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