1. CarotenoidPathway in Capsicum AnnuumLaura Rodriguez-UribeDepartment 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/ZeaxanthinLycopeneCapsanthin/Capsorubin Algae Bacteria β-carotene Deinoxanthin

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

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

5. Biological Antioxidants, Vitamin A precursor Nutritional supplements Carotenoidsquench 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 FogoNuMex 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 phytoenesynthase (Psy), lycopene-βcyclase (Lcyb), β-carotenoidhydrolase (CrtZ-2), and casanthin-capsorubinsynthase (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 Ccsgene from Fogo pepper genes.

13. Multiple alignment of the capsanthin- capsorubinsynthase 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 G GO O G GO O G GO O Psy Lcyb CrtZ-2 Green Ccs EtBr Green/Orange Valencia Fogo NM Sunset Orange

15. Approaches to test the functionality of the CCS mutant • Biotechnological: Expression of the wild type and mutant Capsanthin-Capsorubinsynthases 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 Capsanthincapsorubinsynthase in E. coli as a recombinant protein kD 1 2 3 4 5 6 7 75 50 37 25 Ccs 75 75 75 50 37 25 50 37 25 50 37 25

17. Purification of CCS from pepper fruit wall chromoplasts Chromoplast 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 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. 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. CarotenoidChemistry • Two kinds of carotenoids » Non Pro-Vitamin A lycopene » Pro-Vitamin A -carotene