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

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

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

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

Industry New Mexico red pepper Pigments (natural dyes) Cosmetics

Carotenoid Quantities

Carotenoids in Orange Peppers

Carotenoid profile in ripe orange peppers

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

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 Expression of Capsanthin capsorubin synthase in E. coli as a recombinant protein kD 1 2 3 4 5 6 7 75 50 37 25 Ccs 75 75 50 37 25 75 50 37 25 50 37 25

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

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 .

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

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

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