i Gene Expression and Genetic Analysis of Carotenoid Pigment Accumulation in Carrot

269 Pages·2012·5.03 MB·English

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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
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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
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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.
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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.
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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.
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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
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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
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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
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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
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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