Article | . 2017 Vol. 35, Issue. 5
A New Nonsense Mutation in Capsanthin/Capsorubin Synthase Controlling Orange Pepper Fruit

Department of Horticultural Science, Kyungpook National University1
Department of Plant Science, College of Agriculture and Life Sciences, Seoul National University2

2017.. 599:607


Carotenoids are plant pigments that play a major role in conferring fruit color. Carotenoid content is often controlled by genetic variation in the biosynthetic genes. The color of mature pepper fruit is mainly classified as red, orange, and yellow. Orange and yellow fruit colors are determined by mutations in phytoene synthase (Psy) and capsanthin-capsorubin synthase (Ccs), respectively. In contrast to the current fruit color model, we hypothesized that genetic variation in Ccs also controls orange fruit color in pepper. Ripe fruit of Capsicum annuum ‘K146465’ is orange, and its carotenoid profile obtained by HPLC analysis showed a lack of the major pepper carotenoid capsanthin but an abundance of lutein, zeaxanthin, and β-carotene compared to red pepper. cDNA cloning and sequencing analysis detected a new nonsense mutation due to a T1026 insertion in the coding region of Ccs, but no DNA sequence variation in Psy. We developed a derived cleaved amplified polymorphic sequence (dCAPS) marker to distinguish the nonsense mutation in Ccs. Genetic analysis of the F2 population derived from C. annuum ‘Sweet Banana’ (red fruit color) × C. annuum ‘K146465’ revealed that orange fruit color is determined by a single recessive gene. The nonsense mutation in Ccs distinguished by the dCAPS marker co-segregated with orange fruit color in the F2 population. This germplasm, coupled with the dCAPS marker and carotenoid profiling, will facilitate marker-assisted breeding to select orange fruit color and improve lutein, zeaxanthin, and β-carotene levels in pepper.

1. Bartley GE, Scolnik PA (1995) Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell 7:1027-1038. doi:10.1105/tpc.7.7.1027  

2. Borovsky Y, Tadmor Y, Bar E, Meir A, Lewinsohn E, Paran I (2013) Induced mutation in β-CAROTENE HYDROXYLASE results in accumulation of β-carotene and conversion of red to orange color in pepper fruit. Theor Appl Genet 126:557-565.doi:10.1007/s00122-012-2001-9   

3. Bouvier F, Hugueney P, D'Harlingue A, Kuntz M, Camara B (1994) Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6‐epoxycarotenoid into ketocarotenoid. Plant J 6:45-54. doi:10.1046/j.1365- 313X.1994.6010045.x  

4. Britton G (1985) General carotenoid methods. Meth Enzymol 111:113-149. doi:10.1016/S0076-6879(85)11007-4  

5. Cazzonelli CI, Pogson BJ (2010) Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci 15:266-274.doi:10.1016/j.tplants.2010.02.003  

6. Cutler AJ, Krochko JE (1999) Formation and breakdown of ABA. Trends Plant Sci 4:472-478. doi:10.1016/S1360-1385(99)01497-1 Fiedor J, Burda K (2014) Potential role of carotenoids as antioxidants in human health and disease. Nutrients 6:466-488. doi:10.3390/ nu6020466  

7. Gómez-García MdR, Ochoa-Alejo N (2013) Biochemistry and molecular biology of carotenoid biosynthesis in chili peppers (Capsicum spp.). Int J Mol Sci 14:19025-19053. doi:10.3390/ijms140919025  

8. Gupta P, Sreelakshmi Y, Sharma R (2015) A rapid and sensitive method for determination of carotenoids in plant tissues by high performance liquid chromatography. Plant Methods 11:5. doi: 10.1186/s13007-015-0051-0  

9. Guzman I, Hamby S, Romero J, Bosland PW, O’Connell MA (2010) Variability of carotenoid biosynthesis in orange colored Capsicum spp. Plant Sci 179:49-59. doi:10.1016/j.plantsci.2010.04.014  

10. Ha SH, Kim JB, Park JS, Lee SW, Cho KJ (2007) A comparison of the carotenoid accumulation in Capsicum varieties that show different ripening colours: deletion of the capsanthin-capsorubin synthase gene is not a prerequisite for the formation of a yellow pepper. J Exp Bot 58:3135-3144. doi:10.1093/jxb/erm132  

11. Hirschberg J (2001) Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol 4:210-218. doi:10.1016/S1369-5266(00)00163-1  

12. Hugueney P, Badillo A, Chen H-C, Klein A, Hirschberg J, Camara B, Kuntz M (1995) Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts. Plant J 8:417-424. doi:10.1046/j.1365-313X.1995.08030417.x  

13. Huh JH, Kang BC, Nahm SH, Kim S, Ha KS, Lee MH, Kim BD (2001) A candidate gene approach identified phytoene synthase as the locus for mature fruit color in red pepper (Capsicum spp.). Theor Appl Genet 102:524-530. doi:10.1007/s001220051677  

14. Hurtado-Hernandez H, Smith PG (1985) Inheritance of mature fruit color in Capsicum annuum L. J Hered 76:211-213. doi:10.1093/oxfordjournals.jhered.a110070  

15. Kim JS, An CG, Park JS, Lim YP, Kim S (2016) Carotenoid profiling from 27 types of paprika (Capsicum annuum L.) with different colors, shapes, and cultivation methods. Food Chem 201:64-71. doi:10.1016/j.foodchem.2016.01.041  

16. Kim OR, Cho MC, Kim BD, Huh JH (2010) A splicing mutation in the gene encoding phytoene synthase causes orange coloration in habanero pepper fruits. Mol Cells 30:569-574. doi: 10.1007/s10059-010-0154-4  

17. Krubasik P, Sandmann G (2000) Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups. Biochem Soc Trans 28:806-810. doi:10.1042/bst0280806  

18. Lang YQ, Yanagawa S, Sasanuma T, Sasakuma T (2004) Orange fruit color in Capsicum due to deletion of capsanthin-capsorubin synthesis gene. Breed Sci 54:33-39. doi:10.1270/jsbbs.54.33  

19. Lefebvre V, Kuntz M, Camara B, Palloix A (1998) The capsanthin-capsorubin synthase gene: a candidate gene for the y locus controlling the red fruit colour in pepper. Plant Mol Biol 36:785-789. doi:10.1023/A:1005966313415  

20. Lightbourn GJ, Griesbach RJ, Novotny JA, Clevidence BA, Rao DD, Stommel JR (2008) Effects of anthocyanin and carotenoid combinations on foliage and immature fruit color of Capsicum annuum L. J Hered 99:105-111. doi: 10.1093/jhered/esm108   

21. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321-4325. doi:10.1093/nar/8.19.4321  

22. Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms:experimental applications in Arabidopsis thaliana genetics. Plant J 14:387-392. doi:10.1046/j.1365-313X.1998.00124.x  

23. Nicholas KB (1997) GeneDoc: analysis and visualization of genetic variation. EMB News 4:14   

24. Nwachukwu ID, Udenigwe CC, Aluko RE (2016) Lutein and zeaxanthin: Production technology, bioavailability, mechanisms of action,visual function, and health claim status. Trends Food Sci Technol 49:74-84. doi:10.1016/j.tifs.2015.12.005  

25. Rodriguez-Uribe L, Guzman I, Rajapakse W, Richins RD, O’Connell MA (2012) Carotenoid accumulation in orange-pigmented Capsicum annuum fruit, regulated at multiple levels. J Exp Bot 63:517-526. doi: 10.1093/jxb/err302  

26. Ronen G, Carmel-Goren L, Zamir D, Hirschberg J (2000) An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc Natl Acad Sci USA 97:11102-11107. doi: 10.1073/pnas.190177497  

27. Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54:733-749.doi: 10.1111/j.1365-313X.2008.03447  

28. Taylor M, Ramsay G (2005) Carotenoid biosynthesis in plant storage organs: recent advances and prospects for improving plant food quality. Physiol Plant 124:143-151. doi: 10.1111/j.1399-3054.2005.00509  

29. Thorup T, Tanyolac B, Livingstone K, Popovsky S, Paran I, Jahn M (2000) Candidate gene analysis of organ pigmentation loci in the Solanaceae. Proc Natl Acad Sci USA 97:11192-11197. doi: 10.1073/pnas.97.21.11192  

30. Tian SL, Li L, Chai WG, Shah SNM, Gong ZH (2014) Effects of silencing key genes in the capsanthin biosynthetic pathway on fruit color of detached pepper fruits. BMC Plant Biol 14:314. doi: 10.1186/s12870-014-0314-3  

31. Yonekura L, Nagao A (2007) Intestinal absorption of dietary carotenoids. Mol Nutr Food Res 51:107-115. doi: 10.1002/mnfr.200600145  

32. Yoo HJ, Park WJ, Lee GM, Oh CS, Yeam IH, Won DC, Kim CK, Lee JM (2017) Inferring the genetic determinants of fruit colors in tomato by carotenoid profiling. Molecules 22:764. doi:10.3390/molecules22050764