Abstract
Chalcone isomerase (CHI, EC 5.5.1.6) is one of the essential enzymes in the anthocyanin biosynthetic pathway catalyzing the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones. A full-length cDNA of CHI (844 bp in length) was isolated from purple-fleshed sweet potato [Ipomoea batatas (L.) Lam] cv. Yamakawamurasaki, designated IbCHI, containing a 732-bp open reading frame encoding a 243 amino acid polypeptide. Sequence alignment showed that the coding region of IbCHI gene is highly homologous with CHIs from other plant species and has four conserved amino acid residues (Thr50, Tyr108, Asn115, and Ser192) essential for CHI active sites. Phylogenetic analysis revealed that IbCHI was clustered into type I CHI group. Southern blotting showed that there were multicopies of IbCHI gene in I. batatas genome and IbCHI gene showed significant restriction fragment length polymorphism among different I. batatas cultivars. Overexpression of the IbCHI gene in Arabidopsis tt5 mutant fully complemented the pigmentation phenotype of the seed coat, cotyledon, and hypocotyl, indicating that the IbCHI gene encoded a fully functional CHI protein for anthocyanin and proanthocyanidin biosynthesis. The expression patterns of IbCHI and other anthocyanin biosynthesis-related genes (IbCHS, IbF3H, IbDFR, and IbANS) matched the accumulation patterns of anthocyanin in the thick roots and mature storage roots of different cultivars of sweet potato. However, in the fibrous roots, only IbCHI expressed obviously higher in purple-fleshed cultivar than white-fleshed cultivar, while IbCHS and IbDFR exhibited the opposite expression trends, and IbF3H and IbANS expressed almost equally in purple- and white-fleshed cultivars. The findings suggest that IbCHI is a key enzyme in the anthocyanin biosynthetic pathway of sweet potato, which is responsible for the activation of anthocyanin biosynthesis in the early stage of root development.
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Aza-González C, Herrera-Isidrón L, Núñez-Palenius HG, Martínez De La Vega O, Ochoa-Alejo N (2013) Anthocyanin accumulation and expression analysis of biosynthesis-related genes during chili pepper fruit development. Biol Plant 57:49–55. doi:10.1007/s10535-012-0265-1
Chen PN, Chu SC, Chiou LH, Kuo WH, Chiang CL, Hsieh YS (2006) Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line. Cancer Lett 235:248–259. doi:10.1016/j.canlet.2005.04.033
Cheng H, Li L, Cheng S, Cao F, Wang Y, Yuan H (2011) Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba. Plant Cell Rep 30:49–62. doi:10.1007/s00299-010-0943-4
Cretu GC, Morlock GE (2014) Analysis of anthocyanins in powdered berry extracts by planar chromatography linked with bioassay and mass spectrometry. Food Chem 146:104–112. doi:10.1016/j.foodchem.2013.09.038
Dixon RA, Blyden ER, Robhins MP, van Tunen AJ, Mol JN (1988) Comparative biochemistry of chalcone isomerases. Phytochemistry 27:2801–2808. doi:10.1016/0031-9422(88)80666-6
Dong X, Braun EL, Grotewold E (2001) Functional conservation of plant secondary metabolic enzymes revealed by complementation of Arabidopsis flavonoid mutants with maize genes. Plant Physiol 127:46–57. doi:10.1104/pp. 127.1.46
Druka A, Kudrna D, Rostoks N, Brueggeman R, Von Wettstein D, Kleinhofs A (2003) Chalcone isomerase from rice (0ryza sativa) and barley (Hordeum vulgare): physical, genetic and mutation mapping. Gene 302:171–178. doi:10.1016/S0378-1119(02)01105-8
Felsenstein J (1992) Estimating effective population size from samplesof sequences: a bootstrap Monte Carlo integration method. Genet Res 60:209–220. doi:10.1017/S0016672300030962
Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53:814–827. doi:10.1111/j.1365-313X.2007.03373.x
Grotewold E, Peterson T (1994) Isolation and characterization of a maize gene encoding chalcone flavonone isomerase. Mol Gen Genet 242:1–8. doi:10.1007/BF00277341
Guo HW, Ling WH, Wang Q, Liu C, Hu Y, Xia M, Feng X, Xia X (2007) Effect of anthocyanin-rich extract from black rice on hyperlipidemia and insulin resistance in fructose-fed rats. Plant Food Hum Nutr 62:1–6. doi:10.1007/s11130-006-0031-7
Holton TA, Cornish E (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083. doi:10.1105/tpc.7.7.1071
Jeong ST, Goto-Yamamoto N, Hashizume K, Esaka M (2008) Expression of multi-copy flavonoid pathway genes coincides with anthocyanin, flavonol and flavan-3-ol accumulation of grapevine. Vitis 47:135–140
Jez JM, Noel JP (2002) Reaction mechanism of chalcone isomerase. pH dependence, diffusion control, and product binding differences. J Biol Chem 277:1361–1369. doi:10.1074/jbc.M109224200
Jez JM, Bowman ME, Dixon RA, Noel JP (2000) Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nat Struct Biol 7:786–791. doi:10.1038/79025
Jez JM, Bowman ME, Noel JP (2002) Role of hydrogen bonds in the reaction mechanism of chalcone isomerase. Biochemistry 41:5168–5176. doi:10.1021/bi0255266
Kim SH, Hamada T (2005) Rapid and reliable method of extracting DNA and RNA from sweetpotato, Ipomoea batatas (L). Lam. Biotechnol Lett 27:1841–1845. doi:10.1007/s10529-005-3891-2
Kowalczyk E, Krzesinski P, Kura M, Szmigiel B, Blaszczyk J (2003) Anthocyanins in medicine. Pol J Pharmacol 55:699–702
Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245. doi:10.1093/bioinformatics/17.12.1244
Li F, Jin Z, Qu W, Zhao D, Ma F (2006) Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco. Plant Physiol Biochem 44:455–461. doi:10.1016/j.plaphy.2006.08.006
Li J, Li XD, Zhang Y, Zheng ZD, Qu ZY, Liu M, Zhu SH, Liu S, Wang M, Qu L (2013) Identification and thermal stability of purple-fleshed sweet potato anthocyanins in aqueous solutions with various pH values and fruit juices. Food Chem 136:1429–1434. doi:10.1016/j.foodchem.2012.09.054
Mano H, Ogasawara F, Sato K, Higo H, Minobe Y (2007) Isolation of a regulatory gene of anthocyanin biosynthesis in tuberous roots of purple-fleshed sweet potato. Plant Physiol 143:1252–1268. doi:10.1104/pp. 106.094425
Mckhann HI, Hirsch AM (1994) Isolation of chalcone synthase and chalcone isomerase cDNAs from alfalfa (Medicago sativa L.): highest transcript levels occur in young roots and root tips. Plant Mol Biol 24:767–777. doi: 10.1007/BF00029858
Mol J, Grotewold E, Koes R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217. doi:10.1016/S1360-1385(98)01242-4
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Satué-Gracia MT, Heinonen N, Frankel EN (1997) Anthocyanins as antioxidants on human low density lipoprotein and lecithin liposome systems. J Agric Food Chem 45:3362–3367. doi:10.1021/jf970234a
Shimada N, Aoki T, Sato S, Nakamura Y, Tabata S, Ayabe S (2003) A cluster of genes encodes the two types of chalcone isomerase involved in the biosynthesis of general flavonoids and legume-specific 5-deoxy(iso)flavonoids in Lotus japonicus. Plant Physiol 131:941–951. doi:10.1104/pp. 004820
Shoeva OY, Khlestkina EK, Berges H, Salina EA (2014) The homoeologous genes encoding chalcone–flavanone isomerase in Triticum aestivum L.: structural characterization and expression in different parts of wheat plant. Gene 538:334–341. doi:10.1016/j.gene.2014.01.008
Springob K, Nakajima J, Yamazaki M, Saito K (2003) Recent advances in the biosynthesis of anthocyanins. Nat Prod Rep 20:288–303. doi:10.1039/b109542k
Van Tunen AJ, Koes RE, Spelt CE, Van der Krol AR, Stuitje AR, Mol JN (1988) Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMBO J 7:1257–1263
Wang H, Fan W, Li H, Yang J, Huang J, Zhang P (2013) Functional characterization of dihydroflavonol-4-reductase in anthocyanin biosynthesis of purple sweet potato underlies the direct evidence of anthocyanins function against abiotic stresses. PLoS One 8:e78484. doi:10.1371/journal.pone.0078484
Wei YZ, Hu FC, Hu GB, Li XJ, Huang XM, Wang HC (2011) Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn. PLoS One 6:e19455. doi:10.1371/journal.pone.0019455
Weisel T, Baum M, Eisenbrand G, Dietrich H, Frank W, Stockis JP, Kulling S, Rüfer C, Johannes C, Janzowski C (2006) An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands. Biotechnol J 1:388–397. doi:10.1002/biot.200600004
Wood AJ, Davies E (1994) A cDNA encoding chalcone isomerase from aged pea epicotyls. Plant Physiol 104:1465–1466. doi:10.1104/pp. 104.4.1465
Wu DM, Lu J, Zheng YL, Zhou Z, Shan Q, Ma DF (2008) Purple sweet potato color repairs D-galactose-induced spatial learning and memory impairment by regulating the expression of synaptic proteins. Neurobiol Learn Mem 90:19–27. doi:10.1016/j.nlm.2008.01.010
Yang Y, Zhao G, Yue W, Zhang S, Gu C, Wu J (2013) Molecular cloning and gene expression differences of the anthocyanin biosynthesis-related genes in the red/green skin color mutant of pear (Pyrus communis L.). Tree Genet Genomes 9:1351–1360. doi:10.1007/s11295-013-0644-6
Yoshimoto M, Okuno S, Yoshinaga M, Yamakawa O, Yamaguchi M, Yamada J (1999) Antimutagenicity of sweetpotato (Ipomoea batatas) roots. Biosci Biotechnol Biochem 63:537–541. doi:10.1271/bbb.63.537
Yuan Y, Ma X, Shi Y, Tang D (2013) Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana. Sci Hortic-Amst 153:93–102. doi:10.1016/j.scienta.2013.02.008
Zhao D, Tao J, Han C, Ge J (2012) Flower color diversity revealed by differential expression of flavonoid biosynthetic genes and flavonoid accumulation in herbaceous peony (Paeonia lactiflora Pall.). Mol Biol Rep 39:11263–11275. doi:10.1007/s11033-012-2036-7
Zhou W, Huang C, Gong Y, Feng Q, Gao F (2010) Molecular cloning and expression analysis of an ANS gene encoding anthocyanidin synthase from Purple-fleshed sweet potato [Ipomoea batatas(L.) Lam]. Plant Mol Biol Rep 28:112–121. doi:10.1007/s11105-009-0133-0
Zhou W, Gong Y, Lu X, Huang C, Gao F (2012) Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas). Mol Biol Rep 39:295–302. doi:10.1007/s11033-011-0738-x
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This work was funded by China National Natural Science Foundation (31171601)
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Guo, J., Zhou, W., Lu, Z. et al. Isolation and Functional Analysis of Chalcone Isomerase Gene from Purple-Fleshed Sweet Potato. Plant Mol Biol Rep 33, 1451–1463 (2015). https://doi.org/10.1007/s11105-014-0842-x
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DOI: https://doi.org/10.1007/s11105-014-0842-x