Date of Award
9-18-2013
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Fabricio Medina-Bolivar
Committee Members
Argelia Lorence; Carole Cramer; Jennifer Bouldin; Mariya Khodakovskaya
Abstract
Stilbenoids are polyphenolic compounds that are believed to protect plants against abiotic and biotic stresses. Importantly, an increasing number of these specialized plant metabolites have been associated with potential health benefits in humans. Therefore, understanding the molecular mechanisms involved in their biosynthesis is of importance to enhance the levels of these beneficial compounds in plants. In this research we studied the biosynthesis of stilbenoids in two economically important food crops: muscadine grape and peanut. Hairy root cultures were used as biological systems because their genetic and biochemical stability facilitates the study of specialized metabolism. Selected hairy root cultures from muscadine grape cvs. Fry and Noble and peanut cv. Hull were treated with the oxidative stressor hydrogen peroxide (H2O2) or the stress signaling molecule methyl jasmonate (MeJA) and the effects on stibenoid accumulation, expression of sitlbenoid-related genes, and antioxidant capacity were studied. First, the effects of growth regulators (3-indolebutyric acid and 6-benzylaminopurine) on muscadine hairy roots growth and stilbenoid production were assessed alone and in combination with MeJA. High-Performance Liquid Chromatography analysis showed that growth regulators had no effect on stilbenoid accumulation but MeJA treatment led to increased levels of these compounds. In muscadine grape most of the stilbenoids including resveratrol, piceatannol, piceid, and å-viniferin were retained in the tissue at 12 h post-treatment whereas in peanut, resveratrol and its analogues arachidin-1 and arachidin-3 were secreted into the culture medium between 12 and 24 h after treatment. Furthermore, quantitative analyses of transcript expression levels of stilbenoid-related genes were measured. To achieve this, reference genes for qPCR in muscadine grape (VATP16, ACT7, GAPDH, SAND, and 60SRP) were selected for the first time. Genes involved in the regulation of phenolic biosynthesis were targeted in muscadine grape (PAL, STS family, and CHS). In peanut, expression analyses were performed on six members of the stilbene synthase family using previously described reference genes (ACT7, TBP2, and EF1-á). Our observations indicated an induction between 3 and 6 h post treatment for all the genes under MeJA and H2O2, except for CHS in muscadine grape when H2O2 acted as stressor. Transcript accumulation of STS in peanut peaks within 6 h after MeJA treatment whereas H2O2 induced an earlier accumulation peak at 3 h. Secondary peaks were observed at 48 h with MeJA and at 72 h with H2O2. On the other hand, the antioxidant capacities of stilbenoid standards and hairy root extracts were determined. Analysis performed with purified standards showed that piceatannol had the highest antioxidant activity among the studied stilbenoids with an EC50 of 69 ìM followed by arachidin-1 with 105 ìM. In hairy roots, a correlation between metabolite accumulation and antioxidant capacity was observed on muscadine grape medium and tissue extracts. On the other hand, the antioxidant capacities obtained from peanut culture extracts showed increasing trends in medium extracts but did not show a direct correlation with the quantified levels of stilbenoids which decreased during the time course. This suggests that other compounds with antioxidant activity could be contributing to the total antioxidant capacity of the peanut extracts. This research provides insights on how stilbenoid-producing species respond to compounds that are known to be involved in signaling processes in the cell and at the same time provides new tools for research on gene expression of the STS family of genes and opens a new door for further understanding on how the stilbenoid biosynthetic pathway is regulated. Data obtained from this research could be applied to develop new means to manipulate the levels of stilbenoids in plants in efforts to increase disease resistance in peanut and muscadine grape and to develop functional foods or research ingredients for the pharmaceutical and nutraceutical industries.
Rights Management
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Nopo-Olazabal, Cesar Augusto, "Differential biosynthesis of antioxidant stilbenoids in hairy root cultures of muscadine grape and peanut treated with methyl jasmonate and hydrogen peroxide" (2013). Student Theses and Dissertations. 822.
https://arch.astate.edu/all-etd/822
