AJPS  Vol.6 No.8 , May 2015
A Comt1 Loss of Function Mutation Is Insufficient for Loss of Pungency in Capsicum
Abstract: The participation of O-methyltransferase (COMT) in phenylpropanoid-mediated capsaicinoid biosynthesis has long been proposed. Ferulic acid, a phenylpropanoid intermediate, is a precursor of capsaicinoid biosynthesis and is produced from caffeic acid by the action of COMT. As previously reported that silencing Comt expression caused a drastic decrease in capsaicinoid accumulation, it was presumed that a Comt loss-of-function mutation would cause loss of pungency in Capsicum. This hypothesis was tested by cloning Comt1 and Comt2 from the placenta tissue of the pungent cultivar Habanero. The phylogenetic analysis and comparison of critical amino-acid residues for enzyme function showed that the two COMTs had high similarity with the COMTs of other plant species. Moreover, as the two Comts were both expressed in placenta tissue and expressed prior to the accumulation of capsaicinoids, the two genes could be candidates for capsaicinoid biosynthesis. Second, Comt1 loss-of-function mutants were screened from the germplasm. A truncated Comt1 transcript was expressed in non-pungent pepper No.3341 caused by deletion of the genomic region. The predicted No.3341 COMT1 lacked His-265, which was absolutely necessary for enzymatic activity. Contrary to our expectations, the Comt1 mutation was not related to non-pungency of No.3341, as the deletion of Comt1 did not co-segregate with non-pungency in the F2 population obtained from crossing No.3341 with Habanero. This result was confirmed by screening several pungent accessions harboring the same Comt1 deletion mutation. Although the participation of COMT in phenylpropanoid-mediated capsaicinoid biosynthesis has long been proposed, our present study shows that Comt1 can not be a target for controlling fruit pungency.
Cite this paper: Koeda, S. , Sato, K. , Tanaka, Y. , Takisawa, R. and Kitajima, A. (2015) A Comt1 Loss of Function Mutation Is Insufficient for Loss of Pungency in Capsicum. American Journal of Plant Sciences, 6, 1243-1255. doi: 10.4236/ajps.2015.68127.

[1]   Singh, R.J. (2007) Genetic Resources, Chromosome Engineering, and Crop Improvement. Vol. 3. Taylor and Francis CRC Press, Boca Raton.

[2]   Perry, L., Dickau, R., Zarrillo, S., Holst, I., Pearsall, D.M., Piperno D.R., Berman, M.J., Cooke, R.G., Rademaker, K., Ranere, A.J., Raymond, J.S., Sandweiss, D.H., Scaramelli, F., Tarble, K. and Zeidler, J.A. (2007) Starch Fossils and the Domestication and Dispersal of Chili Peppers (Capsicum spp. L.) in the Americas. Science, 315, 986-988.

[3]   Leete, E. and Louden, M. (1968) Biosynthesis of Capsaicin and Dihydrocapsaicin in Capsicum frutescens. Journal of the American Chemical Society, 90, 6837-6841.

[4]   Suzuki, T., Kawada, T. and Iwai, K. (1981) Biosynthesis of Acyl Moieties of Capsaicin and Its Analogues from Valine and Leucine in Capsicum Fruits. Plant and Cell Physiology, 22, 23-32.

[5]   Sukrasno, N. and Yeoman, M.M. (1993) Phenylpropanoid Metabolism during Growth and Development of Capsicum frutescens Fruits. Phytochemistry, 32, 839-844.

[6]   Abraham-Juarez, M.D., Rocha-Granados, M.D., López, M.G., Rivera-Bustamante, R.F. 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.

[7]   Curry, J., Aluru, M., Mendoza, M., Nevarez, J., Melendrez, M. and O’Connell, M.A. (1999) Transcripts for Possible Capsaicinoid Biosynthetic Genes Are Differentially Accumulated in Pungent and Non-Pungent Capsicum spp. Plant Science, 148, 47-57.

[8]   Aluru, M.R., Mazourek, M., Landry, L.G., Curry, J., Jahn, M. and O’Connell, M.A. (2003) Differential Expression of Fatty Acid Synthase Genes, Acl, Fat and Kas in Capsicum Fruit. Journal of Experimental Botany, 54, 1655-1664.

[9]   Mazourek, M., Pujar, A., Borovsky, Y., Paran, I., Mueller, L. and Jahn, M.M. (2009) A Dynamic Interface for Capsaicinoid Systems Biology. Plant Physiology, 150, 1806-1821.

[10]   Boswell, V.R. (1937) Improvement and Genetics of Tomatoes, Peppers, and Eggplant. In: Wallace, H.A., Ed., Yearbook of Agriculture, United States Government Printing Office, Washington DC, 176-206.

[11]   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 Journal, 42, 675-688.

[12]   Lang, Y.Q., Kisaka, H., Sugiyama, R., Nomura, K., Morita, A., Watanabe, T., Tanaka, Y., Yazawa, S. and Miwa, T. (2009) Functional Loss of pAMT Results in Biosynthesis of Capsinoids, Capsaicinoid Analogs, in Capsicum annuum cv. CH-19 Sweet. Plant Journal, 59, 953-961.

[13]   Tanaka, Y., Hosokawa, M., Miwa, T., Watanabe, T. and Yazawa, S. (2010) Newly Mutated Putative-Aminotransferase in Nonpungent Pepper (Capsicum annuum) Results in Biosynthesis of Capsinoids, Capsaicinoid Analogues. Journal of Agricultural and Food Chemistry, 58, 1761-1767.

[14]   Stellari, G.M., Mazourek, M. and Jahn, M.M. (2010) Contrasting Modes for Loss of Pungency between Cultivated and Wild Species of Capsicum. Heredity, 104, 460-471.

[15]   Tanaka, Y., Hosokawa, M., Miwa, T., Watanabe, T. and Yazawa, S. (2010) Novel Loss-of-Function Putative Aminotransferase Alleles Cause Biosynthesis of Capsinoids, Nonpungent Capsaicinoid Analogues, in Mildly Pungent Chili Peppers (Capsicum chinense). Journal of Agricultural and Food Chemistry, 58, 11762-11767.

[16]   Koeda, S., Sato, K., Tomi, K., Tanaka, Y., Takisawa, R., Hosokawa, M., Doi, M., Nakazaki, T. and Kitajima, A. (2014) Analysis of Non-Pungency, Aroma, and Origin of a Capsicum chinense Cultivar from a Caribbean Island. Journal of the Japanese Society for Horticultural Science, 83, 244-251.

[17]   Fujiwake, H., Suzuki, T. and Iwai, K. (1982) Intracellular Distribution of Enzymes and Intermediates Involved in Biosynthesis of Capsaicin and Its Analogues in Capsicum Fruits. Agricultural and Biological Chemistry, 46, 2685-2689.

[18]   Fujiwake, H., Suzuki, T. and Iwai, K. (1982) Capsaicinoid Formation in the Protoplast from Placenta of Capsicum Fruits. Agricultural and Biological Chemistry, 46, 2591-2592.

[19]   Gang, D.R., Lavid, N., Zubieta, C., Chen, F., Beuerle, T., Lewinsohn, E., Noel, J.P. and Pichersky, E. (2002) Characterization of Phenylpropene O-Methyltransferases from Sweet Basil: Facile Change of Substrate Specificity and Convergent Evolution within a Plant O-Methyltransferase Family. The Plant Cell Online, 14, 505-519.

[20]   Zubieta, C., He, X.-Z., Dixon, R.A. and Noel, J.P. (2001) Structures of Two Natural Product Methyltransferases Reveal the Basis for Substrate Specificity in Plant O-Methyltransferase. Nature Structural and Molecular Biology, 8, 271- 279.

[21]   Lee, B., Choi, D. and Lee, K.W. (1998) Isolation and Characterization of O-Diphenol-O-Methyltransferase cDNA Clone in Hot Pepper (Capsicum annuum L.). Journal of Plant Biology, 41, 9-15.

[22]   Koeda, S., Hosokawa, M., Saito, H. and Doi, M. (2013) Temperature-Sensitive Phenotype Caused by Natural Mutation in Capsicum Latescent in Two Tropical Regions. Journal of Plant Research, 126, 675-684.

[23]   Rice, P., Longden, I. and Bleasby, A. (2000) EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics, 16, 276-277.

[24]   Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic Local Alignment Search Tool. Journal of Molecular Biology, 215, 403-410.

[25]   Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30, 2725-2729.

[26]   Koeda, S., Sato, K., Takisawa, R. and Kitajima, A. (2015) Inheritance of the Non-Pungency in ‘No.3341’ (Capsicum chinense). The Horticulture Journal, in Press.

[27]   Kim, S., Park, M., Yeom, S., Kim, Y.-M., Lee, J.M., Lee, H.-A., Seo, E., Choi, J., Cheong, K., Kim, K.-T., Jung, K., Lee, G.-W., Oh, S.-G., Bae, C.-Y., Kim, S.-B., Lee, H.-Y., Kim, S.-Y., Kim, M.-S., Kang, B.-C., Jo, Y.D., Yang, H.-B., Jeong, H.-J., Kang, W.-H., Kwon, J.-K., Shin, C., Lim, J.Y., Park, J.H., Huh, J.H., Kim, J.-S., Kim, B.-D., Cohen, O., Paran, I., Suh, M.C., Lee, S.B., Kim, Y.-K., Shin, Y., Noh, S.-J., Park, J., Seo, Y.S., Kwon, S.-Y., Kim, H.A., Park, J.M., Kim, H.-J., Choi, S.-B., Bosland, P.W., Reeves, G., Jo, S.-W., Lee, B.-W., Cho, H.-T., Choi, H.-S., Lee, M.-S., Yu, Y., Choi, Y.D., Park, B.-S., van Deynze, A., Ashrafi, H., Hill, T., Kim, W.T., Pai, H.-S., Ahn, H.K., Yeam, I., Giovannoni, J.J., Rose, J.K.C., Sorensen, I., Lee, S.-J., Kim, R.W., Choi, I.-Y., Choi, B.-S., Lim, J.-S., Lee, Y.-H. and Choi, D. (2014) Genome Sequence of the Hot Pepper Provides Insights into the Evolution of Pungency in Capsicum Species. Nature Genetics, 46, 270-278.

[28]   Joshi, C.P. and Chiang, V.L. (1998) Conserved Sequence Motifs in Plant S-Adenosyl-L-Methionine-Dependent Methyltransferases. Plant Molecular Biology, 37, 663-674.

[29]   Ibrahim, R.K., Bruneau, A. and Bantignies, B. (1998) Plant O-Methyltransferases: Molecular Analysis, Common Signature and Classification. Plant Molecular Biology, 36, 1-10.

[30]   Scalliet, G., Journot, N., Jullien, F., Baudino, S., Magnard, J.L., Channelière, S., Vergne, P., Dumas, C., Bendahmane, M., Cock, J.M. and Hugueney, P. (2002) Biosynthesis of the Major Scent Components 3,5-Dimethoxytoluene and 1,3,5-Trimethoxybenzene by Novel Rose O-Methyltransferases. FEBS Letters, 523, 113-118.

[31]   Kothari, S.L., Joshi, A., Kachhwaha, S. and Ochoa-Alejo, N. (2010) Chilli Peppers—A Review on Tissue Culture and Transgenesis. Biotechnology Advances, 28, 35-48.

[32]   Napoli, C., Lemieux, C. and Jorgensen, R. (1990) Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in Trans. The Plant Cell Online, 2, 279-289.

[33]   Van Der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N.M. and Stuitje, A.R. (1990) Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression. The Plant Cell Online, 2, 291-299.

[34]   Fukusaki, E.I., Kawasaki, K., Kajiyama, S., An, C.I., Suzuki, K., Tanaka, Y. and Kobayashi, A. (2004) Flower Color Modulations of Torenia hybrida by Downregulation of Chalcone Synthase Genes with RNA Interference. Journal of Biotechnology, 111, 229-240.

[35]   Kurauchi, T., Matsumoto, T., Taneda, A., Sano, T. and Senda, M. (2009) Endogenous Short Interfering RNAs of Chalcone Synthase Genes Associated with Inhibition of Seed Coat Pigmentation in Soybean. Breed Science, 59, 419-426.

[36]   Tuteja, J.H., Zabala, G., Varala, K., Hudson, M. and Vodkin, L.O. (2009) Endogenous, Tissue-Specific Short Interfering RNAs Silence the Chalcone Synthase Gene Family in Glycine max Seed Coats. Plant Cell, 21, 3063-3077.

[37]   Ohno, S., Hosokawa, M., Kojima, M., Kitamura, Y., Hoshino, A., Tatsuzawa, F., Doi, M. and Yazawa, S. (2011) Simultaneous Post-Transcriptional Gene Silencing of Two Different Chalcone Synthase Genes Resulting in Pure White Flowers in the Octoploid Dahlia. Planta, 234, 945-958.