Date of Award

10-8-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Science

First Advisor

Mariya Khodakovskaya

Abstract

Inositol- (1,4,5) triphosphate (InsP3) is known as an important signaling factor and a regulator of plant responses to environmental and stress conditions. Overexpression of the InsP 5-ptase gene is found to enhance tolerance of transgenic tomato plants to abiotic stress and the induction of lycopene synthesis in InsP 5-ptase transgenic plants. Here, we demonstrated that the genetic reduction of InsP3 affected the overall phosphoinositol pathway, flavonoid metabolism, and light signaling in InsP 5-ptase transgenic plants. Transgenic plants that overexpress the InsP 5-ptase gene demonstrate an increase in the expression of major light signaling transcription factors (LeHY5, SIMYB12, LeELIP). We found that production of metabolites/antioxidants (flavonoids, ascorbic acid) that can be regulated by light was increased in transgenic tomato fruits. The enhanced biosynthesis of such metabolites was in positive correlation with activation of key genes of phenylpropanoid metabolism (CHS1, HCT) and ascorbic acid metabolism (MIOX, GLDH) in transgenic tomato lines. Additionally, it was found that transgenic tomato lines are more tolerant to oxidative stress imposed by light. This fact can be explained by the experimentally documented upregulation of gene expression of antioxidant enzymes (LeAPX1, SICAT2, LeSOD) which serve as scavengers of reactive oxygen species (ROS) produced during stress conditions. Our study confirmed the correlation between modification of the phosphoinositol signaling pathway with increased tolerance to oxidative stress caused by light and activation of enzymatic and non-enzymatic antioxidants in transgenic tomato plants. We have revealed the pharmacological potential of InsP 5-ptase fruits. Since the InsP 5-ptase transgenic plants accumulated higher amounts of lycopene, vitamin C, and flavonoids with potential antioxidant and anticancer properties, an in vitro experiment with cancer cells was performed. Phenolic extracts from InsP 5-ptase expressing tomato fruits exhibited an anticancer effect on a pancreatic cancer cell line (Panc-1) and a breast cancer cell line (MCF-7) by reducing the proliferation of these cancer cell lines. Hypothesis: We hypothesized that genetic reduction of InsP3 affects overall phosphoinositol metabolism, response to light stress, and also secondary metabolite biosynthesis in InsP 5-ptase transgenic tomato plants. It was also hypothesized that an increase in biosynthesis of health-promoting, light-regulated secondary metabolites in transgenic plants would increase the antioxidant properties of transgenic fruits and possibly lead to enhancement of anticancer activity of the extracts derived from InsP 5-ptase fruits. To test our hypothesis we established the following aims: * Investigate the effect of genetic modification of InsP 5-ptase plants on metabolites of the phosphoinositol pathway, * Study how modification of the phosphoinositol pathway affected the production of secondary metabolites and key regulators of light signaling, * Investigate the response of InsP 5-ptase expressing tomato plants to oxidative stress caused by light, * Determine if InsP 5-ptase fruits can affect proliferation of cancer cells

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