Access Type

Open Access Dissertation

Date of Award

January 2020

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Edward M. Golenberg

Abstract

While unisexual flowers have evolved repeatedly throughout angiosperm families, the actual identity of sex determining genes has been elusive, and their regulation within populations remains largely undefined. Additionally, sex liability is often observed in unisexual plants and has been correlated to external and internal cues, suggesting that the genes responsible for unisexual morphology are not necessarily segregating but rather differentially regulated. Understanding these processes will be of significant theoretical and agronomical importance.

Cultivated spinach is a dioecious species in which an individual will bear alternative sexual organs. Previous work has identified spinach B class floral organ identity genes, SpAP3 and SpPI, to have a novel function resulting in the suppression of gynoecium development. We begin by testing the mechanism of the feminization pathway and its relationship to masculinization. Our results confirm earlier observations that exogenous applications of the plant hormone GA masculinize female flowers. Furthermore, inhibition of GA production and of proteasome activity feminizes male flowers. These observations are consistent with the role of the GA in spinach sexual development however, when assessing the GA content of male and female inflorescences and flowers we observe no significant difference between the sexes. We isolate and describe a single DELLA gene (SpGAI) in spinach. DELLA proteins are repressive transcription factors responsive to GA. Gene silencing of SpGAI in females allows activation of B class floral identity genes, and hence masculinization of female flowers. Additionally, SpGAI is differentially expressed in female versus male flowers. These results strongly implicate the role of SpGAI as a feminizing factor in spinach and suggest that the feminizing pathway is epistatic to the masculinizing pathway. We present a unified model for alternative sexual development in spinach and discuss the implications of such a model to established theory.

Our model predicts an interaction between SpGAI and SpLFY, a key transcription regulator involved in the transition from vegetative to floral growth. To explore this potential interaction, we used Yeast 2 Hybrid in vivo and Bimolecular Complementation in planta to screen for physical interaction. Preliminary results indicate a physical interaction occurs between SpGAI and SpLFY. The aforementioned genes and processes address the initial steps of sex determination in spinach. To begin characterization of genes that are important for morphogenesis of unisexual flowers, we generate a transcriptome from male and female inflorescences. Analysis revealed 165 differentially expressed transcripts, of which 88 could be identified by BLAST. Candidate genes were chosen, and differential expression was confirmed with qRT-PCR analysis. Loop-mediated isothermal amplification (LAMP) based in situ observation of genes identified as male specific as well as female specific were performed on spinach inflorescence sections. The sex specific expression detected with in silico analysis was confirmed in vitro with qRT-PCR and in planta with LAMP mediated gene expression observation.

These studies represent important contributions to our understanding of sexual development in unisexual angiosperms. There has been much difficulty identifying sex determining genes and despite great effort no such genes have been described in spinach. Our work identified differential SpGAI expression as critical for unisexual development and altering that expression through various methods has predictable results. Combined with data previously generated in our lab we present a mechanism linking SpGAI expression and flower organ identity gene expression resulting in unisexual flowers. The transcriptome and list of differentially expressed genes will be a useful resource to identify the genes responsible for the morphological differentiation between the sexes. In the future candidate genes will be selected for functional testing using the VIGS based approach developed previously in our lab.

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