Comparative transcriptome analysis of purple-fleshed sweet potato provides insights into the molecular mechanism of anthocyanin biosynthesis

Abstract

Sweet potato, Ipomoea batatas, is a globally important food crop. The purple-fleshed sweet potato, rich in anthocyanins, has great potential for both nutritional and pharmaceutical uses. In this study, we characterized the root transcriptomes of the purple-fleshed sweet potato cv. Jingshu 6 and its mutant JS6-5 with high anthocyanin content by high-throughput RNA sequencing. A total of 22873364 and 27955097 high quality reads were obtained from Jingshu 6 and JS6-5, respectively, and assembled into 35592 unigenes. In all, we obtained 1566 differentially expressed genes (DEGs). Among them, 994 were upregulated and 572 were downregulated in JS6-5 compared to the expression in Jingshu 6. A total of 1436 DEGs were annotated, in which 847 DEGs had gene ontology (GO) terms and 329 DEGs were assigned to 84 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Most importantly, 23 differentially expressed genes and 24 transcription factors were identified as candidate genes involved in anthocyanin biosynthesis. In addition, 2349 SSRs were detected. This study not only provides the candidate genes but also provides insights into the molecular mechanism of anthocyanin biosynthesis in sweet potato.

Keywords

anthocyanin / gene expression / mutant / purple-fleshed sweet potato / transcriptome

References

[1] Loebenstein G, Thottappilly G. The Sweetpotato. 11nd ed. Berlin: Springer Netherlands, 2009, 189–234

[2] Holton T A, Cornish E C. Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell, 1995, 7(7): 1071–1083

[3] Wang L, Zhao Y, Zhou Q, Luo C L, Deng A P, Zhang Z C, Zhang J L. Characterization and hepatoprotective activity of anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8). Journal of Food and Drug Analysis, 2017, 25(3): 607–618

[4] Yoshimoto M, Okuno S, Yoshinaga M, Yamakawa O, Yamaguchi M, Yamada J. Antimutagenicity of sweetpotato (Ipomoea batatas) roots. Nippon Nogeikagaku Kaishi, 1999, 63(3): 537–541

[5] Matsui T, Ebuchi S, Kobayashi M, Fukui K, Sugita K, Terahara N, Matsumoto K. Anti-hyperglycemic effect of diacylated anthocyanin derived from Ipomoea batatas cultivar Ayamurasaki can be achieved through the alpha-glucosidase inhibitory action. Journal of Agricultural and Food Chemistry, 2002, 50(25): 7244–7248

[6] Johnson M, Pace R D. Sweet potato leaves: properties and synergistic interactions that promote health and prevent disease. Nutrition Reviews, 2010, 68(10): 604–615

[7] Lattanzio V, Cardinali A, Linsalata V. Recent Advances in Polyphenol Research, Volume 3. 6nd ed. New Jersey: Wiley-Blackwell, 2012, 143–160

[8] Borevitz J O, Xia Y, Blount J, Dixon R A, Lamb C. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell, 2000, 12(12): 2383–2394

[9] Gonzalez A, Zhao M, Leavitt J M, Lloyd A M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant Journal, 2008, 53(5): 814–827

[10] Winkel-Shirley B. Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiologia Plantarum, 2002, 107(1): 142–149

[11] Guo J, Zhou W, Lu Z, Li H, Li H, Gao F. Isolation and functional analysis of chalcone isomerase gene from purple-fleshed sweet potato. Plant Molecular Biology Reporter, 2015, 33(5): 1451–1463

[12] Zhou W, Gong Y, Lu X, Huang C, Gao F. Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas). Molecular Biology Reports, 2012, 39(1): 295–302

[13] Wang H, Fan W, Li H, Yang J, Huang J, Zhang P. 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, 2013, 8(11): e78484

[14] Zhou W, Huang C, Gong Y, Feng Q, Gao F. Molecular cloning and expression analysis of an ANS gene encoding anthocyanidin synthase from purple-fleshed sweet potato (Ipomoea batatas (L.) Lam). Molecular Biology Reports, 2009, 37(5): 2525–2531

[15] Tanaka M, Takahata Y, Kurata R, Nakayama H, Yoshinaga M. Structural and functional characterization of IbMYB1 genes in recent Japanese purple-fleshed sweetpotato cultivars. Molecular Breeding, 2012, 29(3): 565–574

[16] Dong W, Niu L, Gu J, Gao F. Isolation of a WD40-repeat gene regulating anthocyanin biosynthesis in storage roots of purple-fleshed sweet potato. Acta Physiologiae Plantarum, 2014, 36(5): 1123–1132

[17] Lalusin A G, Nishita K, Kim S H, Ohta M, Fujimura T. A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin. Molecular Genetics and Genomics, 2006, 275(1): 44–54

[18] Tao X, Gu Y H, Wang H Y, Zheng W, Li X, Zhao C W, Zhang Y Z. Digital gene expression analysis based on integrated de novo transcriptome assembly of sweet potato [Ipomoea batatas (L.) Lam]. PLoS One, 2012, 7(4): e36234

[19] Tao X, Gu Y H, Jiang Y S, Zhang Y Z, Wang H Y. Transcriptome analysis to identify putative floral-specific genes and flowering regulatory-related genes of sweet potato. Bioscience, Biotechnology, and Biochemistry, 2013, 77(11): 2169–2174

[20] Wang Z, Fang B, Chen X, Liao M, Chen J, Zhang X, Huang L, Luo Z, Yao Z, Li Y. Temporal patterns of gene expression associated with tuberous root formation and development in sweetpotato (Ipomoea batatas). BMC Plant Biology, 2015, 15(1): 180–192

[21] Qin Z, Li A, Hou F, Wang Q, Dong S, Zhang L. Gene identification using RNA-seq in two sweetpotato genotypes and the use of mining to analyze carotenoid biosynthesis. South African Journal of Botany, 2017, 109: 189–195

[22] Wang Z, Fang B, Chen J, Zhang X, Luo Z, Huang L, Chen X, Li Y. De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas). BMC Genomics, 2010, 11(1): 726–739

[23] Kou M, Xu J L, Li Q, Liu Y J, Wang X, Tang W, Yan H, Zhang Y G, Ma D F. Development of SNP markers using RNA-seq technology and tetra-primer ARMS-PCR in sweetpotato. Journal of Integrative Agriculture, 2017, 16(2): 464–470

[24] Xie F, Burklew C E, Yang Y, Liu M, Xiao P, Zhang B, Qiu D. De novo sequencing and a comprehensive analysis of purple sweet potato (Impomoea batatas L.) transcriptome. Planta, 2012, 236(1): 101–113

[25] Ma P, Bian X, Jia Z, Guo X, Xie Y. De novo sequencing and comprehensive analysis of the mutant transcriptome from purple sweet potato (Ipomoea batatas L.). Gene, 2016, 575(2): 641–649

[26] Grabherr M G, Haas B J, Yassour M, Levin J Z, Thompson D A, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren B W, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology, 2011, 29(7): 644–652

[27] Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J. WEGO: a web tool for plotting GO annotations. Nucleic Acids Research, 2006, 34(Web Server issue): W293–W297

[28] Conesa A, Götz S, García-Gómez J M, Terol J, Talón M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 2005, 21(18): 3674–3676

[29] Schmittgen T D, Livak K J. Analyzing real-time PCR data by the comparative C_T method. Nature Protocols, 2008, 3(6): 1101–1108

[30] Yang Q, Reinhard K, Schiltz E, Matern U. Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L. Plant Molecular Biology, 1997, 35(6): 777–789

[31] Hu X M, Shi C Y, Liu X, Jin L F, Liu Y Z, Peng S A. Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization. Molecular Genetics and Genomics, 2015, 290(1): 29–38

[32] Su D, Asard H. Three mammalian cytochromes b561 are ascorbate-dependent ferrireductases. FEBS Journal, 2006, 273(16): 3722–3734

[33] Page M, Sultana N, Paszkiewicz K, Florance H, Smirnoff N. The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis. Plant, Cell & Environment, 2012, 35(2): 388–404

[34] Jaakola L, Poole M, Jones M O, Kämäräinen-Karppinen T, Koskimäki J J, Hohtola A, Häggman H, Fraser P D, Manning K, King G J, Thomson H, Seymour G B. A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant Physiology, 2010, 153(4): 1619–1629

[35] Zhou H, Lin-Wang K, Wang H, Gu C, Dare A P, Espley R V, He H, Allan A C, Han Y. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors. Plant Journal, 2015, 82(1): 105–121

[36] Morishita T, Kojima Y, Maruta T, Nishizawa-Yokoi A, Yabuta Y, Shigeoka S. Arabidopsis NAC transcription factor, ANAC078, regulates flavonoid biosynthesis under high-light. Plant & Cell Physiology, 2009, 50(12): 2210–2222

[37] Yao G, Ming M, Allan A C, Gu C, Li L, Wu X, Wang R, Chang Y, Qi K, Zhang S, Wu J. Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis. Plant Journal, 2017, 92(3): 437–451

[38] Shin D H, Choi M, Kim K, Bang G, Cho M, Choi S B, Choi G, Park Y I. HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis. FEBS Letters, 2013, 587(10): 1543–1547

[39] Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Molecular Breeding, 1996, 2(3): 225–238

Supplementary materials

The online version of this article at https://doi.org/10.15302/J-FASE-2018219 contains supplementary materials (Tables S1–S2; Figs. S1–S2).

Acknowledgements

This work was supported by National Natural Science Foundation of China (31271777; 31461143017), China Agriculture Research System (CARS-11), and Science and Technology Planning Project of Guangdong Province, China (2015B020202008).

Compliance with ethics guidelines

Hongyuan Zhao, Shanshan Zhang, Feibing Wang, Ning Zhao, Shaozhen He, Qingchang Liu, and Hong Zhai declare that they have no conflicts of interest or financial conflicts to disclose.

This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2018. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)