Advertisement

Theoretical and Applied Genetics

, Volume 132, Issue 1, pp 65–80 | Cite as

Mutation in the putative ketoacyl-ACP reductase CaKR1 induces loss of pungency in Capsicum

  • Sota KoedaEmail author
  • Kosuke Sato
  • Hiroki Saito
  • Atsushi J. Nagano
  • Masaki Yasugi
  • Hiroshi Kudoh
  • Yoshiyuki Tanaka
Original Article
  • 452 Downloads

Abstract

Key message

A putative ketoacyl-ACP reductase (CaKR1) that was not previously known to be associated with pungency of Capsicum was identified from map-based cloning and functional characterization.

Abstract

The pungency of chili pepper fruits is due to the presence of capsaicinoids, which are synthesized through the convergence of the phenylpropanoid and branched-chain fatty acid pathways. The extensive, global use of pungent and non-pungent peppers underlines the importance of understanding the genetic mechanism underlying capsaicinoid biosynthesis for breeding pepper cultivars. Although Capsicum is one of the earliest domesticated plant genera, the only reported genetic causes of its loss of pungency are mutations in acyltransferase (Pun1) and putative aminotransferase (pAMT). In this study, a single recessive gene responsible for the non-pungency of pepper No.3341 (C. chinense) was identified on chromosome 10 using an F2 population derived from a cross between Habanero and No.3341. Five candidate genes were identified in the target region, within a distance of 220 kb. A candidate gene, a putative ketoacyl-ACP reductase (CaKR1), of No.3341 had an insertion of a 4.5-kb transposable element (TE) sequence in the first intron, resulting in the production of a truncated transcript missing the region coding the catalytic domain. Virus-induced gene silencing of CaKR1 in pungent peppers resulted in the decreased accumulation of capsaicinoids, a phenotype consistent with No.3341. Moreover, GC–MS analysis of 8-methyl-6-nonenoic acid, which is predicted to be synthesized during the elongation cycle of branched-chain fatty acid biosynthesis, revealed that its deficiency in No.3341. Genetic, genomic, transcriptional, silencing, and biochemical precursor analyses performed in combination provide a solid ground for the conclusion that CaKR1 is involved in capsaicinoid biosynthesis and that its disruption results in a loss of pungency.

Notes

Acknowledgements

The authors would like to thank Daiki Matsumoto (Yamagata University) for help and advice with the experiments. We would also like to thank Katsuki Ijichi, Ayaka Asami, and Maiko Sofue (Kindai University) for technical assistance with VIGS. This work was supported by JSPS KAKENHI Grant Numbers 25850018, 16K07605, and the Kyoto University research fund for young scientists: Start-up to SK.

Author contribution statement

SK designed the experiments; performed physical mapping, gene expression analysis, GC–MS, and VIGS; analyzed the data; and interpreted the results and wrote the manuscript. KS cultivated and performed the phenotypic evaluation of F1 and F2 populations. HS performed linkage analysis of RAD-seq data. AJN, MY, and HK performed RAD-seq. YT performed HPLC analysis and sequence analysis of transposable element. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Accession numbers

Accession numbers for each of the gene sequences referred to in this work are as follows: genomic sequences of CaKR1 of Habanero (LC379873) and No.3341 (LC379874), and mRNA sequences of Habanero (LC379875) and No.3341 (LC379876).

Supplementary material

122_2018_3195_MOESM1_ESM.pdf (3.3 mb)
Supplementary material 1 (PDF 3387 kb)

References

  1. Abraham-Juarez MD, Rocha-Granados MD, López MG, Rivera-Bustamante RF, 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 227:681–695CrossRefGoogle Scholar
  2. Aluru MR, Mazourek M, Landry LG, Curry J, Jahn M, O’Connell MA (2003) Differential expression of fatty acid synthase genes, Acl, Fat and Kas, in Capsicum fruit. J Exp Bot 54:1655–1664CrossRefGoogle Scholar
  3. Andrews J (1984) Peppers: the domesticated Capsicums. University of Texas Press, AustinGoogle Scholar
  4. Arce-Rodríguez ML, Ochoa-Alejo N (2017) An R2R3-MYB transcription factor regulates capsaicinoid biosynthesis. Plant Physiol 174:1359–1370CrossRefGoogle Scholar
  5. Aza-Gonzalez C, Nunez-Palenius HG, Ochoa-Alejo N (2011) Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.). Plant Cell Rep 30:695–706CrossRefGoogle Scholar
  6. Bachem CW, Horvath B, Trindade L, Claassens M, Davelaar E, Jordi W, Visser RG (2001) A potato tuber-expressed mRNA with homology to steroid dehydrogenases affects gibberellin levels and plant development. Plant J 25:595–604CrossRefGoogle Scholar
  7. Bennett DJ, Kirby GW (1968) Constitution and biosynthesis of capsaicin. J Chem Soc C 4:442–446CrossRefGoogle Scholar
  8. Bhattacharyya MK, Smith AM, Ellis THN et al (1990) The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell 60:115–122CrossRefGoogle Scholar
  9. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 30:2114–2120CrossRefGoogle Scholar
  10. Borovsky Y, Oren-Shamir M, Ovadia R, De Jong W, Paran I (2004) The A locus that controls anthocyanin accumulation in pepper encodes a MYB transcription factor homologous to Anthocyanin2 of Petunia. Theor Appl Genet 109:23–29CrossRefGoogle Scholar
  11. Bosland PW, Votava EJ (2000) Peppers: vegetable and spice capsicums. CABI Publishing, New YorkGoogle Scholar
  12. Brosché M, Strid A (1999) Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses. Plant Physiol 121:479–487CrossRefGoogle Scholar
  13. Calderon-Urrea A, Dellaporta SL (1999) Cell death and cell protection genes determine the fate of pistils in maize. Development 126:435–441PubMedGoogle Scholar
  14. Carrizo García C, Barfuss MH, Sehr EM, Barboza GE, Samuel R, Moscone EA, Ehrendorfer F (2016) Phylogenetic relationships, diversification and expansion of chili peppers (Capsicum, Solanaceae). Ann Bot 118:35–51CrossRefGoogle Scholar
  15. Catchen JM, Amores A, Hohenlohe P, Cresko W, Postlethwait JH (2011) Stacks: building and genotyping loci De Novo from short-read sequences. G3 1:171–182CrossRefGoogle Scholar
  16. Chung E, Seong E, Kim YC, Chung EJ, Oh SK, Lee S, Park JM, Joung YH, Choi D (2004) A method of high frequency virus-induced gene silencing in chili pepper (Capsicum annuum L. cv. Bukang). Mol Cells 17:377–380PubMedGoogle Scholar
  17. Curry J, Aluru M, Mendoza M, Nevarez J, Melendrez M, O’Connell MA (1999) Transcripts for possible capsaicinoid biosynthetic genes are differentially accumulated in pungent and non-pungent Capsicum spp. Plant Sci 148:47–57CrossRefGoogle Scholar
  18. DeLong A, Calderon-Urrea A, Dellaporta SL (1993) Sex determination gene TASSELSEED2 of maize encodes a short-chain alcohol dehydrogenase required for stage-specific floral organ abortion. Cell 74:757–768CrossRefGoogle Scholar
  19. Fernandez-Pozo N, Rosli HG, Martin GB, Mueller LA (2015) The SGN VIGS tool: user-friendly software to design virus-induced gene silencing (VIGS) constructs for functional genomics. Mol Plant 8:486–488CrossRefGoogle Scholar
  20. Fisher M, Kroon JT, Martindale W, Stuitje AR, Slabas AR, Rafferty JB (2000) The X-ray structure of Brassica napus beta-keto acyl carrier protein reductase and its implications for substrate binding and catalysis. Structure 15:339–347CrossRefGoogle Scholar
  21. Govindarajan VS (1985) Capsicum: production, technology, chemistry and quality. 1. History, botany, cultivation and primary processing. Crit Rev Food Sci Nutr 22:109–176CrossRefGoogle Scholar
  22. Hadacek F (2002) Secondary metabolites as plant traits: current assessment and future perspective. Crit Rev Plant Sci 21:273–322CrossRefGoogle Scholar
  23. Han K, Lee HY, Ro NY, Hur OS, Lee JH, Kwon JK, Kang BC (2018) QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum. Plant Biotechnol J.  https://doi.org/10.1111/pbi.12894 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Jacobsen SE, Olszewski NE (1996) Gibberellins regulate the abundance of RNAs with sequence similarity to proteinase inhibitors, dioxygenases and dehydrogenases. Planta 198:78–86CrossRefGoogle Scholar
  25. Kavanagh KL, Jörnvall H, Persson B, Oppermann U (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families: the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Cell Mol Life Sci 65:3895–3906CrossRefGoogle Scholar
  26. Kim M, Kim S, Kim S, Kim BD (2001) Isolation of cDNA clones differentially accumulated in the placenta of pungent pepper by suppression subtractive hybridization. Mol Cells 11:213–219PubMedGoogle Scholar
  27. Kim S, Park M, Yeom SI et al (2014) Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet 46:270–278CrossRefGoogle Scholar
  28. Kim J, Park M, Jeong ES, Lee JM, Choi D (2017) Harnessing anthocyanin-rich fruit: a visible reporter for tracing virus-induced gene silencing in pepper fruit. Plant Methods 13:3CrossRefGoogle Scholar
  29. Kirii E, Goto T, Yoshida Y, Yasuba K, Tanaka Y (2017) Non-pungency in a Japanese chili pepper landrace (Capsicum annuum) is caused by a novel loss-of-function Pun1 allele. Hort J 86:61–69CrossRefGoogle Scholar
  30. Koeda S, Sato K, Tomi K, Tanaka Y, Takisawa R, Hosokawa M, Doi M, Nakazaki T, Kitajima A (2014) Analysis of non-pungency, aroma, and origin of a Capsicum chinense cultivar from a Caribbean island. J Jpn Soc Hort Sci 83:244–251CrossRefGoogle Scholar
  31. Koeda S, Sato K, Takisawa R, Kitajima A (2015a) Inheritance of non-pungency in ‘No.3341’ (Capsicum chinense). Hort J 84:323–326CrossRefGoogle Scholar
  32. Koeda S, Sato K, Tanaka Y, Takisawa R, Kitajima A (2015b) A Comt1 loss of function mutation is insufficient for loss of pungency in Capsicum. Am J Plant Sci 6:1243–1255CrossRefGoogle Scholar
  33. Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli peppers—a review on tissue culture and transgenesis. Biotechnol Adv 28:35–48CrossRefGoogle Scholar
  34. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  35. Lang Y, Kisaka H, Sugiyama R, Nomura K, Morita A, Watanabe T, Tanaka Y, Yazawa S, Miwa T (2009) Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 sweet. Plant J 59:953–961CrossRefGoogle Scholar
  36. Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefGoogle Scholar
  37. Lee JM, Kim S, Lee JY, Yoo JY, Cho MC, Cho MR, Kim BD, Bahk YY (2006) A differentially expressed proteomic analysis in placental tissues in relation to pungency during the pepper fruit development. Proteomics 6:5248–5259CrossRefGoogle Scholar
  38. Leete E, Louden MCL (1968) Biosynthesis of capsaicin and dihydrocapsaicin in Capsicum frutescens. J Am Chem Soc 9:6837–6841CrossRefGoogle Scholar
  39. Liu W, Parrott WA, Hildebrand DF, Collins GB, Williams EG (1990) Agrobacterium induced gall formation in bell pepper (Capsicum annuum L.) and formation of shoot-like structures expressing introduced genes. Plant Cell Rep 9:360–364PubMedGoogle Scholar
  40. Liu S, Li W, Wu Y, Chen C, Lei J (2013) De Novo transcriptome assembly in chili pepper (Capsicum frutescens) to identify genes involved in the biosynthesis of capsaicinoids. PLoS ONE 8:e48156CrossRefGoogle Scholar
  41. Liu L, Venkatesh J, Jo YD, Koeda S, Hosokawa M, Kang JH, Goritschnig S, Kang BC (2016) Fine mapping and identification of candidate genes for the sy-2 locus in a temperature-sensitive chili pepper (Capsicum chinense). Theor Appl Genet 129:1541–1556CrossRefGoogle Scholar
  42. Maligeppagol M, Manjula R, Navale PM, Babu KP, Kumbar BM, Laxman RH (2016) Genetic transformation of chilli (Capsicum annuum L.) with Dreb1A transcription factor known to impart drought tolerance. Indian J Biotechnol 15:17–24Google Scholar
  43. Mazourek M, Pujar A, Borovsky Y, Paran I, Mueller L, Jahn MM (2009) A dynamic interface for capsaicinoid systems biology. Plant Physiol 150:1806–1821CrossRefGoogle Scholar
  44. Nakatsuka T, Nishihara M, Mishiba K, Hirano H, Yamamura S (2006) Two different transposable elements inserted in flavonoid 3′,5′-hydroxylase gene contribute to pink flower coloration in Gentian scabra. Mol Genet Genom 275:231–241CrossRefGoogle Scholar
  45. Narasimha Prasad BC, Gururaj HB, Kumar V, Giridhar P, Parimalan R, Sharma A, Ravishankar GA (2006) Influence of 8-methyl-nonenoic acid on capsaicin biosynthesis in in vivo and in vitro cell cultures of Capsicum spp. J Agric Food Chem 54:1854–1859CrossRefGoogle Scholar
  46. Ogawa K, Murota K, Shimura H, Furuya M, Togawa Y, Matsumura T, Masuta C (2015) Evidence of capsaicin synthase activity of the Pun1-encoded protein and its role as a determinant of capsaicinoid accumulation in pepper. BMC Plant Biol 15:93.  https://doi.org/10.1186/s12870-015-0476-7 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Park YJ, Nishikawa T, Minami M, Nemoto K, Iwasaki T, Matsushima K (2015) A low-pungency S3212 genotype of Capsicum frutescens caused by a mutation in the putative aminotransferase (p-AMT) gene. Mol Genet Genom 290:2217–2224CrossRefGoogle Scholar
  48. Perry L, Dickau R, Zarrillo S et al (2007) Starch fossils and the domestication and dispersal of chili peppers (Capsicum spp. L.) in the Americas. Science 315:986–988CrossRefGoogle Scholar
  49. Persson B, Kallberg Y, Oppermann U, Jörnvall H (2003) Coenzyme-based functional assignments of short-chain dehydrogenases/reductases (SDRs). Chem Biol Interact 143–144:271–278CrossRefGoogle Scholar
  50. Pickersgill B (1997) Genetic resources and breeding of Capsicum spp. Euphytica 96:129–133CrossRefGoogle Scholar
  51. Picton S, Gray J, Barton S, AbuBakar U, Lowe A, Grierson D (1993) cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant. Plant Mol Biol 23:193–207CrossRefGoogle Scholar
  52. Qin C, Yu CS, Shen YO et al (2014) Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc Natl Acad Sci USA 111:5135–5140CrossRefGoogle Scholar
  53. Sakaguchi S, Sugino T, Tsumura Y et al (2015) High-throughput linkage mapping of Australian white cypress pine (Callitris glaucophylla) and map transferability to related species. Tree Genet Genom 11:121CrossRefGoogle Scholar
  54. Sakurai N, Ara T, Enomoto M, Motegi T, Morishita Y, Kurabayashi A, Iijima Y, Ogata Y, Nakajima D, Suzuki H, Shibata D (2014) Tools and databases of the KOMICS web portal for preprocessing, mining, and dissemination of metabolomics data. Biomed Res Int 194812Google Scholar
  55. Steinitz B, Wolf D, Matzevitch-Josef T, Zelcer A (1999) Regeneration in vitro and genetic transformation of pepper (Capsicum spp.): the current state of the art. Capsicum Eggplant Plant Newsl 18:9–15Google Scholar
  56. Stellari GM, Mazourek M, Jahn MM (2010) Contrasting modes for loss of pungency between cultivated and wild species of Capsicum. Heredity 104:460–471CrossRefGoogle Scholar
  57. Stewart C Jr, Kang BC, Liu K, Mazourek M, Moore SL, Eun YY, Kim BD, Paran I, Jahn MM (2005) The Pun1 gene for pungency in pepper encodes a putative acyltransferase. Plant J 42:675–688CrossRefGoogle Scholar
  58. Stewart C Jr, Mazourek M, Stellari GM, O’Connell M, Jahn M (2007) Genetic control of pungency in C. chinense via the Pun1 locus. J Exp Bot 58:979–991CrossRefGoogle Scholar
  59. Tanaka Y, Hosokawa M, Miwa T, Watanabe T, Yazawa S (2010a) Newly mutated putative-aminotransferase in non-pungent pepper (Capsicum annuum) results in biosynthesis of capsinoids, capsaicinoid analogues. J Agric Food Chem 58:1761–1767CrossRefGoogle Scholar
  60. Tanaka Y, Hosokawa M, Miwa T, Watanabe T, Yazawa S (2010b) Novel loss-of-function putative aminotransferase alleles cause biosynthesis of capsinoids, non-pungent capsaicinoid analogues, in mildly pungent chili peppers (Capsicum chinense). J Agric Food Chem 58:11762–11767CrossRefGoogle Scholar
  61. Tanaka Y, Sonoyama T, Muraga Y, Koeda S, Goto T, Yoshida Y, Yasuba K (2015) Multiple loss-of-function putative aminotransferase alleles contribute to low pungency and capsinoid biosynthesis in Capsicum chinense. Mol Breed 35:142CrossRefGoogle Scholar
  62. Tanaka Y, Fukuta S, Koeda S, Goto T, Yoshida Y, Yasuba K (2018) Identification of a novel mutant pAMT allele responsible for low-pungency and capsinoid production in chili pepper accession ‘No.4034’ (Capsicum chinense). Hort J 87:222–228CrossRefGoogle Scholar
  63. Vitte C, Fustier MA, Alix K, Tenaillon MI (2014) The bright side of transposons in crop evolution. Brief Funct Genom 13:276–295CrossRefGoogle Scholar
  64. Wessler SR, Baran G, Varagona M, Dellaporta SL (1986) Excision of Ds produces waxy proteins with a range of enzymatic activities. EMBO J 5:2427–2432CrossRefGoogle Scholar
  65. White SW, Zheng J, Zhang YM, Rock CO (2005) The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem 74:791–831CrossRefGoogle Scholar
  66. Wu X, Knapp S, Stamp A, Stammers DK, Jörnvall H, Dellaporta SL, Oppermann U (2007) Biochemical characterization of TASSELSEED 2, an essential plant short-chain dehydrogenase/reductase with broad spectrum activities. FEBS J 274:1172–1182CrossRefGoogle Scholar
  67. Yazawa S, Ueda M, Suetome N, Namiki T (1989) Capsaicinoids content in the fruit of interspecific hybrids in Capsicum. J Jpn Soc Hort Sci 58:353–360CrossRefGoogle Scholar
  68. Zhang ZX, Zhao SN, Liu GF, Huang ZM, Cao ZM, Cheng SH, Lin SS (2016) Discovery of putative capsaicin biosynthetic genes by RNA-Seq and digital gene expression analysis of pepper. Sci Rep 6:38081CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of AgricultureKindai UniversityNaraJapan
  2. 2.Experimental Farm, Graduate School of AgricultureKyoto UniversityKizugawaJapan
  3. 3.Tropical Agriculture Research FrontJapan International Research Center for Agricultural SciencesIshigakiJapan
  4. 4.Faculty of AgricultureRyukoku UniversityOtsuJapan
  5. 5.Faculty of EngineeringUtsunomiya UniversityUtsunomiyaJapan
  6. 6.Center for Ecological ResearchKyoto UniversityOtsuJapan
  7. 7.Graduate School of Environmental and Life ScienceOkayama UniversityOkayamaJapan

Personalised recommendations