Full Text - Isolation of a UDP-glucose: Flavonoid5-O-glucosyltransferase gene and expression analysis of anthocyanin biosynthetic genes in herbaceous peony (Paeonia lactiflora Pall.)

Isolation of a UDP-glucose: Flavonoid 5-O-glucosyltransferase gene and expression analysis of anthocyanin biosynthetic genes in herbaceous peony (Paeonia lactiflora Pall.)

¹Yangzhou University, College of Horticulture and Plant Protection, Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou, P.R. China

Financial support: This work was supported by Agricultural Science & Technology Independent Innovation Fund of Jiangsu Province (CX[10]114, CX[11]3015, CX[11]1017, CX[12]4052), Agricultural Science & Technology Support Project of Jiangsu Province (BE2011325) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Herbaceous peony (Paeonia lactiflora Pall.) is an excellent material for studying the formation of flower colour because of its abundant colour. The full-length cDNA of a UDP-glucose: Flavonoid 5-O-glucosyltransferase gene (UF5GT) containing 1629 bp nucleotides was obtained from P. lactiflora. The expression patterns of nine related anthocyanin biosynthetic genes (PlPSY, PlCHS, PlCHI, PlF3H, PlF3'H, PlDFR, PlANS, PlUF3GT and PlUF5GT) in diurnal variation petals showed that their expression peaks were basically at 15:00 and the expression patterns were consistent with the trend of sampling conditions except individual gene. And the highest expression levels were in PlCHS, PlDFR and PlUF3GT, which could be the candidates to regulate P. lactiflora flower colour by means of genetic engineering.

Flower pigmentation is caused by the accumulation of pigments within the epidermal cells, which contains flavonoid, carotenoid and betacyanin (Mol et al. 1998). Among those, flavonoid is comprised of anthoxanthin (flavone and flavonol) and anthocyanin (Qi, 1989). And anthocyanin can assume a wide range of colours, therefore, more attention is paid by biochemists and breeders (Tanaka and Ohmiya, 2008; Tanaka et al. 2010). Till now, hundreds of different forms of anthocyanin have been isolated, and their structures have also been identified (Harborne and Williams, 2000; Fukuchi-Mizutani et al. 2003). Meanwhile, the anthocyanin biosynthetic pathway and related structural genes have been studied in many flower plants, such as azalea (Nakatsuka et al. 2008a), chrysanthemum (Chen et al. 2010), sunflower (Zhang et al. 2009) and so on, which provide part explanations for extensive variation of flower colours.

In anthocyanin biosynthetic pathway, L-phenylalanine is converted to naringenin by phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI). And then, the next pathway is catalyzed the formation of complex aglycone and anthocyanin composition by flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucoside: flavonoid glucosyltransferase (UFGT) and methyl transferase (MT). Among those, UFGT is divided into UF3GT and UF5GT, which are responsible for the glucosylation of anthocyanin to produce stable molecules (Li et al. 2001; Yamazaki et al. 2002). And the best-studied UFGT is only UF3GT, which is regarded as a key enzyme in anthocyanin biosynthesis (Kobayashi et al. 2001; Castellarin et al. 2007a, Castellarin et al. 2007b; Fukuchi-Mizutani et al. 2011; Hu et al. 2011). In contrast, there are only few reports on UF5GT. Studies show that UF5GT catalyzes glucosylation at the 5-hydroxyl position of anthocyanidin 3-O-glycoside, thereafter, it has been isolated from solanaceae (Vogt et al. 1999), asteraceae (Ogata et al. 2001), gentian (Nakatsuka et al. 2008b), etc.

Herbaceous peony (Paeonia lactiflora Pall.), a traditional famous flower in China, contains nine flower colours, which makes it an excellent material for studying the formation of flower colours (Wang and Zhang, 2005). In the studies of P. lactiflora anthocyanin compositions, Jia et al. (2008a) and Jia et al. (2008b) have identified eight anthocyanin including peonidin-3,5-di-O-glucoside, pelargonidin-3,5-di-O-glucoside, cyanidin-3,5-di-O-glucoside, peonidin-3-O-glucoside, cyanidin-3-O-glucoside, peonidin-3-O-gluco-side-5-O-arabinoside, cyanidin-3-O-glucoside-5-O-galactoside and pelargonidin-3-O-glucoside-5-O-galactoside. These compositions demonstrate that UF5GT is absolutely necessary in P. lactiflora. In the studies of P. lactiflora molecular biology, almost all of the structural genes involved in anthocyanin biosynthetic pathway have been isolated, but an important gene encoding UDP-glucose: Flavonoid 5-O-glucosyltransferase remains to be identified. In this study, the full-length sequence of UF5GT was isolated, and the diurnal expression patterns of nine anthocyanin biosynthetic genes in petals were examined. These would provide a basis for flower colour modification by engineering of the anthocyanin biosynthetic pathway.

Herbaceous peony was grown in the germplasm repository of Horticulture and Plant Protection College, Yangzhou University, Jiangsu Province, P.R. China (32º30' N, 119º25' E). The petals of P. lactiflora (cv. ‘Hongyanzhenghui') were sampled every 3 hrs from 6:00 to 21:00 in the flowering stage to study diurnal expression patterns of anthocyanin biosynthetic genes, and the sampling conditions were illustrated in Figure 1 which was measured by LI-6400 (LI-COR, USA). All samples were immediately frozen in liquid nitrogen, and then stored at -80ºC until analysis.

Total RNA was extracted according to a modified CTAB extraction protocol (Zhao et al. 2011). Prior to reverse-transcription, RNA samples were treated with DNase using DNase I kit (TaKaRa, Japan), according to the manufacturer's guidelines.

3' rapid-amplification of cDNA ends (RACE) primers were designed according to the retrieved UF5GT cDNA sequences of other plants from GenBank (Outer primer: 5'-CACAAGGCCACATAAACCC-3'; Inner primer: 5'-TGGATTGAACCGGCTAC-3'). And then on the basis of the 3' cDNA sequence, 5' RACE primer was designed (5'-TGAGAAACTGAGTCGCCACCGAAAGAA-3'). In gene expression analysis, the P. lactiflora Actin was used as an internal control (Zhao et al. 2012), and other gene expression analysis primers were together listed in Table 1.

Isolation of cDNA was performed by 3' full RACE Core Set Ver. 2.0 (TaKaRa, Japan) and SMARTer^TM RACE cDNA Amplification Kit (Clontech, Japan), and the specific operations were performed according to the manufacture's guidelines. PCR products were cloned and sent Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) to sequence.

Real-time quantitative polymerase chain reaction (Q-PCR) was performed on a BIO-RAD CFX96^TM Real-Time System (C1000^TM Thermal Cycler) (Bio-Rad, USA). The cDNA was synthesized from 1 µg RNA using PrimeScript^® RT reagent Kit With gDNA Eraser (TaKaRa, Japan). Q-PCR was carried out using the SYBR^® Premix Ex Taq^TM (Perfect Real Time) (TaKaRa, Japan) and gene relative expression levels were calculated by the 2^-△△Ct comparative threshold cycle (Ct) method (Schmittgen and Livak, 2008).

In this study, 3' and 5' RACE strategies using gene-specific primers for UF5GT gene resulted in an approximate 850 bp band of 3' and 5' cDNA ends, respectively. The spliced results showed that UF5GT cDNA had 1629 bp nucleotides in length which contained a 5'-untranslated region (UTR) of 106 bp, an open reading frame (ORF) of 1398 bp started with an ATG initiation codon and ended with a TGA stop codon, a 3'-UTR of 125 bp and a poly (A) tail, encoding 465 amino acid residues. In addition, this gene had been submitted to GenBank with accession number JQ070807.

Amino acid sequence analysis of UF5GT in P. lactiflora demonstrated that the putative molecular weight was 51.92 kDa, theoretical isoelectric point (pI) was 5.40, and total number of negatively charged residues (Asp + Glu) and positively charged residues (Arg + Lys) was 64 and 47, respectively. Its instability index (II) was computed to be 50.22 which classified this protein as unstable. This protein was comprised of several phosphorylation sites of cAMP- and cGMP-dependent protein kinase, protein kinase C, casein kinase II and Tyrosine kinase, other sites of N-glycosylation and N-myristoylation, and a UDP-glucosyltransferase signature. NCBI search in conserved domain database showed that this protein had a conserved domain of Glycosyltransferase_GTB_type superfamily. Meanwhile, homology analysis revealed that the similarities of amino acid sequences between P. lactiflora and other plants including Petunia x hybrida (BAA89009), Perilla frutescens (BAA36422.1), Glandularia x hybrida (BAA36423), Arabidopsis thaliana (NP_193146.1), Solanum melongena (BAF03079.1) were 56%, 54%, 53%, 51% and 50%, which were in conformity with homology matrix of 6 sequences (Table 2). And low similarity was also presented in other plants (Yamazaki et al. 2002; Nakatsuka et al. 2008b). Therefore, this sequence could be confirmed UF5GT gene in P. lactiflora, which was appointed PlUF5GT.

Phylogenetic tree shown in Figure 2 was drawn according to multiple sequence alignment of PlUF5GT and other UFGT genes by MEGA software. These genes could be classified into two types according to species: plant and bacterium, and plant also could be divided into UF3GT and UF5GT groups. Among those, PlUF5GT belonged to UF5GT group which had close relationship with Eustoma grandiflorum. These results were concordant with traditional classification.

Previous studies had shown that structural genes in plant anthocyanin biosynthesis pathway were specifically expressed which could be genetically manipulated to modify flower colour (Hu et al. 2009; Tanaka et al. 2010). In present study, in order to examine diurnal variation expression patterns of anthocyanin biosynthetic genes, i.e. PlPSY, PlCHS, PlCHI, PlF3H, PlF3'H, PlDFR, PlANS, PlUF3GT and PlUF5GT which had been submitted to GenBank by our laboratory, Q-PCR technology was performed using total RNA obtained from ‘Hongyanzhenghui' petals of 6 time points in the flowering stage. According to the expression patterns, these nine genes could be divided into three groups: the first group (PlPSY, PlCHS, PlCHI, PlF3H and PlANS) gradually increased with the elapse of time until 15:00, and then declined; the second group (PlF3'H and PlUF5GT) was throughout the whole day with a low level; the third group (PlDFR and PlUF3GT) was highly expressed in general, and that of afternoon was higher than that of forepart. When the relative expression level was concerned, the highest levels were in PlCHS, PlDFR and PlUF3GT genes, while the lowest levels were in PlF3'H and PlUF5GT (Figure 3).

CHS was the first key gene catalyzing the formation of entry point chalcone in plant anthocyanin pathway (Nakatsuka et al. 2008a; Park et al. 2011). Noda et al. (2004) found CHS was highly expressed in petals and sepals of lisianthus, Zhou et al. (2010) and Zhou et al. (2011) discovered that the highest transcript abundance of CHS1 isolated from tree peony was in petals. In present study, PlCHS was abundantly expressed which was in conformity with previous reporters (Noda et al. 2004; Zhou et al. 2010; Zhou et al. 2011), these revealed that PlCHS played an essential role in the formation of P. lactiflora flower colour. In addition, DFR, ANS, UF3GT and UF5GT were also the key genes in the anthocyanin pathway. DFR catalyzed the reaction from dihydroﬂavonol to colourless leucoanthocyanidin, which was catalyzed by ANS to the coloured anthocyanidin. And they were all glucosylated to produce stable molecules by UF3GT and UF5GT (Yamazaki et al. 2002; Nakatsuka et al. 2005). In this study, the expression levels of DFR and UF3GT were higher than that of ANS, which was consistent with previous reports in fully opened ﬂowers of gentian plants (Nakatsuka et al. 2005). Meanwhile, the expression level of UF5GT was very low. Hence an inference could be drawn from these results that DFR, ANS and UF3GT were more important than UF5GT. Considering the nine genes, their expression peaks were basically at 15:00, the expression patterns were consistent with the trend of sampling temperature, and lagged behind the trend of light intensity except individual gene, which indicated that the formation of flower colour was affected by external environmental conditions (Miller et al. 2011).

CASTELLARIN, S.D.; MATTHEWS, M.A.; DI GASPERO, G. and GAMBETTA, G.A. (2007a). Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta, vol. 227, no. 1, p. 101-112. [CrossRef]

CASTELLARIN, S.D.; PFEIFFER, A.; SIVILOTTI, P.; DEGAN, M.; PETERLUNGER, E. and DI GASPERO, G. (2007b). Transcriptional regulation of anthocyanin biosynthesis in ripening fruits of grapevine under seasonal water deficit. Plant, Cell & Environment, vol. 30, no. 11, p. 1381-1399. [CrossRef]

CHEN, S.M.; ZHU, X.R.; CHEN, F.D.; LUO, H.L.; LV, G.S.; FANG, W.M. and ZHANG, Z. (2010). Expression profiles of anthocyanin biosynthetic genes in chrysanthemum cultivars with different flower colors. Acta Botanica Boreali-Occidentalia Sinica, vol. 30, no. 3, p. 453-458.

FUKUCHI-MIZUTANI, M.; OKUHARA, H.; FUKUI, Y.; NAKAO, M.; KATSUMOTO,K.; YONEKURA-SAKAKIBARA, K.; KUSUMI, T.; HASE, T. and TANAKA, Y. (2003). Biochemical and molecular characterization of a novel UDP-Glucose: Anthocyanin 3'-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian. Plant Physiology, vol. 132, no. 3, p. 1652-1663. [CrossRef]

FUKUCHI-MIZUTANI, M.; AKAGI, M.; ISHIGURO, K.; KATSUMOTO, Y.; FUKUI, Y.; TOGAMI, J.; NAKAMURA, N. and TANAKA, Y. (2011). Biochemical and molecular characterization of anthocyanidin/ﬂavonol 3-glucosylation pathways in Rosa x hybrida. Plant Biotechnology, vol. 28, no. 2, p. 239-244. [CrossRef]

HARBORNE, J.B. and WILLIAMS, C.A. (2000). Advances in flavonoid research since 1992. Phytochemistry, vol. 55, no. 6, p. 481-504. [CrossRef]

HU, K.; MENG, L.; HAN, K.T.; SUN, Y. and DAI, S.L. (2009). Isolation and expression analysis of key genes involved in anthocyanin biosynthesis of cineraria. Acta Horticulturae Sinica, vol. 36, no. 7, p. 1013-1022.

HU, C.; GONG, Y.; JIN, S. and ZHU, Q. (2011). Molecular analysis of a UDP-glucose: Flavonoid 3-O-glucosyltransferase (UFGT) gene from purple potato (Solanum tuberosum). Molecular Biology Reports, vol. 38, no. 1, p. 561-567. [CrossRef]

JIA, N.; SHU, Q.Y.; WANG, L.S.; DU, H.; XU, Y.J. and LIU, Z.A. (2008a). Analysis of petal anthocyanins to investigate coloration mechanism in herbaceous peony cultivars. Scientia Horticulturae, vol. 117, no. 2, p. 167-173. [CrossRef]

JIA, N.; SHU, Q.Y.; WANG, D.H.; WANG, L.S.; LIU, Z.A.; REN, H.X.; XU, Y.J.; TIAN, D.K. and TILT, K.M. (2008b). Identiﬁcation and characterization of anthocyanins by high-performance liquid chromatography-electrospray ionization-mass spectrometry in herbaceous peony species. Journal of the American Society for Horticultural Science, vol. 133, no. 3, p. 418-426.

KOBAYASHI, S.; ISHIMARU, M.; DING, C.K.; YAKUSHIJI, H. and GOTO, N. (2001). Comparison of UDP-glucose: Flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Science, vol. 160, no. 3, p. 543-550. [CrossRef]

LI, Y.; BALDAUF, S.; LIM, E.K. and BOWLES, D.J. (2001). Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. The Journal of Biological Chemistry, vol. 276, p. 4338-4343. [CrossRef]

MILLER, R.; OWENS, S.J. and RØRSLETT, B. (2011). Plants and colour: Flowers and pollination. Optics & Laser Technology, vol. 43, no. 2, p. 282-294. [CrossRef]

MOL, J.; GROTEWOLD, E. and KOES, R. (1998). How genes paint ﬂowers and seeds. Trends in Plant Science, vol. 3, no. 6, p. 212-217. [CrossRef]

NAKATSUKA, T.; NISHIHARA, M.; MISHIBA, K. and YAMAMURA, S. (2005). Temporal expression of ﬂavonoid biosynthesis-related genes regulates ﬂower pigmentation in gentian plants. Plant Science, vol. 168, no. 5, p. 1309-1318. [CrossRef]

NAKATSUKA, A.; MIZUTA, D.; KII, Y.; MIYAJIMA, I. and KOBAYASHI, N. (2008a). Isolation and expression analysis of ﬂavonoid biosynthesis genes in evergreen azalea. Scientia Horticulturae, vol. 118, no. 4, p. 314-320. [CrossRef]

NAKATSUKA, T.; SATO, K.; TAKAHASHI, H.; YAMAMURA, S. and NISHIHARA, M. (2008b). Cloning and characterization of the UDP-glucose: Anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian. Journal of Experimental Botany, vol. 59, no. 6, p. 1241-1252. [CrossRef]

NODA, N.; KANNO, Y.; KATO, N.; KAZUMA, K. and SUZUKI, M. (2004). Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum). Physiologia Plantarum, vol. 122, no. 3, p. 305-313. [CrossRef]

OGATA, J.; SAKAMOTO, T.; YAMAGUCHI, M.; KAWANOBU, S. and YOSHITAMA, K. (2001). Isolation and characterization of anthocyanin 5-O-glucosyltransferase from flowers of Dahlia variabilis. Journal of Plant Physiology, vol. 158, no. 6, p. 709-714. [CrossRef]

PARK N.I.; XU, H.; LI, X.; JANG, I.H.; PARK, S.; AHN, G.H.; LIM, Y.P.; KIM, S.J. and PARK, S.U. (2011). Anthocyanin accumulation and expression of anthocyanin biosynthetic genes in radish (Raphanus sativus). Journal of Agricultural and Food Chemistry, vol. 59, no. 11, p. 6034-6039. [CrossRef]

QI, A.T. (1989). Physiology and biochemistry of flower color. China Forestry Publishing House, Beijing. 324 p. ISBN 7-5038-0565-X.

SCHMITTGEN, T.D. and LIVAK, K.J. (2008). Analyzing real-time PCR data by the comparative C_T method. Nature Protocols, vol. 3, no. 6, p. 1101-1108. [CrossRef]

TAMURA, K.; PETERSON, D.; PETERSON, N.; STECHER, G.; NEI, M. and KUMAR, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, vol. 28, no. 10, p. 2731-2739. [CrossRef]

TANAKA, Y. and OHMIYA, A. (2008). Seeing is believing: Engineering anthocyanin and carotenoid biosynthetic pathways. Current Opinion in Biotechnology, vol. 19, no. 2, p. 190-197. [CrossRef]

TANAKA, Y.; BRUGLIERA, F.; KALC, G.; SENIOR, M.; DYSON, B.; NAKAMURA, N.; KATSUMOTO, Y. and CHANDLER, S. (2010). Flower color modification by engineering of the flavonoid biosynthetic pathway: Practical perspectives. Bioscience, Biotechnology, and Biochemistry, vol. 74, no. 9, p. 1760-1769. [CrossRef]

VOGT, T.; GRIMM, R. and STRACK, D. (1999). Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae. The Plant Journal, vol. 19, no. 5, p. 509-519. [CrossRef]

WANG, J.G. and ZHANG, Z.S. (2005). Herbaceous peonies of China. China Forestry Publishing House, Beijing. 310 p. ISBN 7-5038-3961-9.

YAMAZAKI, M.; YAMAGISHI, E.; GONG, Z.; FUKUCHI-MIZUTANI, M.; FUKUI, Y.; TANAKA, Y.; KUSUMI, T.; YAMAGUCHI, M. and SAITO, K. (2002). Two ﬂavonoid glucosyltransferases from Petunia hybrida: Molecular cloning, biochemical properties and developmentally regulated expression. Plant Molecular Biology, vol. 48, no. 4, p. 401-411. [CrossRef]

ZHANG, J.L.; PAN, D.R.; ZHOU, Y.F.; WANG, Z.C.; HUA, S.M.; HOU, L.L. and SUI, F.F. (2009). Cloning and expression of genes involved in anthocyanins synthesis in ornamental sunflower. Acta Horticulturae Sinica, vol. 36, no. 1, p. 73-80.

ZHAO, D.; ZHOU, C. and TAO, J. (2011). Carotenoid accumulation and carotenogenic genes expression during two types of persimmon fruit (Diospyros kaki L.) development. Plant Molecular Biology Reporter, vol. 29, no. 3, p. 646-654. [CrossRef]

ZHAO, D.; TAO, J.; HAN, C. and GE, J. (2012). An actin gene as the internal control for gene expression analysis in herbaceous peony (Paeonia lactiflora Pall.). African Journal of Agricultural Research, vol. 7, no. 14, p. 2153-2159.

ZHOU, L.; WANG, Y. and PENG, Z. (2010). Isolation and tissue-specific expression of chalcone synthase gene Ps-CHS1 in tree peony. Acta Horticulturae Sinica, vol. 37, no. 8, p. 1295-1302.

ZHOU, L.; WANG, Y. and PENG, Z. (2011). Molecular characterization and expression analysis of chalcone synthase gene during flower development in tree peony (Paeonia suffruticosa). African Journal of Biotechnology, vol. 10, no. 8, p. 1275-1284.

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