Abstract
Sweet potato [Ipomoea batatas (L.) Lam.] is a vegetable crop with significant economic value that is utilized both as a staple food and as an industrial raw material. Sweet potato possesses a variety of healthful beneficial secondary metabolites, which also contribute towards its adaptation to the environment and capacity to withstand stresses. Cinnamate 4-hydroxylase constitutes a primary enzyme in the phenylpropanoid pathway that is involved in the biosynthesis of numerous secondary metabolites. Elucidating the molecular and expressional characteristics of C4H is essential for understanding the functional genetic diversity of sweet potato germplasms. Based on the transcriptome analysis of five sweet potato genotypes with different anthocyanin contents, a cinnamate 4-hydroxylase (C4H) gene, termed IbC4H, was isolated from a purple sweet potato cv. “Xuzishu 3”. The cDNA sequence of IbC4H contained a 1518-bp ORF encoding a 505 amino acid protein with a molecular weight of 58.15 kDa. Genomic DNA analysis revealed that the IbC4H gene possessed three exons and two introns. The IbC4H protein shared 86.34–97.82% sequence identity with that reported for other plants. A significant positive correlation between the expression of IbC4H and phenolic acid content suggested that IbC4H might be integral to the regulation of phenolic biosynthesis in sweet potato. Furthermore, IbC4H was significantly upregulated in the young leaves and roots of cv. “Xuzishu 3” and was strongly induced when subjected to drought stress. Drought stress tolerance in transgenic tobacco plants was significantly improved by the heterologous expression of the transgene IbC4H, which was simultaneously associated with the increased expression of stress-related genes, higher superoxide dismutase (SOD) activity, and proline (Pro) content, along with lower malondialdehyde (MDA) production. Collectively, these results highlight the important role of IbC4H in the phenylpropanoid pathway and support the protective function of polyphenols in the improved scavenging of reactive oxygen species of plants under stress.
Similar content being viewed by others
References
An CH et al (2015) Heterologous expression of IbMYB1a by different promoters exhibits different patterns of anthocyanin accumulation in tobacco. Plant Physiol Biochem 89C:1–10
Artés-Hernández F, Rivera-Cabrera F, Kader AA (2007) Quality retention and potential shelf-life of fresh-cut lemons as affected by cut type and temperature. Postharvest Biol Technol 43:245–254
Bassard JE et al (2012) Protein–protein and protein–membrane associations in the lignin pathway. Plant Cell 24:4465–4482
Bursal E, Gülçin İ (2011) Polyphenol contents and in vitro antioxidant activities of lyophilised aqueous extract of kiwifruit (Actinidia deliciosa). Food Res Int 44:1482–1489
Cheng YJ, Kim MD, Deng XP, Kwak SS, Chen W (2013) Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor. J Microbiol Biotechnol 23:1737–1746
Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7:1085–1097
Dixon RA, Achnine L, Kota P, Liu CJ, Reddy M, Wang L (2002) The phenylpropanoid pathway and plant defence—a genomics perspective. Mol Plant Pathol 3:371–390
Dong W, You Y, Niu L, Gao F (2014) Isolation and analysis of the promoter of an anthocyanin synthase gene from purple-fleshed sweet potato tubers. Acta Physiol Plant 36:2637–2649
Gotoh O (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 267:83–90
Gowri G, Bugos RC, Campbell WH, Maxwell CA, Dixon RA (1991) Stress responses in alfalfa (Medicago sativa L.) X. Molecular cloning and expression of S-adenosyl-l-methionine: caffeic acid 3-O-methyltransferase, a key enzyme of lignin biosynthesis. Plant Physiol 97:7–14
Gravot ALR, Hehn A, Lievre K, Gontier E, Goergen JBF (2004) Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation. Arch Biochem Biophys 422:71–80
Guo J, Zhou W, Lu Z, Li H, Li H, Gao F (2015) Isolation and functional analysis of chalcone isomerase gene from purple-fleshed sweet potato. Plant Mol Biol Report 33:1451–1463
Han C, Li J, Jin P, Li X, Wang L, Zheng Y (2017) The effect of temperature on phenolic content in wounded carrots. Food Chem 215:116–123
Hotze M, Schröder G, Schröder J (1995) Cinnamate 4-hydroxylase from Catharanthus roseus and a strategy for the functional expression of plant cytochrome P 450 proteins as translational fusions with P 450 reductase in Escherichia coli. FEBS Lett 374:345–350
Hu M, Lu Z, Guo J, Luo Y, Li H, Li L, Gao F (2016) Cloning and characterization of the cDNA and promoter of UDP-glucose: flavonoid 3-O-glucosyltransferase gene from a purple-fleshed sweet potato. South Afr J Bot 106:211–220
Hundertmark M, Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genom 9:1–22
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Report 5:387–405
Ji CY et al (2016) Molecular characterization of tocopherol biosynthetic genes in sweet potato that respond to stress and activate the tocopherol production in tobacco. Plant Physiol Biochem 106:118–128
Jia L et al (2012) Proanthocyanidins inhibit seed germination by maintaining a high level of abscisic acid in Arabidopsis thaliana. J Integr Plant Biol 54:663–673
Kim SH, Hamada T (2005) Rapid and reliable method of extracting DNA and RNA from sweet potato, Ipomoea batatas (L). Lam. Biotechnol Lett 27:1841–1845
Kim J, Choi B, Natarajan S, Bae H (2013) Expression analysis of kenaf cinnamate 4-hydroxylase (C4H) ortholog during developmental and stress responses. Plant Omics 6:65–72
Koopmann E, Logemann E, Hahlbrock K (1999) Regulation and functional expression of cinnamate 4-hydroxylase from parsley. Plant Physiol 119:49–56
Kumar S, Omer S, Patel K, Khan BM (2013) Cinnamate 4-hydroxylase (C4H) genes from Leucaena leucocephala: a pulp yielding leguminous tree. Mol Biol Rep 40:1265–1274
Kumar R et al (2016) RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua. Sci Rep 6:e26458–e26470
Kusumi K, Mizutani A, Nishimura M, Iba K (1997) A virescent gene V1 determines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice. Plant J 12:1241–1250
Lalusin AG, Nishita K, Kim SH, Ohta M, Fujimura T (2006) A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin. Mol Genet Genom 275:44–54
Li JB, Luan YS, Jin H (2012) The tomato SlWRKY gene plays an important role in the regulation of defense responses in tobacco. Biochem Biophys Res Commun 427:671–676
Li W, Yang L, Jiang L, Zhang G, Luo Y (2016) Molecular cloning and functional characterization of a cinnamate 4-hydroxylase-encoding gene from Camptotheca acuminata. Acta Physiol Plant 38:256
Liu K, Wang L, Xu Y, Chen N, Ma Q, Li F, Chong K (2007) Overexpression of OsCOIN, a putative cold inducible zinc finger protein, increased tolerance to chilling, salt and drought, and enhanced proline level in rice. Planta 226:1007–1016
Liu D, Wang L, Liu C, Song X, He S, Zhai H, Liu Q (2014) An Ipomoea batatas iron-sulfur cluster scaffold protein gene, IbNFU1, is involved in salt tolerance. PLoS One 9:e93935
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time puantitative PCR and the 2−ΔΔCT method. Methods 25:402–408
Luo C, Wang X, Gao G, Wang L, Li Y, Sun C (2013) Identification and quantification of free, conjugate and total phenolic compounds in leaves of 20 sweet potato cultivars by HPLC-DAD and HPLC-ESI-MS/MS. Food Chem 141:2697–2706
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
Matsui T, Ebuchi S, Kobayashi M, Fukui K, Sugita K, Terahara N, Matsumoto K (2002) Anti-hyperglycemic effect of diacylated anthocyanin derived from Ipomoea batatas cultivar Ayamurasaki can be achieved through the alpha-glucosidase inhibitory action. J Agric Food Chem 50:7244–7248
Millar DJ et al (2007) Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids. Phytochemistry 68:1497–1509
Mizutani M, Ohta D (2010) Diversification of P450 genes during land plant evolution. Plant Biol 61:291–315
Mizutani M, Ward E, Dimaio J, Ohta D, Ryals J, Sato R (1993) Molecular cloning and sequencing of a cDNA encoding mung bean cytochrome P450 (P450C4H) possessing cinnamate 4-hydroxylase activity. Biochem Biophys Res Commun 190:875–880
Mizutani M, Ohta D, Sato R (1997) Isolation of a cDNA and a genomic clone encoding cinnamate 4-hydroxylase from Arabidopsis and its expression manner in planta. Plant Physiol 113:755–765
Nelson D, Werck-Reichhart D (2011) A P450-centric view of plant evolution. Plant J Cell Mol Biol 66:194–211
Ortiz DM, Paul R (1996) Book reviews: cytochrome P450. Structure, mechanism, and biochemistry. Science 271:1507–1508
Park JH, Park NI, Xu H, Park SU (2010) Cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase and pyranocoumarin biosynthesis in Angelica gigas. J Nat Prod 73:1394–1397
Park SC et al (2015) Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity. Physiol Plant 153:525–537
Pei X, Guo H, Wan F, Cheng H (2012) Phenylalanine ammonia-lyase and cinnamate 4-hydroxylase genes’ response to HHO in Eupatorium adenophorum. Weed Biol Manag 12:177–182
Pereira DM, Valentão P, Pereira JA, Andrade PB (2009) Phenolics: from chemistry to biology. Molecules 14:2202–2211
Phimchan P, Chanthai S, Bosland PW, Techawongstien S (2014) Enzymatic changes in phenylalanine ammonia-lyase, cinnamic-4-hydroxylase, capsaicin synthase, and peroxidase activities in capsicum under drought stress. J Agric Food Chem 62:7057–7062
Ro DK, Mah N, Ellis BE, Douglas CJ (2001) Functional characterization and subcellular localization of poplar (Populus trichocarpa x Populus deltoides) cinnamate 4-hydroxylase. Plant Physiol 126:317–329
Runeckles V, Conn E (1974) Metabolism and regulation of secondary plant products. Recent Adv Phytochem 8:249
Sadeghi M, Dehghan S, Fischer R, Wenzel U, Vilcinskas A, Kavousi HR, Rahnamaeian M (2013) Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress, wounding, and salicylic acid treatment in. Plant Signal Behav 8:2420–2423
Sakaguchi M, Mihara K, Sato R (1987) A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. EMBO J 6:2425–2431
Schilmiller AL, Stout J, Weng JK, Humphreys J, Ruegger MO, Chapple C (2009) Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis. Plant J 60:771–782
Shekhar S, Mishra D, Buragohain AK, Chakraborty S, Chakraborty N (2015) Comparative analysis of phytochemicals and nutrient availability in two contrasting cultivars of sweet potato (Ipomoea batatas L.). Food Chem 173:957–965
Singh K, Kumar S, Rani A, Gulati A, Ahuja PS (2009) Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) and catechins (flavan-3-ols) accumulation in tea. Funct Integr Genom 9:125–134
Suda I et al (2002) Direct absorption of acylated anthocyanin in purple-fleshed sweet potato into rats. J Agric Food Chem 50:1672–1676
Torres-Contreras AM, Nair V, Cisneros-Zevallos L, Jacobo-Velázquez DA (2014) Plants as biofactories: stress-induced production of chlorogenic acid isomers in potato tubers as affected by wounding intensity and storage time. Ind Crops Prod 62:61–66
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–e78484
Wise AA, Liu Z, Binns AN (2006) Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol Biol 343:43–54
Xu H, Park NI, Li X, Kim YK, Lee SY, Park SU (2010) Molecular cloning and characterization of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis. Biores Technol 101:9715–9722
Yang CQ, Lu S, Mao YB, Wang LJ, Chen XY (2010) Characterization of two NADPH: cytochrome P450 reductases from cotton (Gossypium hirsutum). Phytochemistry 71:27–35
Yoshimoto M, Okuno S, Yoshinaga M, Yamakawa O, Yamaguchi M, Yamada J (1999) Antimutagenicity of sweet potato (Ipomoea batatas) roots. Biosci Biotechnol Biochem 63:537–541
Yue Y, Zhang M, Zhang J, Tian X, Duan L, Li Z (2012) Overexpression of the AtLOS5 gene increased abscisic acid level and drought tolerance in transgenic cotton. J Exp Bot 63:3741–3748
Zhai H et al (2016) A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato. Plant Biotechnol J 14:592–602
Zhao Z, Moghadasian MH (2008) Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review. Food Chem 109:691–702
Zhao Z, Egashira Y, Sanada H (2003) Ferulic acid sugar esters are recovered in rat plasma and urine mainly as the sulfoglucuronide of ferulic acid. J Nutr 133:1355–1361
Ziebell A, Gjersing E, Hinchee M, Katahira R, Sykes R, Johnson D, Davis M (2015) Down-regulation of p-coumaroyl quinate/shikimate 3′-hydroxylase (C3′ H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla × E. grandis leads to improved sugar release. Biotechnol Biofuels 8(1):128
Acknowledgements
We appreciate Dr. Dapeng Zhang (The U.S. Department of Beltsville Agricultural Research Center) for reading and editing this manuscript. This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Research Innovation Program for College Graduates of Jiangsu Province (Grant no. KYZZ15-0388), the China Agriculture Research System (CARS-11), the Key Research and Development Plan of Jiangsu Province (BE2015313).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by R. Baczek-Kwinta.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wang, A., Zhu, M., Luo, Y. et al. A sweet potato cinnamate 4-hydroxylase gene, IbC4H, increases phenolics content and enhances drought tolerance in tobacco. Acta Physiol Plant 39, 276 (2017). https://doi.org/10.1007/s11738-017-2551-1
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11738-017-2551-1