Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Characterization of Putative Capsaicin Synthase Promoter Activity

  • Kim, June-Sik (Department of Plant Science, and Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Science, Seoul National University) ;
  • Park, Minkyu (Department of Interdisciplinary Program of Agriculture Biotechnology, College of Agriculture and Life Science, Seoul National University) ;
  • Lee, Dong Ju (Department of Plant Science, and Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Science, Seoul National University) ;
  • Kim, Byung-Dong (Department of Plant Science, and Research Institute for Agriculture and Life Sciences, College of Agriculture and Life Science, Seoul National University)
  • Received : 2009.02.13
  • Accepted : 2009.08.18
  • Published : 2009.10.31
⟨ Previous Next ⟩

Abstract

Capsaicin is a very important secondary metabolite that is unique to Capsicum. Capsaicin biosynthesis is regulated developmentally and environmentally in the placenta of hot pepper. To investigate regulation of capsaicin biosynthesis, the promoter (1,537 bp) of pepper capsaicin synthase (CS) was fused to GUS and introduced into Arabidopsis thaliana (Col-0) via Agrobacterium tumefaciens to produce CSPRO::GUS transgenic plants. The CS was specifically expressed in the placenta tissue of immature green fruit. However, the transgenic Arabidopsis showed ectopic GUS expressions in the leaves, flowers and roots, but not in the stems. The CSPRO activity was relatively high under light conditions and was induced by both heat shock and wounding, as CS transcripts were increased by wounding. Exogenous capsaicin caused strong suppression of the CSPRO activity in transgenic Arabidopsis, as demonstrated by suppression of CS expression in the placenta after capsaicin treatment. Furthermore, the differential expression levels of Kas, Pal and pAmt, which are associated with the capsaicinoid biosynthetic pathway, were also suppressed in the placenta by capsaicin treatment. These results support that capsaicin, a feedback inhibitor, plays a pivotal role in regulating gene expression which is involved in the biosynthesis of capsaicinoids.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation, Korea Ministry of Science and Technology

References

  1. Aluru, M.R., Mazourek, M., Landry, L.G., Curry, J., Jahn, M.M., and O’Connell, M. (2003). Differential expression of fatty acid synthase genes, Acl, Fat and Kas, in Capsicum fruit. J. Exp. Bot. 54, 1655-1664 https://doi.org/10.1093/jxb/erg176
  2. Bennet, D.J., and Kirby, G.W. (1968). Constitution and biosynthesis of capsaicin. J. Chem. Soc. C, 442-449 https://doi.org/10.1039/j39680000442
  3. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  4. Clough, S.J. (2005). Floral dip: agrobacterium-mediated germ linetransformation. Methods Mol. Biol. 286, 91-102
  5. Cotter, D.J. (1980). A review of studies on chile. Agri. Expt. State, NM State Univ., Las Cruces, NM. 673
  6. Curry, J., Aluru, M.R., Mendoza, M., Nevarez, J., Melendrez, M., and O’Connell, M. (1999). Transcripts for possible capsaicinoids biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum ssp. Plant Sci. 148, 47-57 https://doi.org/10.1016/S0168-9452(99)00118-1
  7. Del Rosario Abraham-Juarez, M., Del Carmen Rocha-Granados, M., Lopez, R.F., Rivera-Bustamante, M.G., and Ochoa-Alejo, N. (2008). Virus-induced silencing of Comt, pAmt and Kas genes results in a reduction of capsaicinoid accumulation in chili pepper fruits. Planta 227, 681-695 https://doi.org/10.1007/s00425-007-0651-7
  8. Higo, K., Ugawa, Y., Iwamoto, M., and Korenaga, T. (1999). Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res. 27, 297-300 https://doi.org/10.1093/nar/27.1.297
  9. Holsters, M., de Waele, D., Depicker, A., Messens, E., van Montagu, M., and Schell, J. (1978). Transfection and transformation of Agrobacterium tumefaciens. Mol. Genet. Genetics 163, 181-187 https://doi.org/10.1007/BF00267408
  10. Iwai, K., Lee, K., Kobashi, M., and Suzuki, T. (1977). Formation of pungent principles in fruits of sweet pepper, Capsicum annuum L. var. grossum during post-harvest ripening under continuous light. Agric. Biol. Chem. 41, 1873-1876 https://doi.org/10.1271/bbb1961.41.1873
  11. Iwai, K., Suzuki, T., and Fujiwake, H. (1979). Formation and metabolism of pungent principle of Capsicum fruit IV: Formation and accumulation of pungent principles in Capsicum annuum var. Karayatsubsa at different growth stages after flowering. Agric. Biol. Chem. 43, 2493-2498
  12. Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907
  13. Jeong, C.S., Yeuong, Y.R., Yun, H.K., Yoo, K.C., and Nagaoka, M. (1996). Effect of light intensities and temperature on capsaicin and sugar contents of each growth stage in Capsicum annuum L.. J. Agr. Sci. Inst. Agri. Sci. Kangwon. Nat. Univ. 7, 69-72
  14. Kim, K.M. (2004). Comparison of capsaicinoids analytical methods and changes of capsaicinoid precursors in hot pepper fruits, MS thesis, Seoul National University, Korea
  15. Kim, K.S., Kim, S.D., Park, J.R., Roh, S.M., and Yoon, T.H. (1978). The effect of light quality on the major components of hot pepper plant (Capsicum annuum L.) grown in polyethylene film house. Kor. J. Food Sci. Tech. 10, 8-10
  16. Kim, S., Kim, Y.H., Lee, Z.W., Kim, B.D., and Ha, K.S. (1997). Analysis of chemical constituents in fruits of red pepper (Capsicum anuum L. cv. Bugang). J. Kor. Soc. Hort. Sci. 38, 384-390
  17. Kim, M., Kim, S., and Kim, B.D. (2001). Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization. Mol. Cells 11, 213-219
  18. Knotkova, H., Pappagallo, M., and Szallasi, A. (2008). Capsaicin (TRPV1 Agonist) therapy for pain relief: farewell or revival? Clin. J. Pain 24, 142-154 https://doi.org/10.1097/AJP.0b013e318158ed9e
  19. Kopp, B., and Jurenitsch, J. (1981). Biosynthesis of capsaicinoids in Capsicum annuum L. var. annuum. Planta Med. 43, 272-279 https://doi.org/10.1055/s-2007-971508
  20. Lee, J.Y. (2006). Identification and molecular analysis of candidate genes related to capsaicinoid biosynthesis pathway, MS Thesis, Seoul National University, Korea
  21. Lee, K.B., Engler, C., Yang, J.E., Lee, S.W., and Park, Y.H. (2001). Effect of light, temperature, and shaking speed on production of capsaicin in suspension-cultured Jalapeno pepper (Capsicum annuum L.). Agro. Chem. Biotech. 44, 84-86
  22. Lee, C.J., Yoo, E., Shin, J., Lee, J., Hwang, H.S., and Kim, B.D. (2005). Non-pungent Capsicum contains a deletion in the capsaicinoid synthetase gene, which allows early detection of pungency with SCAR markers. Mol. Cells 19, 262-267
  23. Leete, E., and Louden, M.C. (1968). Biosynthesis of capsaicin and dihydrocapsaicin in Capsicum frutescens. J. Am. Chem. Soc. 90, 6837-6841 https://doi.org/10.1021/ja01026a049
  24. Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau, Y., van de Peer, Y., Rouze, P., and Rombauts, S. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325-327 https://doi.org/10.1093/nar/30.1.325
  25. Leung, F.W. (2008). Capsaicin-sensitive intestinal mucosal afferent mechanism and body fat distribution. Life Sci. 83, 1-5 https://doi.org/10.1016/j.lfs.2008.04.018
  26. Lindsay, K., and Bosland, P.W. (1995). A field study of environmental interaction on pungency. Capsicum Eggplant Newslett. 14, 36-38
  27. Lu, S., Gu, H., Yuan, X., Wang, X., Wu, A.M., Qu, L., and Liu, J.Y. (2007). The GUS reporter-aided analysis of the promoter activities of a rice metallothionein gene reveals different regulatory regions responsible for tissue-specific and inducible expression in transgenic Arabidopsis. Transgenic Res. 16, 177-191 https://doi.org/10.1007/s11248-006-9035-1
  28. Murakami, K., Ido, M., and Masuda, M. (2006). Fruit pungency of ‘Shishito’ pepper as affected by a dark interval in continuous fluorescent illumination with temperature alteration. J. Soc. High Tech. Agric. 18, 284-289 https://doi.org/10.2525/shita.18.284
  29. Park, C.R., and Kim, S.D. (1975). The effect of light on the matured hot green pepper fruits during the after-ripening period. Kor. J. Nut. 8, 27-30
  30. Prandl, R., Kloske, E., and Schöffl, F. (1995). Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants. Plant Mol. Biol. 28, 73-82 https://doi.org/10.1007/BF00042039
  31. Stewart, C., Kang, B.C., Liu, K., Mazourek, M., Moore, S.L., Yoo, E.Y., Kim, B.D., Paran, I., and Jahn, M.M. (2005). The Pun1 gene for pungency in pepper encodes a putative acyltransferase. Plant J. 42, 675-688 https://doi.org/10.1111/j.1365-313X.2005.02410.x
  32. Stewart, C., Mazourek, M., Stellari, G.M., O’Connell, M., and Jahn, M.M. (2007). Genetic control of pungency in C. chinense via the Pun1 locus. J. Exp. Bot. 58, 979-991 https://doi.org/10.1093/jxb/erl243
  33. Sukrasno, N., and Yeoman, M.M. (1993). Phenylpropanoid metabolism during growth and development of capsaicin-frutescens fruits. Phytochemistry 32, 839-844 https://doi.org/10.1016/0031-9422(93)85217-F
  34. Surh, Y.J. (2003). Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer 3, 768-780 https://doi.org/10.1038/nrc1189
  35. Thresh, J.C. (1876). Capsaicin, the active principle of Capsicum fruits. Pharm. J. 7, 21
  36. Vaishnava, P., and Wang, D.H. (2003). Capsaicin sensitive-sensory nerves and blood pressure regulation. Curr. Med. Chem. 1, 177-188
  37. Wierenga, P.J., and Withrow, R.B. (1985). Yield and quality of trickle-irrigated chile peppers. Agric. Water Manag. 9, 339-356 https://doi.org/10.1016/0378-3774(85)90043-5
  38. Xu, Q., Barrios, C.A., Cutright, T., and Newby, B.M. (2005). Assessment of antifouling effectiveness of two natural product antifoulants by attachment study with freshwater bacteria. Environ. Sci. Pollut. Res. 12, 278-284 https://doi.org/10.1065/espr2005.04.244
  39. Yoo, E.Y., Kim, S., Kim, J.Y., and Kim, B.D. (2001). Construction and characterization of a bacterial artificial chromosome library from chili pepper. Mol. Cells 12, 117-120
  40. Yoo, E.Y., Kim, S., Kim, Y.H., Lee, C.J., and Kim, B.D. (2003). Construction of a deep coverage BAC library from Capsicum annuum, 'CM334'. Theor. Appl. Genet. 107, 540-543 https://doi.org/10.1007/s00122-003-1279-z
  41. Yun, H.K., Kim, K.Y., Kim, Y.C., Lee, J.W., Kim, I.S., Yoo, K.C., and Higashio, H. (2002a). Change of some constituents along with the fruit maturity in Capsicum species. J. Kor. Soc. Hort. Sci. 43, 39-42
  42. Yun, H.K., Kim, K.Y., Kim, Y.C., Seo, T.C., Kim, I.S., Yoo, K.C., and Higashio, H. (2002b). Changes of color and fruit components by temperature treatment after harvest of unripened fruit in hot pepper. J. Kor. Soc. Hort. Sci. 43, 289-229

Cited by

  1. Biosynthesis of capsinoid is controlled by the Pun1 locus in pepper vol.31, pp.3, 2009, https://doi.org/10.1007/s11032-012-9811-y
  2. Molecular and cellular control of cell death and defense signaling in pepper vol.241, pp.1, 2009, https://doi.org/10.1007/s00425-014-2171-6
  3. Silencing AT3 gene reduces the expression of pAmt, BCAT, Kas, and Acl genes involved in capsaicinoid biosynthesis in chili pepper fruits vol.59, pp.3, 2009, https://doi.org/10.1007/s10535-015-0525-y
  4. Phenylalanine biosynthesis and its relationship to accumulation of capsaicinoids during Capsicum chinense fruit development vol.60, pp.3, 2016, https://doi.org/10.1007/s10535-016-0608-4
  5. Tissue-Preferential Activity and Induction of the Pepper Capsaicin Synthase PUN1 Promoter by Wounding, Heat and Metabolic Pathway Precursor in Tobacco and Tomato Plants vol.60, pp.3, 2009, https://doi.org/10.1007/s12033-018-0060-0
  6. QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum vol.16, pp.9, 2018, https://doi.org/10.1111/pbi.12894
  7. Biochemistry and molecular biology of capsaicinoid biosynthesis: recent advances and perspectives vol.38, pp.9, 2019, https://doi.org/10.1007/s00299-019-02406-0
  8. Jasmonate-Inducible R2R3-MYB Transcription Factor Regulates Capsaicinoid Biosynthesis and Stamen Development in Capsicum vol.67, pp.39, 2009, https://doi.org/10.1021/acs.jafc.9b04978
  9. Gene expression related to the capsaicinoids biosynthesis in the Capsicum genus: Molecular and transcriptomic studies vol.43, pp.1, 2020, https://doi.org/10.1007/s40415-019-00575-6