1. Carotenoid Pathway in Capsicum Annuum Laura Rodriguez-Uribe Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, NM USA

2. 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
β-carotene Lutein/Zeaxanthin Lycopene Capsanthin/Capsorubin
Algae Bacteria
β-carotene Deinoxanthin

3. Carotenoid biosynthetic pathway in Capsicum
Peppers possess the most evolved
carotenoid pathway

4. Carotenoid biosynthesis and accumulation
Fruit wall
Chromoplasts
Carotenoid composition changes during
fruit ripening

5. Biological Antioxidants, Vitamin A precursor
Nutritional supplements
Biological Antioxidants, Vitamin A precursor
Carotenoids 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

6. Industry
New Mexico red pepper
Pigments (natural dyes) Cosmetics

7. Carotenoid Quantities

8. Carotenoids in Orange Peppers

9. Carotenoid profile in ripe orange peppers

10. Thin layer chromatography with the carotenoid composition at different ripening stages
G GO O G GO O G GO O Cs Cr V
Green
Green/Orange
Valencia Fogo NuMex Standards
Sunset
Orange

11. Objectives
Investigate the β-carotene content of orange fruited capsicum varieties to correlate the relationship between fruit color and β-carotene content

12. 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.

13. Multiple alignment of the capsanthin- capsorubin synthase protein
Multiple alignment with the Ccs amino acid sequences from
Yolo wonder, Valencia, and Fogo.

14. 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

15. 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

16. Expression of Capsanthin capsorubin synthase in E
Expression of Capsanthin capsorubin synthase in E. coli as a recombinant protein kD 75 50 37 25
Ccs 75 75 50 37 25 75 50 37 25 50 37 25

17. Purification of CCS from pepper fruit wall chromoplasts
Percoll density
gradient
Fruit wall
Isolation and Purification
of CCS protein

18. Conclusions 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 .

19. Overall Significance Nutrient + Pigment Rich Peppers ↑ β-Carotene
NMSU Research
Metabolomics / Breeding
New Mexico
Breeders
World Nutrition
Nutrient + Pigment Rich Peppers
↑ β-Carotene
↑ Capsanthin/Capsorubin
↑ Quality in New Mexico
Improvement of other agronomically important crops

20. NM Agricultural Experiment Station and
Thank You!
Dr. Mary A. O’Connell Dr. Paul Bosland
Dr. Richard D. Richins Chile Pepper Institute
Lab members
Supported in part by
NM Agricultural Experiment Station and
NIH grant R25 GM61222

21. Carotenoid Chemistry Two kinds of carotenoids » Non Pro-Vitamin A
lycopene
» Pro-Vitamin A
-carotene