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. GoldmanJournal of Agricultural and Food Chemistry 53: 325-331 (2005)

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• Lycopenered• Anthocyaninspurple• 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)

BIOSYNTHESIS PATHWAY

GGPP = geranylgeranyl-pyrophosphate

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

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)

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

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]

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]

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

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

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]

BIOSYNTHESIS PATHWAY

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

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

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.

rprp vs. RpRp

Carotenoids[rprp] = 0.04*[RpRp]

Phytoene[rprp] = 476.36mAu

[RpRp] = not detectable

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

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)

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

RESEARCH GOALS

• Identify putative carotenoid biosynthetic gene sequences in carrot

• Place as STS markers on carrot linkage map from Santos and Simon (2004)

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

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

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

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

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

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