Enhancing beta-carotene content in potato by rnai-mediated silencing of the beta-carotene hydroxylase gene

  • J. Van Eck
  • B. Conlin
  • D. F. Garvin
  • H. Mason
  • D. A. Navarre
  • C. R. Brown


Plant carotenoids are lipid soluble pigments that play key roles in numerous plant functions. They also play significant roles in the human diet by serving as precursors for vitamin A synthesis and by reducing the occurrence of certain diseases. The purpose of this work was to identify novel methods for enhancing betacarotene content in potato, a major staple food crop. In particular, we used RNA interference (RNAi) to silence the beta-carotene hydroxylase gene (bch), which converts beta-carotene to zeaxanthin. Agrobacterium tumefaciens-mediated transformation was employed to introduce two RNAi constructs into three different potato lines (‘Yema de Huevo’, 91E22, and ‘Desiree’). One construct contained the tuber-specific granulebound starch synthase (GBSS) promoter, and the other contained the strong constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter. Eighty-six percent of the silenced lines had altered carotenoid profiles, as revealed by HPLC. Beta-carotene content was increased from trace amounts in wild type tubers up to 331 μg 100 g1 fresh weight. In addition, some transformants exhibited a significant decrease in zeaxanthin content and/or an increase in lutein. In general, transformants derived from the GBSS construct contained more beta-carotene than CaMV 35S transformants. Reverse-transcriptase PCR (RT-PCR) analysis of bch RNA abundance in tubers demonstrated that the extent of bch silencing varied between transformants, and was in most cases associated with the level of beta-carotene. Similarly, RT-PCR showed that bch silencing also occurred in leaves, but primarily in the CaMV 35S lines. These results demonstrate that silencing bch has the potential to increase the content of two health-promoting carotenoids, betacarotene and lutein, in potato.

Additional Key Words

carotenoids beta-carotene Solanum tuberosum provitamin A gene silencing metabolic engineering 


Los carotenoides vegetales son pigmentos solubles que juegan un rol importante en numerosas funciones de la planta. También juegan un rol significativo en la dieta humana, pues sirven como precursores de la síntesis de vitamina A y reducen la presencia de ciertas enfermedades. El propósito de este trabajo fue de identificar métodos nuevos para incrementar el contenido de betacaroteno en papa, un cultivo alimenticio importante de consumo diario. Particularmente hemos utilizado la RNA interferencia (RNAi) para silenciar el gen betacaroteno hidroxilasa (bch), el cual convierte el betacaroteno en zeaxantina. Se empleó la transformacion mediada por Agrobactrium tumefaciens para introducir dos construcciones de RNAi en tres diferentes líneas de papa (‘Yema de huevo’, 91E22 y ‘Desiree’). Una construcción contenía el promotor específico del tubérculo ligado a gránulo de sintasa del almidón (GBSS) y el otro contenía el promotor constitutive del mosaico de la coliflor 35S (CaMV 35S). El 85% de las líneas silenciadas tuvieron carotenoides de perfiles alterados, tal como lo revelado por HPCL. El contenido de beta-caroteno se incrementó de trazas en tubérculos tipo silvestre hasta 33.1 ¼g/100 g-1 de peso fresco. Además, algunos transformantes exhibieron una significativa disminucion en el contenido de zeaxantina y/o un aumento en luteína. En general, los transformantes derivados del GBSS, contenían más beta-caroteno que los de CaMV 35S. El análisis reverso transcriptasa PCR (RT-PCR) de abundancia de bch RNA en tubérculos demostró que la cantidad de silenciamiento bch varió entre transformantes y fue en muchos casos asociado con el nivel de beta-caroteno. Similarmente, el RT-PCR mostró que el silenciamiento bch también ocurrió en las hojas, pero principalmente en las líneas CaMV 35S. Estos resultados demuestran que el silenciar el bch tiene potencial para incrementar dos carotenoides promotores de la salud, el betacaroteno y la luteína, en papa.

Literature cited

  1. Alba R, P Payton, Z Fei, R McQuinn, P Debbie, GB Martin, SD Tanksley and JJ Giovannoni. 2005. Transcriptome and selected metabolite analyses reveal multiple points of ethylene control during tomato fruit development. Plant Cell 17:2954–2965.PubMedCrossRefGoogle Scholar
  2. Barber N. 2003. The tomato: An important part of the urologist’s diet? BJU Int 91:307–309.PubMedCrossRefGoogle Scholar
  3. Becker D, E Kemper, J Schell and 0R Masterson. 1992. New plant binary vectors with selectable markers located proximal to the left TDNA border. Plant Mol Biol 20:1195–1197.PubMedCrossRefGoogle Scholar
  4. Bonierbale MW, RL Plaisted and SD Tanksley. 1988. RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics 120:1095–1103.PubMedGoogle Scholar
  5. Breithaupt DE and A Bamedi. 2002. Carotenoids and carotenoid esters in potatoes(Solanum tuberosum L.): New insights into an ancient vegetable. J Agric Food Chem 50:7175–7181.PubMedCrossRefGoogle Scholar
  6. Breithaupt DE, P Weller, M Wolters and A Hahn. 2003. Plasma response to a single dose of dietary β-cryptoxanthin esters from papaya(Carica papaya L.) or non-esterified β-cryptoxanthin in adult human subjects: A comparative study. Br J Nutr 52:575–581.Google Scholar
  7. Brown CR, CG Edwards, C-P Yang and BB Dean. 1993. Orange flesh trait in potato: Inheritance and carotenoid content. J Amer Soc HortSci 118:145–150.Google Scholar
  8. Brown CR, TS Kim, Z Ganga, K Haynes, D De Jong, M Jahn, I Paran and W De Jong. 2006. Segregation of total carotenoid in high level potato germplasm and its relationship to beta-carotene hydroxlyase polymorphism. Amer J Potato Res 83:365–372.CrossRefGoogle Scholar
  9. Carrington JC, DD Freed and AJ Leinicke. 1991. Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein. Plant Cell 3:953–962.PubMedCrossRefGoogle Scholar
  10. Chang S, J Puryear and J Cairney. 1993. Simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116.CrossRefGoogle Scholar
  11. Chuang C and EM Meyerowitz. 2000. Specific and heritable genetic interference by double-stranded RNA inArabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990.PubMedCrossRefGoogle Scholar
  12. Combs GF Jr. 1992. The Vitamins: Fundamental Aspects in Nutrition and Health. Academic Press, Inc., San Diego.Google Scholar
  13. Cunningham FX and E Gantt. 1998. Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Bio 49:557–583.CrossRefGoogle Scholar
  14. Diretto G, R Tavazza, R Welsch, D Pizzichini, G Mourgues, V Papacchioli, P Beyer and G Giuliano. 2006. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase. BMC Plant Biol 6:1–21.CrossRefGoogle Scholar
  15. Ducreux LJ, WL Morris, PE Hedley, T Shepherd, HV Davies, S Millam and MA Taylor. 2005. Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein. J Exp Bot 56:81–89.PubMedGoogle Scholar
  16. Fishwick MJ and A Wright. 1980. Isolation and characterization of amyloplast envelope membranes fromSolarium tuberosum. Phytochemistry 19:55–59.CrossRefGoogle Scholar
  17. Fraser PD and PM Bramley. 2004. The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265.PubMedCrossRefGoogle Scholar
  18. Giovannucci E, A Ascherio, EB Rimm, MJ Stampfer, GA Colditz and WC Willett. 1995. Intake of carotenoids and retinol in relation to risk of prostate cancer. J Natl Cancer Inst 87:1767–1776.PubMedCrossRefGoogle Scholar
  19. Goodwin TW. 1980. Carotenoids.In: E Bell, BV Charlwood (eds), Encyclopedia of Plant Physiology. Springer-Verlag, New York, pp 257- 287.Google Scholar
  20. Hirshberg J. 1998. Molecular biology of carotenoid biosynthesis.In: G Britton, S Liaaen-Jensen, H Pfander (eds), Carotenoids. Birkhaeuser Verlag, Berlin, pp 149–194.Google Scholar
  21. Krinsky NI, JT Landrum and RA Bone. 2003. Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu Rev Nutr 23:171–201.PubMedCrossRefGoogle Scholar
  22. Kuipers AG, WJ Soppe, E Jacobsen and RG Visser. 1994. Field evaluation of transgenic potato plants expressing an antisense granulebound starch synthase gene: Increase of the antisense effect during tuber growth. Plant Mol Biol 26:1759–1773.PubMedCrossRefGoogle Scholar
  23. Marano MR, EC Serra, EG Orellano and N Carrillo. 1993. The path of chromoplast development in fruits and flowers. Plant Science 94:1–17.CrossRefGoogle Scholar
  24. Mayne ST. 1996. Beta-carotene, carotenoids, and disease prevention in humans. Faseb J 10:690–701.PubMedGoogle Scholar
  25. Murashige T and F Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497.CrossRefGoogle Scholar
  26. Odell JT, F Nagy and NH Chua. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812.PubMedCrossRefGoogle Scholar
  27. Osganian SK, MJ Stampfer, E Rimm, D Spiegelman, JE Manson and WC Willett. 2003. Dietary carotenoids and risk of coronary artery disease in women. Am J Clin Nutr 77:1390–1399.PubMedGoogle Scholar
  28. Rock CL, RA Jacob and PE Bowen. 1996 Update on the biological characteristics of the antioxidant micronutrients: Vitamin C, vitamin E, and the carotenoids. J Am Diet Assoc 9:693–702.CrossRefGoogle Scholar
  29. Romer S, J Lubeck, F Kauder, S Steiger, C Adomat and G Sandmann. 2002. Genetic engineering of a zeaxanthin-rich potato by antisense inactivation and co-suppression of carotenoid epoxidation. Metabolic Engineering 4:263–272.PubMedCrossRefGoogle Scholar
  30. Ruperti B, C Bonghi, A Rasori, A Ramina and P Tonutti. 2001. Characterization and expression of two members of the peach 1- aminocyclopropane-1-carboxylate oxidase gene family. Physiol Plant 111:336–344.PubMedCrossRefGoogle Scholar
  31. Seddon J, UA Ajani, RD Sperduto, R Hiller, N Blair, TC Burton, MD Farber, ES Gragoudas, J Haller and DT Miller. 1994. Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. Eye Disease Case-Control Study Group. JAMA 272:1413–1420.PubMedCrossRefGoogle Scholar
  32. Thorup TA, B Tanyolac, KD Livingstone, S Popovsky, I Paran and M Jahn. 2000. Candidate gene analysis of organ pigmentation loci in the Solanaceae. Proc Nati Acad Sci USA 97:11192–11197.CrossRefGoogle Scholar
  33. Steege van der G, M Nieboer, J Swaving and MJ Tempelaar. 1992. Potato granule-bound starch synthase promoter-controlled GUS expression: Regulation of expression after transient and stable transformation. Plant Mol Biol 20:19–30.PubMedCrossRefGoogle Scholar
  34. Visser RG, I Somhorst, GJ Kuipers, NJ Ruys, WJ Feenstra and E Jacobsen. 1991. Inhibition of the expression of the gene for granulebound starch synthase in potato by antisense constructs. Mol Gen Genet 225:289–296.PubMedCrossRefGoogle Scholar
  35. Visser RG, A Stolte and E Jacobsen. 1991. Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants. Plant Mol Biol 17:691–699.PubMedCrossRefGoogle Scholar
  36. Waterhouse PM and CA Helliwell. 2003. Exploring plant genomes by RNA-induced gene silencing. Nat Rev Genet 4:29–38.PubMedCrossRefGoogle Scholar
  37. West CE, A Eilander and M vanLieshout. 2002. Consequences of revised estimates of carotenoid bioefficacy for dietary control of vitamin A deficiency in developing countries. J Nutr 132:2920- 2926.Google Scholar
  38. West KP Jr. 2002. Extent of vitamin A deficiency among preschool children and women of reproductive age. J Nutr 132:2857–2866.Google Scholar

Copyright information

© Springer 2007

Authors and Affiliations

  • J. Van Eck
    • 1
  • B. Conlin
    • 1
  • D. F. Garvin
    • 2
  • H. Mason
    • 3
  • D. A. Navarre
    • 4
  • C. R. Brown
    • 4
  1. 1.The Boyce Thompson Institute for Plant ResearchIthacaUSA
  2. 2.USDA/ARS Plant Science Research Unit and Deptof Agronomy and Plant GeneticsSaint PaulUSA
  3. 3.Department of BiologyArizona State UniversityTempeUSA
  4. 4.USDA/ARSProsserUSA

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