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Carotenoid Pathway in Capsicum Annuum
Laura Rodriguez-Uribe
Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM USA
Carotenoids
•Most abundant natural pigments, more than 700 carotenoids have been identified in photosynthetic organisms such as plants, algae, and bacteria
Carrot Corn Tomato Pepper
Algae Bacteria β-carotene Deinoxanthin
β-carotene Lutein/Zeaxanthin Lycopene Capsanthin/Capsorubin
Carotenoid biosynthetic pathway in Capsicum
Peppers possess the most evolved carotenoid pathway
Carotenoid biosynthesis and accumulation
Fruit wall
Chromoplasts
Carotenoid composition changes duringfruit ripening
Biological Antioxidants, Vitamin A precursorCarotenoids quench free radicals, prevent protein oxidation and DNA damage
Sources of Free Radicals: Effects:
Cigarette smoke Cancers
Environmental pollutants Atherosclerosis
Radiation Damaged DNA
Ultraviolet light Heart disease
Pesticides Rheumatoid arthritis
Nutritional supplements
New Mexico red pepper
Pigments (natural dyes) Cosmetics
Industry
1
Species Cultiv ar Phenot ype -car o tene Capsanthin Total C arotenoid
C. annuu m Hung a rian Apple Red 0.137 ± 0 0.310 ± 0.05 1.524 ± 0.18 Andy Red 0.415 ± 0.01 0.747 ± 0.04 5.558 ± 0.52 Indian PC -1 Red 1.361 ± 0.11 0.443 ± 0.12 3.756 ± 0.83 Big Red Cayenne Red 0.405 ± 0.07 0.230 ± 0 1.597 ± 0.15 Blackbird Red 0.093 ± 0.02 0.755 ± 0.27 2.105 ± 0.42 Giant Thai Red 1.319 ± 0.26 1.151 ± 0.05 6.551 ± 0.34 NuMex Garnet Red 1.158 ± 0.05 0.655 ± 0.45 5.342 ±1.38 Pimiento Red 0.175 ± 0.03 2.9 76 ± 0.77 5.675 ± 0.46 Sandia Red 0.391 ± 0.05 0.558 ± 0.03 3.348 ± 0.56 Sweet Banana Red 0.315 ± 0.09 0.094 ± 0.04 0.693 ± 0.39 NuMex Centennial Red 0.121 ± 0.10 0.036 ± 0.02 0.213 ± 0.08 Nambe Red 0.577 ± 0.22 3.028 ± 0.64 10.755 ± 2.54 NuMex Nematador Red 0.563 ± 0.40 4.014 ± 2.86 9.073 ± 2 Alba Orange 0.014 ± 0 0.047 ± 0 0.151 ± 0.01 Blushing Beauty Orange 0.000 ± 0 0.142 ± 0.05 0.311 ± 0.01 Costeno Amarillo Orange 1.239 ± 0.04 0.303 ± 0.04 3.515 ± 0.50 Early Sunsation Orange 0.116 ± 0.06 0.132 ± 0.07 0.461 ± 0.23 Orange Thai Orange 0.822 ± 0.07 0.322 ± 0.07 2.446 ± 0.43 Oriole Orange 0.277 ± 0.10 0.601 ± 0.12 1.442 ± 0.42 Gourmet Rainbow Orange 0.503 ± 0 0.954 ± 0 3.666 ± 0 Mandarin Orange 0.037 ± 0.01 0.075 ± 0.02 0.130 ± 0.01 NuMex Thanksgiving Orange 0.007 ± 0 0.028 ± 0.01 0.048 ± 0.017 . St Patri ’cks Day Orange 0 ± 0 0.011 ± 0.01 0.011 ± 0.01 NuMex Sunglo Yellow 0 ± 0 0.119 ± 0 0.301 ± 0 Yellow Cheese Pimiento Yellow 0.039 ± 0.04 0.097 ± 0 0.313 ± 0 Dove White 0.000 ± 0 0.007 ± 0 0.011 ± 0
. C baccatum Bolivian Yellow Orange 0.204 ± 0.18 0.924 ± 0.86 2.497 ± 1.20 . C chinense Aj i Dulce Red 0.228 ± 0.04 0.417 ± 0.22 1.103 ± 0.24
NuMex Suave Orange Orange 0 ± 0 0 ± 0 0.011 ± 0 J amaican Hot Chocolate Brown 0.204 ± 0.18 0.102 ± 0.04 0.891 ± 0.43 J amaican Yellow Yellow 0.280 ± 0.13 0.207 ± 0.09 1.478 ± 0.70
2
Carotenoid Quantities
Carotenoids in Orange Peppers
Carotenoid profile in ripe orange peppers
Thin layer chromatography with the carotenoid composition at different ripening stages
Valencia Fogo NuMex Standards Sunset
G GO O G GO O G GO O Cs Cr V
Green
Green/Orange
Orange
Objectives
• Investigate the β-carotene content of orange fruited capsicum varieties to correlate the relationship between fruit color and β-carotene content
Carotenoid biosynthetic genes in orange colored peppers
• Carotenoid pathway genes phytoene synthase (Psy), lycopene-β cyclase (Lcyb), β-carotenoid hydrolase (CrtZ-2), and casanthin-capsorubin synthase (Ccs), from six orange fruited peppers were cloned and sequenced.
• Mutations in the Lcyb of the six orange peppers, and a new version of the Ccs gene from Fogo pepper genes.
Multiple alignment of the capsanthin- capsorubin synthase protein
Multiple alignment with the Ccs amino acid sequences from Yolo wonder, Valencia, and Fogo.
Expression of carotenoid biosynthetic genes during ripening in orange peppers
EtBr
Ccs
CrtZ-2
Psy
Lcyb
G GO O G GO O G GO O
Valencia Fogo NM Sunset
Green
Green/Orange
Orange
Approaches to test the functionality of the CCS mutant
• Biotechnological: Expression of the wild type and mutant Capsanthin-Capsorubin
synthases as recombinant proteins in an heterologous system such as Escerichia. Coli (E.coli) and assay its activity.
• Biochemical: Purification and characterization of the Capsanthin-Capsorubin
from Valencia and Fogo peppers chromoplasts
Expression of Capsanthin capsorubin synthase in E. coli as a recombinant protein
kD 1 2 3 4 5 6 7
755037
25
Ccs
Purification of CCS from pepper fruit wall chromoplasts
Fruit wallPercoll density gradient
Chromoplast
Isolation and Purification of CCS protein
Carotenoid composition differs among peppers
The six orange peppers in this study have a new version of the Lcyb gene A novel version of the Ccs gene was found in the Fogo cultivar
Differences in gene expression in the carotenoid pathway might determine the carotenoid composition in mature peppers .
Conclusions
Overall SignificanceNMSU Research
Metabolomics / Breeding
New MexicoBreeders
Nutrient + Pigment Rich Peppers↑ β-Carotene
↑ Capsanthin/Capsorubin↑ Quality in New Mexico
Improvement of other agronomically important crops
World Nutrition
Thank You!
Dr. Mary A. O’Connell Dr. Paul BoslandDr. Richard D. Richins Chile Pepper InstituteLab members
Supported in part by NM Agricultural Experiment Station and
NIH grant R25 GM61222
Carotenoid Chemistry
Two kinds of carotenoids
» Non Pro-Vitamin A
lycopene
» Pro-Vitamin A
-carotene