University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2016 Genetic Basis of Biosynthesis and Cytotoxic Activity of Medicago truncatula Triterpene Saponins Brynn Kathleen Lawrence University of Arkansas, Fayetteville Follow this and additional works at: http://scholarworks.uark.edu/etd Part of the Plant Biology Commons, Plant Breeding and Genetics Commons, and the Plant Pathology Commons Recommended Citation Lawrence, Brynn Kathleen, "Genetic Basis of Biosynthesis and Cytotoxic Activity of Medicago truncatula Triterpene Saponins" (2016). Theses and Dissertations. 1687. http://scholarworks.uark.edu/etd/1687 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected], [email protected]. Genetic Basis of Biosynthesis and Cytotoxic Activity of Medicago truncatula Triterpene Saponins A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Cell and Molecular Biology By Brynn Kathleen Lawrence Ouachita Baptist University Bachelor of Science in Biology, 2014 August 2016 University of Arkansas This thesis is approved for recommendation to the Graduate Council. ______________________________________ Dr. Kenneth L. Korth Thesis Director ______________________________________ ______________________________________ Dr. Sun-Ok Lee Dr. David S. McNabb Committee Member Committee Member Abstract Saponins are a large family of specialized metabolites produced in many plants. They can have negative effects on a number of plant pests and are thought to play a role in plant defense. With current and possible future uses in industry and agriculture, saponins have also been shown to be hypocholesterolemic, hypoglycemic, immunostimulatory, antioxidative, anti-inflammatory, and cytotoxic. In spite of their usefulness, our understanding of the genetic basis for saponin biosynthesis is still incomplete. We generated recombinant populations with parents from genetically distinct accessions of Medicago truncatula, with either high or low accumulation and varying profiles of saponins. Primers for a PCR-based parental test were developed from single-nucleotide polymorphisms in the sequence encoding CYP72A68, a cytochrome P450 enzyme involved in the biosynthesis of M.truncatula sapogenins. Comparison of translated CYP72A68 amino acid sequences across accessions revealed high similarity, and comparison with CYP72A proteins from other plant species suggests similar protein functioning across the accessions. The F2 generation plants from one recombinant population were screened for hemolytic saponin accumulation levels in leaf extracts by measuring cleared zones in blood agar plates. Three distinct phenotypes were observed. Plants in the F2 generation showed either a high or low hemolysis phenotype matching those of the parent plants, or an intermediate level of hemolysis. The high:medium:low phenotypic ratio for 141 plants was 1:3:1. Quantitative RT-PCR showed a correlation between expression of CYP716A12, CYP72A67, and CYP72A68 genes, all encoding cytochrome P450 enzymes involved in synthesis of hemolytic sapogenins, and the three hemolysis phenotypes. Concurrently, we found that treatment of Caco-2 human colon cancer cells with saponin extracts from four M. truncatula accessions resulted in decreased cell proliferation over time, and that this effect did not appear to be mediated through apoptosis induction. The saponin extracts were analyzed by HPLC-MS to identify individual saponins that could contribute to cytotoxic activity. Therefore, accession differences in M. truncatula saponin accumulation result from differential regulation of saponin synthesis gene expression and inheritance of these differences depends on more than a single gene with dominant and recessive alleles. Saponin extracts are shown to have a negative impact on cancer cell lines. Acknowledgements I am extremely grateful to my advisor Dr. Ken Korth for his guidance and mentorship that have pushed me to grow as a scientist and made completion of this thesis possible. I would also like to thank my committee members Dr. Sun-Ok Lee and Dr. David McNabb for always making themselves available to provide additional direction and support. I thank my current and past lab members for their friendship and for being excellent people who were a joy to work with. Special thanks go to Xuan Gu, Cindi Brownmiller, and Lacy Nelson for patiently training me, contributing to my research, and working so hard to make sure I always had the supplies and support I needed to do my best work. Thank you to my former professors at Ouachita Baptist University, especially Dr. Ruth Plymale and Dr. Nathan Reyna, for so excellently preparing me for graduate level work and continuing to be such helpful mentors. Finally, I am extremely grateful to my family and friends, and especially my incredible husband, for their constant encouragement and for giving me the confidence and motivation to undertake such a challenging task. Table of Contents Chapter 1 Medicago truncatula triterpene saponins……………………………………………………... 1 I. Introduction……………………………………………………………………………………………. 1 i. Specialized metabolism in plants ii. Saponins: basic structures and properties iii. Applications of saponins: industry iv. Applications of saponins: agriculture v. Applications of saponins: human health vi. Cytotoxicity of saponins vii. Medicago truncatula viii. Triterpene saponin biosynthesis in M. truncatula ix. Caco-2 cell culture x. Concluding statements II. Objectives……………………………………………………………………………………………. 23 Chapter 2 Genetic basis for triterpene saponin biosynthesis in Medicago truncatula………………. 24 I. Introduction………………………………………………………………………………………….. 24 II. Materials and Methods……………………………………………………………………………... 27 a. Plant maintenance b. Crosses c. RNA extraction d. Sequencing e. DNA extraction f. Confirmation of crosses g. Saponin hemolysis assay h. Gene expression studies using qPCR i. Statistical analysis III. Results……………………………………………………………………………………………….. 34 a. Sequence of CYP72A68 cDNA is highly similar across M. truncatula accessions b. Confirmation of the presence of male parent DNA in putative cross progeny c. Cross progeny and parent accession leaf extracts display varying levels of hemolytic activity d. Expression of saponin synthesis genes correlates with A17 x ESP105 F2 hemolysis phenotypes IV. Discussion…………………………………………………………………………………………… 48 Chapter 3 Cytotoxic activity of Medicago truncatula triterpene saponins…………………………….. 58 I. Introduction………………………………………………………………………………………….. 58 II. Materials and Methods……………………………………………………………………………... 62 a. Plant maintanence b. High performance liquid chromatography/mass spectrometry c. Preparation saponin extracts for cell treatment d. Cell maintenance e. Cell proliferation assay f. Caspase-3 assay g. Statistical analysis III. Results……………………………………………………………………………………………….. 68 a. M. truncatula accessions accumulate varying levels and types of saponins in leaf tissue b. Treatment with M. truncatula saponin extracts results in decreased Caco-2 cell proliferation c. Treatment with M. truncatula saponin extracts does not result in increased activation of caspase-3 IV. Discussion…………….……………………………………………………………………………... 77 Literature Cited…………………………………………………………………………………………………… 86 List of Figures Figure 1: Triterpene and steroidal saponin aglycone chemical structures…………………………………… 3 Figure 2: Saponin aglycone biosynthesis in M. truncatula……………………………………………………. 19 Figure 3: Alignment of CYP72A68 cDNA sequences in A17, ESP105, and PRT178 M. truncatula plants……………………………………………………………………………………………………………. 34-36 Figure 4: Phylogenetic relationships among CYP72A proteins in various plant species………………….. 37 Figure 5: Alignment of amino acid sequences for CYP72A proteins in various legumes……………... 38-41 Figure 6: Confirmation of presence of male parent DNA in putative cross progeny……………………….. 42 Figure 7: Segregation of hemolytic saponin levels in A17 x ESP105 F2 plants as demonstrated on blood agar Petri plates…………………………………………………………………………………………………… 43 Figure 8: Relative gene expression of CYP716A12 in M. truncatula leaves subjected to mechanical damage, taken from A17, ESP105, and from A17xESP105 F2 plants with high, medium, and low hemolysis phenotypes……………………………………………………………………………………………. 44 Figure 9: Relative gene expression of CYP72A67 in M. truncatula leaves subjected to mechanical damage, taken from A17, ESP105, and from A17xESP105 F2 plants with high, medium, and low hemolysis phenotypes……………………………………………………………………………………………. 45 Figure 10: Relative gene expression of CYP72A68 in M. truncatula leaves subjected to mechanical damage, taken from A17, ESP105, and from A17 x ESP105 F2 plants with high, medium, and low hemolysis phenotypes……………………………………………………………………………………………. 46 Figure 11: Standard curve for calculation of cell number……………………………………………………... 65 Figure 12: Standard curve for calculation of caspase-3 U/well………………………………………………. 66 Figure 13: Saponin profiles in M. truncatula accessions……………………………………………………… 69 Figure 14: Percent cell viability for Caco-2 cells treated with A17, ESP105, PRT178, and GRC43 saponin extracts at 100 µg/ml 10% FBS media………………………………………………………………………….. 73 Figure 15: Percent cell viability for Caco-2 cells treated with A17, ESP105, PRT178, and GRC43 saponin extracts at 250 µg/ml 10% FBS media………………………………………………………………………….. 74 Figure 16: