Relationship of carotenoids and tecopherols in a sample of carrot root-color accessions and carrot germplasm carrying R

1. Relationship of carotenoids and tecopherols in a sample of carrot root-color accessions and carrot germplasm carrying Rp and rp alleles Koch, T. C. and I. L. Goldman Journal of Agricultural and Food Chemistry 53: 325-331 (2005)

2. CAROTENOIDS & TOCOPHEROLS • Role in plants: • Carotenoids prevent formation of oxygen radicals • Tocopherols protect membranes from oxidative stress • Role in human diet: • Powerful antioxidants that prevent degenerative effects • Some convert to vitamin A • α- and β-carotene  orange-colored roots • Lycopenered • Anthocyaninspurple • Low total carotenoids  white • Tocopherols don’t contribute to color • Carotene: origin 1860-1865 “carrot” + “-ene” (dictionary.com) • Richest source of carotenoids in crude palm oil (wikipedia.org)

3. BIOSYNTHESIS PATHWAY GGPP = geranylgeranyl-pyrophosphate

4. FIELD EXPERIMENT • Assess levels of major carotenoids and tocopherols in carrot roots & leaves • Measure accumulation of compounds along carotenoid & tocopherol biosynthesis pathway • Explain color differences among 8 accessions • 4 replications/accession • 10 samples/replication • 2 locations; 2 growing seasons

5. EIGHT ACCESSIONS • W266Drprp (reduced pigment) • Recessive allele for reduced pigment • Shown to reduce carotenoid conc. by up to 92% • W266DRpRp (orange) • W276B (orange) • Danvers (orange) • HCM (orange) • Beta III (dark orange) • Okuzawa (red) • Yellow type (yellow)

6. RESULTS: FIXED EFFECTS • Accession, year, and location all interacted significantly • Year and location effects: • Mainly resulted in change of data magnitude • Rarely changed accession ranks • For analysis, years and locations were pooled, since ranks were rarely affected

7. RESULTS: α-carotene • W266Drprp (reduced pigment) • W266DRpRp (orange) • W276B (orange) • Danvers (orange) • HCM (orange) • Beta III (dark orange) • Okuzawa (red) • Yellow type (yellow) xylem and phloem: [orange] > [non-orange] [xylem] = 0.69*[phloem] [leaf] = 0.36*[phloem]

8. RESULTS: β-carotene • W266Drprp (reduced pigment) • W266DRpRp (orange) • W276B (orange) • Danvers (orange) • HCM (orange) • Beta III (dark orange) • Okuzawa (red) • Yellow type (yellow) xylem and phloem: [orange] > [non-orange] [xylem] = 0.67*[phloem] [leaf] = 0.33*[phloem]

9. α- and β-carotene • High range within roots • Artificially selected for human consumption • Lower range within leaves • Naturally selected for because prevent photo-oxidative damage in leaves • Lack of artificial selection

10. RESULTS: α-TOCOPHEROL • W266Drprp (reduced pigment) • W266DRpRp (orange) • W276B (orange) • Danvers (orange) • HCM (orange) • Beta III (dark orange) • Okuzawa (red) • Yellow type (yellow) HIGHEST AVERAGE FOR XYLEM AND PHLOEM

11. TOCOPHEROL • No patterns between orange and non-orange • Much higher levels in leaves than in roots • Perhaps it aids in photosynthesis • Surprising ratios of [root] : [leaves]

12. BIOSYNTHESIS PATHWAY

13. PHYTOENE AND LYCOPENE: PRECURSORS TO CAROTENOIDS • W266Drprp (reduced pigment) • W266DRpRp (orange) • W276B (orange)** • Danvers (orange)** • HCM (orange) • Beta III (dark orange) • Okuzawa (red) • Yellow type (yellow) PHYTOENE PHYTOENE, LYCOPENE* PHYTOENE *minimal lycopene detected in all other accessions **minimal phytoene detected in W276B and Danvers

14. PHYTOENE AND LYCOPENE: PRECURSORS TO CAROTENOIDS • Non-orange roots showed increased levels of precursors • Suggests reduction in production/efficiency of enzyme converting to α- and β-carotene • Leaves didn’t contain the precursors • All leaves contained ample α- and β-carotene

15. SUMMARY OF CORRELATIONS • Positive correlation (r=0.92) between α- and β-carotene • May be able to simultaneously select for both • α- and β-carotene negatively correlated with phytoene and lycopene* • Possibly because phytoene and lycopene are precursors to α- and β-carotene • Tycopherol negatively correlated with phytoene and lycopene* • Xylem: tycopherol positively correlated with α- and β-carotene (r=0.65 and r=0.52) • Possibility of selecting for high levels of all three compounds • Leaves: tycopherol positively correlated with α- and β-carotene (r=0.28 and r=0.65) • Possibly due to common origin of biosynthetic pathways *Correlations may be skewed due to small number of accessions with presence of phytoene or lycopene. Require more tests with more non-orange accessions.

16. rprp vs. RpRp Carotenoids [rprp] = 0.04*[RpRp] Phytoene [rprp] = 476.36mAu [RpRp] = not detectable

17. BIOSYNTHESIS PATHWAY • Recessive mutation reported to cause 93% loss of root pigmentation • Simultaneous decrease in levels of α- and β-carotene suggests allele blocks carotenoid pathway at step immediately following phytoene

18. Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization, single nucleotide polymorphism (SNP) markers and genome mapping Just, B.J., C.A.F. Santos, M.E.N. Fonseca, L.S. Boiteux, B.B. Oloizia, and P.W. Simon Theoretical Applied Genetics 114: 693-704 (2007)

19. HISTORY OF CARROT MAPPING: AN OVERVIEW • Genetic linkage maps • Several have been published • Santos and Simon (2004) merged maps for six linkage groups in two populations • PCR-based codominant markers • Several published, but limited usefulness across unrelated populations • STS (sequence tagged sites) markers • Have not been developed for carrot • Used in other crops to create linkage maps from different crosses that can be compared

20. RESEARCH GOALS • Identify putative carotenoid biosynthetic gene sequences in carrot • Place as STS markers on carrot linkage map from Santos and Simon (2004)

21. METHODS • Map population, and extract DNA • B493 x QAL F2 • B493: dark orange inbred, QAL: white wild carrot • F1 plant self-pollinated to produce F2 • 183 F2 plants grown • Target genes, design primers, and amplify initial PCR of putative carotenoid structural gene-containing genomic sequences • Clone and sequence • Design copy-specific primers, and identify polymorphism • Genotype the population at each putative carotenoid biosynthetic gene and Y2mark • Construct linkage map • Added to map consisting mostly of AFLP markers, generated by Santos (2001) • Extracted RNA • Performed RACE PCR and amplified full-length cDNA clones

22. RESULTS: mapping • Placed 24 putative carrot carotenoid biosynthetic structural genes on carrot linkage map • 2 genes omitted because lacked polymorphism or displayed severe segregation distortion • Sequenced full-length transcript for 22 of the genes • 15 new putative genes identified • 24 genes studied are distributed over eight of the nine carrot linkage groups

23. B493 X QAL LINKAGE MAP • QAL and B493 coupling linkage groups shown side by side • Maps positions of putative carotenoid biosynthetic structural genes • Codominant markers are connected with dotted lines between the two maps • Other markers are dominant AFLP fragments from Santos (2001) • Just one codominant marker  ambiguous orientation

24. RESULTS: QTLs • 3 of the markers mapped to region of QTL clustering identified by Santos and Simon (2002) for major carotenoid pigments • Candidate genes for some of the QTLs

25. RESULTS: mRNA • mRNA for all genes present in orange roots • Genes before and after α- and β-carotene in pathway are expressed • Need future research to elucidate extent of pathway regulation at transcription level

26. FUTURE RESEARCH • Mapped genes will aid in identifying homologous groups across studies • Future researchers now have tool to study functionality of the genes by producing their protein products