i Gene Expression and Genetic Analysis of Carotenoid Pigment Accumulation in Carrot (Daucus carota L.) By Megan Joy Bowman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Plant Breeding and Plant Genetics) at the UNIVERSITY OF WISCONSIN-MADISON 2012 Date of final oral examination: 6/7/2012 The dissertation is approved by the following members of the Final Oral Committee: Philipp Simon, Professor, Horticulture Irwin Goldman, Professor, Horticulture Rebecca Harbut, Assistant Professor, Horticulture Patrick Krysan, Associate Professor, Horticulture Patrick Masson, Professor, Genetics i GENE EXPRESSION AND GENETIC ANALYSIS OF CAROTENOID PIGMENT ACCUMULATION IN CARROT (DAUCUS CAROTA L.) Megan Joy Bowman Under the supervision of Professor Philipp W. Simon At the University of Wisconsin-Madison ABSTRACT Carrot (Daucus carota) is an important vegetable crop, providing over 30% of the pro- vitamin A carotenoids in the human diet in the United States. As a major source of these nutriceutical compounds, research efforts have been directed to the regulation of this complex metabolic pathway. This project focused on evaluating gene expression, sequence changes and genetic mapping as it relates to the accumulation of the carotenoid pigments α-carotene, β- carotene, xanthophylls, and lutein. Real-time quantitative PCR (RT-qPCR) was used to determine the level of expression of genes in the carotenoid biosynthesis pathway in different carrot culitvars. Two genes, phytoene synthase 1 (PSY1) and phytoene synthase 2 (PSY2) were found to be expressed at significantly higher levels in orange and dark orange carrot cultivars, whereas the pale orange rp “reduced pigment” mutant exhibited reduced phytoene desaturase (PDS) expression. DNA sequence analysis of putative promoter and coding regions of both PSY1 and PSY2 has identified sequence polymorphisms and large deletions were identified in the putative promoter of PSY2 in the dark orange cultivar B2327. Simple sequence repeat markers (SSRs) were used to create a genetic map from an F2 population segregating for the rp mutation. HPLC (high performance liquid chromatography) analyses of the carotenoids of carrot leaf tissue demonstrated xanthophylls and β-carotene as the carotenoids present in all carrots, but ii significantly higher levels of α-carotene was identified in carrot germplasm with orange and dark orange storage roots. Chlorophyll fluorescence analysis identified genotype specific variation in photosystem II efficiency, significantly impacted by the time of harvest. To identify new candidate genes involved in carotenoid accumulation in carrot storage root, microarray analysis of sibling white and orange inbred lines from a cross of orange X white carrots was completed. Expression of three genes involved in photosynthesis was significantly higher in the orange rooted carrot germplasm. To examine the role that chromoplast biosynthesis may have in the accumulation of carotenoid pigments, the ratio of nuclear to plastid genome was analyzed in carrot germplasm with differing levels of pigmentation. iii ACKNOWLEDGEMENTS There are many people I would like to thank for their support during my time as a graduate student at UW-Madison. I would like to thank my advisor Dr. Philipp Simon for his time and devotion to mentoring me during my graduate studies. When I first arrived at UW, finding my place as a plant breeding and plant genetics student proved to be difficult. Coming from an undergraduate research experience in plant molecular biology I was uncertain that I could be happy pursuing research that involved heading out to the field and getting dirty, very far from the comforts of the lab bench! Dr. Simon instructed me on the importance of both aspects of plant research and gave me the field experience I needed to fully understand the role of molecular biology research in agriculture. Dr. Simon always welcomed creativity in approaching my thesis work and allowed me to pursue opportunities outside of the laboratory to further my research interests. With his help, I have been able to combine my passion for plant molecular biology and plant breeding towards the development of better crops for human health. For that, I am forever grateful. I would like to thank Dr. Warren Gabelman and the Seminis Seed Company for providing me with the University of Wisconsin Gabelman-Seminis Distinguished Graduate Fellowship, which funded the last two years of my graduate work. I would like to thank the International Carotenoid Society for providing a travel scholarship in 2011 and the UW Graduate School for providing a Vilas Research Travel Award so I could pursue research in Krakow, Poland in January 2012. I would like to thank Dr. Irwin Goldman, Dr. Patrick Krysan, Dr. Patrick Masson, Dr. Jean- Michel Ané and Dr. Rebecca Harbut for serving on my thesis committee and administering my preliminary exam. A special thanks to Dr. D. Kyle Willis for all of this help with the RT-qPCR experiments. Thank you in particular to Dr. Doug Senalik for his help with HPLC and most everything that goes on in the Simon Lab. Thank you for not running away every time I came into your office and said “Doug, I have a question.” Also a big thank you to Mr. Rob Kane, who showed me that field work can be fun and the proper growing techniques for carrot. Thank you to the graduate students in the Simon Lab and PBPG program for being a great support during these years at UW. A special thanks to graduate students Josh Parsons and Pim Satapoomin, for their help, friendship and support as I have been finishing school. Thank you to all the undergraduate students that have assisted in this thesis work, including the HPLC and genetic mapping projects. I couldn’t have finished this project without you. To all the past and present members of the Simon Lab, including Dr. Massimo Iorizzo, Dr. Pablo Cavagnaro, Dr. Marina Iovene, Dr. Leah McCann, Dr. Alicja Macko-Podgorni and Dr. Laxmikanta Acharya, thank you for your support and advice. Thank you to Dr. Dariusz Grzebelus at the University of Agriculture in Krakow for his invitation and support to pursue research in his laboratory in Krakow, Poland. iv An especially big thank you goes to my parents, Bud and Ellen Sargent, who have always supported my love of science. Thank you to Bob and Debbie Bowman, who have been an immense help to me during the past five years. Thank you to my wonderful husband Stephen Bowman. To say that I couldn’t have finished graduate school without you is of course an understatement. You have always been my number one cheerleader when I was down and the first person I told when I was excited about my work. Thank you for being my friend, counselor, editor, and my support for the past seven years, and surely more. To my beautiful little girl Madeleine, whom we welcomed during my time at UW-Madison: Becoming your mother has been the most exciting, thrilling, challenging and amazing project I have ever undertaken. Thank you for your smiles, your ‘ugs and ‘mooches when I surely needed them! Thank you for giving me much needed perspective during the hardest times of this graduate school journey. I cannot wait to see what the future holds for our family. v TABLE OF CONTENTS Abstract………………………………………………………………………………………....….i Acknowledgements……….………………………………………………………………...........iii Table of Contents……….…………………………………………………………………...….…v List of Figures……..…..………………………………………………………………..……......vi List of Tables..………………………………………..…………………………………….….….x List of Appendices……..………………………………………………………………….….…xiii Chapter 1: Review of carotenoid biosynthesis, accumulation, and research goals………………1 Chapter 2: Gene expression in the carotenoid biosynthesis pathway of carrots with diverse storage root colors (Daucus carota L.)………………………………………………………..…38 Introduction………………………………………………………………………………………39 Materials and Methods…………………………………………………...………………………41 Results…………………………………………………………………………………………....45 Discussion……………………………………………………………………………..…………51 References………….………………...…….…………………………………………...……......57 Chapter 3: Carotenoid pigment variation and photosystem II efficiency in the leaf tissue of carrot (Daucus carota L.)………………………………………………………………….....….60 Introduction………………………………………………………………………………………61 Materials and Methods…………………………………………………...………………………63 Results…………………………………………………………………………………………....68 Discussion……………………………………………………………………………..…………78 References………….………………...…….…………………………………………...…...…...83 vi Chapter 4: Microarray transcriptome analysis to evaluate whole genome gene expression changes during the accumulation of α and β-carotene in the storage root of carrot (Daucus carota L.)…………………………………………...……………………………………………………86 Introduction………………………………………………………………………………………87 Materials and Methods…………………………………………………...………………………90 Results…………………………………………………………………………………….……...93 Discussion………………………………………………………………………………………104 References………….………………...…….…………………………………………........…...110 Chapter 5: Gene expression analysis of the Rp (reduced pigment) mutant in carrot (Daucus carota L.)…………………………………………………………………………………….…114 Introduction……………………………………………………………………………………..115 Materials and Methods…………………………………………………...………………..……116 Results……………………………………………………………………………………..…....121 Discussion………………………………………………………………………………………132 References………….………………...…….…………………………………………........…...137 Chapter 6: Plastome quantification and plastid gene expression during carotenoid accumulation in carrot (Daucus carota L.)……………………………………………………………...….….141 Introduction……………………………………………………………………………………..142 Materials and Methods…………………………………………………...………………..……145 Results……………………………………………………………………………………..…....147 Discussion………………………………………………………………………………………152 References………….………………...…….…………………………………………........…...157 Chapter 7: Conclusions and future research directions………………………………..………159 Appendices…………………………………………………………………………………......172 vii LIST OF FIGURES Figure 1.1: The carotenoid biosynthesis pathway, adapted from Just et al., 2007. ………...…..26 Figure 1.2: Examples of chemical structures of metabolites in the carotenoid biosynthesis pathway…………………………………………………………………………………………..27 Figure 2.1: RT-qPCR evaluation of gene expression in the carotenoid biosynthetic pathway incarrot storage roots at 11 weeks post planting. The genes phytoene synthase 1 (PSY1) and phytoene synthase 2 (PSY2) are differentially expressed between white and orange pigmented carrot storage root.. ………………………………………………………………………......….47 Figure 2.2: RT-qPCR results of PSY1 and PSY2 at 14 weeks post planting (H4). The genes phytoene synthase 1 (PSY1) and phytoene synthase 2 (PSY2) are differentially expressed between white and orange pigmented carrot storage root……………………………………….48 Figure 2.3: RT-qPCR analysis of PSY1 and PSY2 in carrot leaf tissue, 11 weeks post planting ……………………………………………………………………………………………………49 Figure 2.4: High performance liquid chromatography (HPLC) of storage root tissue in carrot inbred lines with a variety of pigmented storage root. Dark orange inbred line B2327 contains a higher amount of α-β-carotene as compared to the other genotypes evaluated…………………50 Figure 3.1: Chlorophyll fluorescence of leaf tissue in carrot inbred lines with a variety of pigmented storage root…………………………………………………………..…..……….75-76 Figure 3.2: Real-time quantitative PCR (RT-qPCR) analysis of carotenoid biosynthesis geneslycopene ɛ cyclase (LCYE) and lycopene β-cyclase (LCYB) in the leaf tissue of carrot germplasm with a variety of carrot storage root colors………………………………………….77 Figure 4.1: Hierarchical clustering of 59 genes with at least 2-fold difference or greater expression by RMA (Robust Multichip Average) with moderated t-test at p= 0.05 and Benjamini Hochberg False Discovery Rate (FDR) (Benjamini and Hochberg, 1995)…………………...…95 Figure 4.2: Gene ontology description of cellular component of genes identified as differentially expressed in the B493 X QAL white and orange sibling inbred lines…………………………...96 Figure 4.3: RT-qPCR expression of selected genes determined to be differentially expressed by the Medicago Affymetrix GeneChip microarray. RT-qPCR analysis of the B493 X QAL sibling inbred lines B8788 and B8750 and genetically unrelated orange (B2566) and white (B6644) determined that genes were not differentially expressed…………………………………..…….98 Figure 4.4: RT-qPCR expression of selected genes determined to be differentially expressed by the Medicago Affymetrix GeneChip microarray. RT-qPCR analysis of the B493 X QAL sibling inbred lines B8788 and B8750 and genetically unrelated orange (B2566) and white (B6644) determined that these genes were differentially expressed in a color independent manner. …..100 viii Figure 4.5: RT-qPCR expression of selected genes determined to be differentially expressed by the Medicago Affymetrix GeneChip microarray of RILs B8750 and B8788, and confirmed by RT-qPCR analysis of these two RILs and genetically unrelated orange (B2566) and white (B6644) inbreds. The genes dcPSI-N, dcPOR1 and dcLCHA3are significantly differentially expressed higher in orange storage rooted carrot germplasm, while dcExt1 was expressed higher in the white cultivated inbred line and RIL…………………………………………….………102 Figure 5.1: W266rprp X W269 mapping population harvested in Markesan, WI, September 2011……………………………………………………………………………..………………121 Figure 5.2: HPLC analysis of various carotenoid pigments in the root and leaf tissue of the W266rprp mutant, orange inbred B2566 and dark orange inbred B2327 over four harvest periods. H1= 5 weeks H2=8 weeks H3=11 weeks H4=14 weeks post planting………..…122-123 Figure 5.3: HPLC analysis of total carotenoid content in the root and leaf tissue of the W266rprp mutant, orange inbred B2566 and dark orange inbred B2327 over four harvest periods. H1= 5 weeks H2=8 weeks H3=11 weeks H4=14 weeks post planting………….……124 Figure 5.4: RT-qPCR analysis of the relative expression of phytoene desaturase (PDS) in the leaf and root tissue of the W266rprp mutant and W266RpRp wildtype carrot…………...……125 Figure 5.5: Diagram of phytoene desaturase (PDS) NCBI-Spidey mRNA to genomic alignment of whole genome sequence of carrot inbred lines 493A and 493B to the original PDS GenBank reference sequence DQ222429.1 (Just et al., 2007)……………………………………………126 Figure 5.6: Genetic map of W266rprp X W269 population in carrot………………………….131 Figure 6.1: The ratio of plastid to chromosomal genome in various colored carrot storage root tissue, determined by quantitiative real time PCR…………………………………………..….148 Figure 6.2: The ratio of plastid to chromosomal genome in leaf tissue, determined by quantitiative real time PCR…………………………………………………….……………….149 Figure 6.3: RT-qPCR expression analysis of plastid genes AccD, PsaJ and PsbC……………151 Figure 6.4: RT-qPCR expression analysis of the genes Or and Pftf in carrot storage root……152 Figure A 3.1: Diagram of primers used in sequencing the coding region of PSY1 in pigmented carrot germplasm…………………………………………………………………………….…183 Figure A 3.2: Diagram of primers used in sequencing the coding region of PSY2 in pigmented carrot germplasm. ……………………………………………………………………...………185 Figure A 3.3: Diagram of primers used in sequencing the putative promoter of PSY1 in pigmented carrot germplasm………………………………………………………………...….186 Figure A 3.4: Diagram of primers used in sequencing the putative promoter of PSY2 in pigmented carrot germplasm…………………………………………………………...……….187 ix Figure A 13.1: Examples of the original sectored carrot material used in the development of orange and yellow carrot lines………………………………………………………………….240 Figure A 14.1: High-resolution DNA electrophoresis of extracted total RNA from leaf and root tissue from the B493XQAL population, and lines B7262 and B6274…………………...…….243 Figure A 15.1: Gel electrophoresis of PCR to detect hAT transposable element insertion in carotenoid isomerase (CRTISO) in 8 individuals of the carrot cultivar AS07……………...….246 Figure A 15.2: Gel electrophoresis of PCR to detect Tourist transposable element insertion in isopentenyl pyrophosphate isomerase (IPI) in 17 individuals of the carrot cultivar AS22…….246 Figure A 15.3: RT-qPCR results of carotenoid biosynthesis gene carotenoid isomerase (CRTISO) in the leaf tissue of carrot cultivar AS07 (Deep Purple)………………………..…..247 Figure A 15.4: RT-qPCR results of carotenoid biosynthesis gene isopentenyl pyrophosphate isomerase (IPI) in the leaf tissue of carrot cultivar AS22 (Pusa Kesar)……………………..…247 Figure A 17.1: Chlorophyll fluorescence analysis of 19 genotypes of cultivated carrot germplasm and the wild carrot relative QAL (Queen Anne’s Lace) during UV exposure…….252