Madison Dye

Date of Award

2024

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Clore, Amy

Area of Concentration

Biology

Abstract

A fusion event occurs between the carpels of Catharanthus roseus L. involving the dedifferentiation and redifferentiation of epidermal cells into less specialized cells known as parenchyma. This results in a solidly-fused gynoecium. An unknown “factor” exchanged between the contacting carpels mediates this process. Little is known about the identity of the factor and the signaling process that ensues, but there are data in support of a role for brassinosteroids, and a bit of evidence for the involvement of reactive oxygen species (ROS), particularly hydrogen peroxide. The present study was conducted to further investigate the role of additional ROS, namely singlet oxygen and superoxide, using various dyes, an inhibitor, a scavenger, and microscopy techniques, all as outlined in this thesis. In untreated/control sets of carpels, patterns of staining were seen for superoxide and singlet oxygen that were somewhat distinct early in development but were more consistent in later stages. A commonality that did occur relatively early on, however, was that staining for both ROS (using nitroblue tetrazolium, or NBT, for superoxide, and Singlet Oxygen Sensor GreenTM, or SOSG, for singlet oxygen) occurred at and near the fusion plane. Gynoecia that were stained with either dye in the very late post-fusion stage also consistently displayed a “necklace” of circumferential staining at the base of the stigma just above the skirt. After treatment with the inhibitor for ROS generation, salicylhydroxamic acid (SHAM), carpels displayed a noticeable difference in that there was less NBT staining visible on the carpel structures when compared to their controls. Carpels treated with SHAM or the singlet oxygen scavenger, L-histidine, were superficially adhered but separated with ease. However, carpels treated with SHAM remained consistently fused at a single point, allowing them to be opened like a book. This focal site of fusion also displayed NBT staining. In L-histidine-treated carpels that were stained with SOSG, overall fluorescence was decreased relative to controls and not present in some areas. Collectively, these results suggest a role for both superoxide and singlet oxygen in carpel fusion and potentially in tip growth that occurs in elongating papillary cells in the “necklace” region. Future studies should focus on investigating singlet oxygen staining patterns in SHAM-treated carpels, as well as the relationship between ROS and brassinosteroids as discussed herein.

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