bibliographical references (pages 107-116)



Number of Pages



South Dakota State University


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