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Broderick PhD Dissertation.pdf (8.47 MB)

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Pollination-Induced Gene Changes That Lead to Senescence in Petunia × hybrida

Broderick, Shaun Robert
http://rave.ohiolink.edu/etdc/view?acc_num=osu1408958432

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Horticulture and Crop Science.
Flower longevity is a genetically programmed event that ends in flower senescence. Flowers can last from several hours to several months, based on flower type and environmental factors. For many flowers, particularly those that are ethylene-sensitive, longevity is greatly reduced after pollination. Cellular components are disassembled and nutrients are remobilized during senescence, which reduces the net energy expenditures of floral structures. The goal of this research is to identify the genes that can be targeted to extent shelf life by inhibiting pollination-induced senescence. Identifying and characterizing regulatory shelf-life genes will enable breeders to incorporate specific alleles that improve post production quality into ethylene-sensitive crops. Petunia × hybrida is particularly amenable to flower longevity studies because of its large floral organs, predictable flower senescence timing, and importance in the greenhouse industry. A general approach to gene functional analysis involves reducing gene expression and observing the resulting phenotype. Viruses, such as tobacco rattle virus (TRV), can be used to induce gene silencing in plants like petunia. We optimized several parameters that improved virus-induced gene silencing (VIGS) in petunia by increasing the consistency and efficiency of silencing. They included applying inocula to wounded apical meristems, growing petunias at temperatures of 20 °C day/18 °C night, utilizing the cultivar `Picobella Blue’, and inoculating plants at three or four weeks after sowing. As a control for VIGS experiments, an empty vector is frequently used, but severe TRV symptoms often lead to death in petunia. We developed a control construct, which contained a fragment of the green florescent protein. This construct eliminated all severe viral symptoms and served as a better control. This optimized protocol and control construct enabled us to silence many genes and screen for phenotypic results within a few months. To identify candidate, pollination-associated genes for VIGS analysis, RNA-sequencing (RNA-seq) libraries were developed from RNA extracted from pollinated and unpollinated petunia corollas at 12, 18, and 24 h after flower opening. The libraries were sequenced and an expressed sequence tag (EST) library of 33K contigs was generated, which represents the largest petunia corolla transcriptome to date. Differential gene expression analysis and a weighted gene co-expression network analysis (WGCNA) were performed to identify pollination-associated genes. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment provided a biological overview of the gene networks and molecular pathways associated with flower senescence, which included cellular catabolic processes and pathways such as the Regulation of autophagy, Plant hormone signal transduction, and Sucrose & starch metabolism. With this molecular information, better targets can be selected for further studies. Fifteen differentially expressed, pollination-associated genes were phenotypically characterized using VIGS for alterations in flower longevity. Petunias inoculated with a VIGS vector containing a putative EIN3-binding F-box protein (EBF) resulted in accelerated senescence. Two additional PhEBFs were identified from the assembled RNA-seq transcripts. PhEBF2b and PhEIL expression increased sooner in plants inoculated with the CHS-EBF2b construct than in the CHS control. We hypothesize that this work will lead to improved post production quality in floricultural crops.
Michelle Jones (Advisor)
Feng Qu (Committee Member)
Eric Stockinger (Committee Member)
Esther van der Knaap (Committee Member)
213 p.
Bioinformatics; Biology; Cellular Biology; Horticulture; Molecular Biology; Plant Biology; Plant Pathology; Plant Sciences; Pollen; Virology
VIGS; petunia; RNA-seq; transcriptome; WGCNA; KEGG; gene ontology; pollination; autophagy; calcium; tobacco rattle virus; viral symptoms; lesions; necrosis; ethylene; EIN3-binding F-box protein; EIL; EIN3; style; corolla; petal; pollen; empty vector

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Citations

  • Broderick, S. R. (2014). Pollination-Induced Gene Changes That Lead to Senescence in Petunia × hybrida [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1408958432

    APA Style (7th edition)

  • Broderick, Shaun. Pollination-Induced Gene Changes That Lead to Senescence in Petunia × hybrida. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1408958432.

    MLA Style (8th edition)

  • Broderick, Shaun. "Pollination-Induced Gene Changes That Lead to Senescence in Petunia × hybrida." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1408958432

    Chicago Manual of Style (17th edition)

osu1408958432
861
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This open access ETD is published by The Ohio State University and OhioLINK.