Ectopic expression of Lc differentially regulated anthocyanin biosynthesis in the floral parts of tobacco (Nicotiana tobacum L.) plants
© The Author(s) 2016
Received: 21 May 2016
Accepted: 22 August 2016
Published: 8 September 2016
Anthocyanins are the conspicuous pigments of flowering plants and participate in several aspects of plant development and defense, such as seeds and pollens dispersal. Leaf colour (Lc) is the first basic/helix-loop-helix (bHLH) transcription factor controlling anthocyanin biosynthesis isolated from maize (Zea mays L.). Ectopic expression of maize Lc enhanced anthocyanin biosynthesis in many plants including tobacco (Nicotiana tobacum L.). However, the molecular regulatory mechanism of anthocyanin biosynthesis in the different floral parts of tobacco remains largely unknown. Therefore, the molecular and biochemical characterization of anthocyanin biosynthesis were investigated in the flowers of both wild type and Lc-transgenic tobacco plants.
At the reproductive stage, with respect to the different parts of the flowers in wild type SR1, the calyxes and the pistils were green, and the petals and the filaments showed light pink pigmentation; the Lc-transgenic tobacco exhibited light red in calyxes and crimson in petals and in filaments respectively. Correspondingly, the total anthocyanin contents (TAC) in calyxes, petals and filaments of Lc-transgenic plants were much higher than that of the counterparts in SR1. Though the TAC in anthers of Lc-transgenic plants was low, it was still significantly higher than that of SR1. SR1 has almost the same TAC in the pistils as Lc-transgenic plants. Consistent with the intense phenotype and the increased TAC, Lc was weakly expressed in the calyxes and strongly expressed in petals and filaments of Lc-transgenic plants, while Lc was not detected in SR1. The expression level of NtAN2 in petals was similar between SR1 and Lc-transgenic lines. In agreement with the expression profile of Lc, both early (NtCHS) and late anthocyanin-biosynthetic genes (NtDFR, NtF3′H, and NtANS) were coordinately up-regulated in the counterparts of flowers. HPLC analysis demonstrated that the cyanidin (Cya) deposition was mainly responsible for the intense pigmentation of Lc-transgenic tobacco.
Ectopic expression of Lc greatly enhanced both early- and late- anthocyanin-biosynthetic gene expression, and therefore resulted in the Cya-based TAC increase in the calyxes, the filaments and the petals in tobacco plants.
KeywordsAnthocyanin-biosynthetic genes Cyanidin Flower colour Lc Tobacco (Nicotiana tobacum L.)
Anthocyanins represent the most conspicuous class of flavonoids owing to their striking colours ranged from orange to red to blue. The striking colours not only facilitate the pollination and seed dispersal but also contribute to plant adaptation to environmental stresses, such as UV light stress (Campanella et al. 2014). The regulation of anthocyanin biosynthesis is extensively studied in higher plants. Anthocyanin biosynthesis is a branch of flavonoid pathway, which is usually categorized as either early flavonoid biosynthetic genes (EBGs) or late biosynthetic genes (LBGs) (Quattrocchio et al. 1993; Mol et al. 1998). The EBGs encode enzymes such as the chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H) (Quattrocchio et al. 1993). The LBGs lead to the production of the anthocyanins, involving the dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) and UDPG: flavonoid 3-Oglucosyltranferase (UFGT) (Quattrocchio et al. 1993; Mol et al. 1998).
The control of flavonoid biosynthesis pathway is largely at the level of transcription of regulators and of the corresponding biosynthetic genes. Three types of transcription factor, i.e. R2R3 MYB, bHLH and WD40, have been reported for the regulation of anthocyanin biosynthesis at the transcript level (Xie et al. 2016). Lc was the first characterized myc bHLH transcription factor, which was involved in the regulation of anthocyanin biosynthesis. Lc controlled the chalcone synthase gene (CHS) and dihydroflavonol 4-reductase gene (DFR) in maize (Ludwig et al. 1989), and it also regulated the flavonoid pathway in both monocots and dicots. There have been an increasing number of reports on genetic engineering of anthocyanin biosynthesis pathway for floricultural and agricultural purposes.
Heterologous expression of Lc enhanced anthocyanin biosynthesis in tobacco, Petunia and Caladium bicolour at both vegetative stage and floral stage (Lloyd et al. 1992; Bradley et al. 1998; Bovy et al. 2002; Li et al. 2005; Albert et al. 2009). Moreover, expression of Lc in rice (Oryza sativa L.) resulted in red spikelet and caused sterility (Song et al. 2013). Recently, ectopic expression of Lc in cotton (Gossypium hirsutum L.) promoted its anthocyanin biosynthesis in cotton fiber and increased its tolerance to bollworm, while expression of Lc in sweet potato (Ipomoea batatas L.) enhanced anthocyanin and lignin biosynthesis (Fan et al. 2016; Wang et al. 2016). The effects of Lc in different plants might depend on the specificity of plant species and their growth conditions.
Tobacco is a commonly used heterologous system to investigate the gene functions in plants (Pattanaik et al. 2010). Lc was found to be coordinated with some transcription factors such as R2R3 MYB transcription factor to regulate anthocyanin biosynthesis (Franken et al. 1994). In tobacco, two types of transcription factors, bHLH transcription factor (NtAN1a, NtAN1b) and R2R3 MYB (NtAN2) were characterized and expressed predominantly in flowers and in juvenile leaves under low-temperature stress (Bai et al. 2011; Pattanaik et al. 2010; Huang et al. 2012). Though the change of flower colour of Lc-transgenic tobacco has been reported (Lloyd et al. 1992; Yang et al. 2007), the molecular mechanism underlying the enhanced pigmentation in different floral parts of Lc-transgenic tobacco plants remains unclear. Here we investigated the different impacts of heterologous expression of Lc upon anthocyanin biosynthesis in the calyxes, petals, filaments, stamens and pistils of tobacco flowers and identified the specific anthocyanidin responsible for intense pigmentation in Lc-transgenic tobacco plants.
Plant growth conditions and treatments
The production of Lc-transgenic plant lines were described by Huang et al. (2012). Seeds of wild type tobacco SR1 (Nicotiana tobacum L.) and the T2 seeds of transgenic lines were surface-sterilized in 30 % household bleach with 0.01 % of Tween, and then washed with sterilized water for three times. The seeds were sown on the MS medium. After two true leaves emerged, the seedlings were transplanted into sand culture irrigated with Hoagland’s solution with the concentration of 20 μmol/L of Fe-Na2EDTA.
The T2 Lc-transgenic lines were cultivated in the soil pots in greenhouse and they were characterized carefully, especially for the flower organs. At the reproductive stage, the full expanded flowers prior to fertilization were collected for anthocyanin quantification and RNA gel assay (Nishihara et al. 2005). Different parts were from at least 5 intact flowers.
Northern blot analysis
Primers used for amplification of probes in Northern blot assay
Forward primer (5′–3′)
Reverse primer (5′–3′)
Multiplex RT-PCR assay
Taking the petals as samples, ten structural genes (NtPAL, Nt4CL, NtC4H, NtCHS, NtCHI, NtF3H, NtF3'H, NtDFR, NtANS and NtFLS) and two regulatory genes (Lc and NtAN2) were selected and analyzed with multiplex RT-PCR. The sequences of the primer pair and the multiplex RT-PCR procedure were described previously in Huang et al. (2012).
Quantification of total anthocyanin content (TAC) and HPLC assay of cyanidin, delphinidin and pelargonidin
Different parts from at least 5 intact flowers were collected as samples. The total TAC assay was carried out as described in Huang et al. (2012). To investigate the components of the intensified pigmentations in petals of Lc-transgenic tobacco, the anthocyanin was extracted and analyzed from the pool with at least five petals from SR1 and Lc-transgenic plants (Wang et al. 2006). The anthocyanin standards, cyanidin (Cya), delphinidin (Del) and pelargonidin (Pel) (Sigma) were dissolved in methanol to prepare the storage concentration at 0.1, 0.1 and 0.5 mg/mL, respectively. The HPLC procedure was carried out as described by Merken and Beecher (2000) with slight modifications. Briefly, the anthocyanin extracts were separated through a Zorbax Eclipse XDB-C18 column (Hewlett Packard) (250 × 4.6 mm, 5 μm) preceded by a guard column (12.5 × 4.6 mm) with the same stationary phase, and both columns were maintained at 30 °C. The flow rate was 1 mL/min. Each injection volume was 20 μL. Using a gradient of acetonitrile in 0.05 % (w/w) trifluoroacetic acid, the LC system Schimazu 10A vp was used and the consecutive assay from 200 to 600 nm. On the basis of the specific retention time, the Cya, Del and Pel were assayed with a photodiode array detector Merken and Beecher (2000).
Lc-transgenic tobacco lines exhibited more intense pigmentation in floral parts
Cyanidin is responsible for the intense pigmentation in Lc-transgenic tobacco lines
To clarify the major component of anthocyanins responsible for intense pigmentation in Lc-transgenic flowers, three most common anthocyanins, Cya (cyanidin), Pel (pelargonidin), and Del (delphinidin) were further quantified with HPLC. The HPLC extraction and analysis system of the three anthocyanidins were established. The retention times of Cya, Del and Pel standard sample were 32.148, 37.960 and 41.213 min, respectively (Fig. 1C). Only Cya was identified in Lc-transgenic tobacco petals. Moreover, the Cya concentrations of three Lc-transgenic lines were at least 38-fold higher than that of SR1 (Fig. 1D), which demonstrated that the increased cyanidin was responsible for the intense pigmentation in the petals.
Boosted expression of key anthocyanin-biosynthetic genes in Lc-transgenic lines
To elucidate how ectopic expression of Lc enhanced anthocyanin biosynthesis in the different parts of Lc-transgenic flowers, six key anthocyanin biosynthetic genes (NtPAL, Nt4CL, NtCHS, NtDFR, NtANS and NtGST) were analyzed with Northern blot. NtPAL was highly expressed in the petals, filaments and pistils and its expression pattern were similar between SR1 and Lc-transgenic lines. Nt4CL expressed weakly in the calyxes and anthers, strongly in the petals, filaments and pistils in both SR1 and Lc-transgenic lines. NtCHS expressed exclusively in petals of SR1, while it was greatly upregulated in petals and filaments in Lc-transgenic lines. NtDFR expressed highly in the petals, filaments and anthers, low in the calyx, and absent in the pistils of SR1, while it expressed significantly higher in the petals and filaments of Lc-transgenic lines. Similar expression profile was detected for both NtANS and NtCHS, with an exclusive expression in petals in SR1, and a significantly upregulated expression in the petals and filaments in Lc-transgenic lines. NtGST was ubiquitously expressed in the five floral parts of SR1 and Lc-transgenic lines (Fig. 2).
The anthocyanin biosynthesis pathway was enhanced in petals of Lc-transgenic plants
For the anthocyanin regulatory gene, Lc was only detected in Lc-transgenic lines (Fig. 3L) not in SR1. The expression of NtAN2, the partner of Lc-like bHLH proteins in regulating the anthocyanin biosynthesis pathway was not changed in the Lc-transgenic lines (Fig. 3K), which suggested that ectopic expression of Lc rather than NtAN2 is responsible for the up-regulation of anthocyanin biosynthesis genes as well as the anthocyanin accumulation in the Lc-transgenic lines.
Taken together, the four key genes NtCHS, NtF3’H, NtDFR and NtANS were greatly upregulated in petals and filaments of Lc-transgenic lines, which resulted in enhanced anthocyanin biosynthesis and more intense pigmented calyx, petals and filaments in the Lc-transgenic lines.
Great efforts have been made to modify the flower colour by manipulating the structural genes (such as DFR, CHS, and ANS) or their regulatory genes (such as Lc, Pl) (Nishihara and Nakatsuka 2011; Grotewold 2006; Han et al. 2009). The first introduction of maize A1 gene encoding DFR into petunia enhanced the pelargonidin synthesis and resulted in brick red pigment in flower (Meyer et al. 1987). Recently, transcription factors controlling the genes involved in anthocyanin biosynthesis have been characterized not only in model plants, but also in floricultural plants (e.g. petunia, snapdragon) and food crops. The strategy by manipulating the transcription factors can modify multiple anthocyanin biosynthetic genes effectively. Heterologous expression of AtPAR in Taraxacum brevicorniculatum resulted in a red/purple vegetative tissue (Qiu et al. 2014). Ectopic expression of maize Lc greatly boosted the production of anthocyanin in cotton and in sweet potato (Fan et al. 2016; Wang et al. 2016). In this study, heterologous expression maize Lc in tobacco exhibited more intense pigmentation in the floral tissues (Fig. 1A) similar to the previous report by Lloyd et al. (1992) and Yang et al. (2007). The specific expression of Lc in the calyx, petals and filaments increased the transcript abundance of anthocyanin biosynthetic genes (Figs. 2, 3), which resulted in the higher anthocyanin content, and led to the more intense pigmentation in floral parts (Fig. 1).
The more intense pigmentation in floral parts of Lc-transgenic tobacco plants (Fig. 1A) should be due to systematic effects of ectopic expression of Lc. Ectopic expression of Lc in petals enhanced both early flavonoid-biosynthetic (NtCHS and NtF3′H) and late flavonoid biosynthetic genes (NtDFR, NtANS) (Figs. 2, 3), which was different in petunia or rice. In Lc-transgenic petunia, CHS, CHI, F3H was weakly upregulated, while DFR, F3′H, F3′5′H, ANS and UFGT were highly increased; PAL, 3RT, and C4H were not affected (Bradley et al. 1998). In rice, CHS-like gene was increased in Lc-transgenic rice and might lead to sterility (Song et al. 2013). All these studies suggested that ectopic expression of Lc in different species regulated the common targets (such as CHS and DFR genes). However, the effects of ectopic Lc differed in the different plant species or even tissues.
Flavonoids shares a basic C6–C3–C6 skeleton structure in common consisting of two aromatic rings (A and B) and a heterocyclic ring C containing one oxygen atom (Halbwirth et al. 2010). The anthocyanins are one of the major classes of the flavonoids according to the oxidative status of the ring C. Delphinidin, pelargonidin and cyanidin differ in the different positions of hydroxyl of the B-rings. From a biochemical perspective, anthocyanin biosynthesis can be controlled by two processes, one is the number of hydroxyls of B-rings; the other is the substrate specificity of dihydroflavonol 4-reductase (DFR) (Meyer et al. 1987). For the former reaction, dihydrokaempferol (DHK) can be hydroxylated by flavonoid 3′-hydroxylase (F3′H) to produce dihydroquercetin (DHQ) or be changed to dihydromyricetin (DHM) by flavonoid 3′, 5′-hydroxylase (F3′5′H), respectively (Holton and Cornish, 1995). For the latter reaction, DHK cannot be catalyzed by DFR efficiently in some plant species, such as in petunia and tobacco (Forkmann and Ruhnau 1987). In this study, the expression of F3′H in Lc-transgenic lines was significantly increased than that in SR1 (Fig. 3), which might be partly responsible for the increase in Cya content rather than Del or Pel. Aharoni et al. (2001) found that ectopic expression of FaMYB1, a strawberry fruit ripening transcription factor containing the repressor domain, resulted in the reduced Cya content and other flavonoid content, which in turn confirmed that Cya is the major anthocyanin determines the petal colour in tobacco plants.
Taken together, heterologous expression of maize Lc gene enhanced the anthocyanin biosynthesis in the calyx, petals and filaments by the upregulation of both early and late flavonoid biosynthetic genes. Consequently, the anthocyanin accumulated especially in the Cya content.
high performance liquid chromatography
total anthocyanin content
TZ constructed the Lc-pCambia1301 vector. ZAH performed the other Lc-transgenic work and molecular and biochemical assay and wrote the paper. SSZ and NW critically read and revised the manuscript. All authors read and approved the final manuscript.
We sincerely thank Dr Wu Huilan (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for technical assistance in multiplex RT-PCR assay. We are also grateful to Dr Wang Yiqing (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for her technical assistance in HPLC assay of anthocyanin components. This research was supported by National Natural Science Foundation of China (Nos. 31272221).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Aharoni A, De Vos CH, Wein M, Sun Z, Greco R, Kroon A, Mol JN, O’Connell AP (2001) The strawberry FaMYB1 transcription factor suppress anthocyanin and flavonol accumulation in transgenic tobacco. Plant J 28:319–332View ArticlePubMedGoogle Scholar
- Albert NW, Lewis DH, Zhang H, Irving LJ, Jameson PE, Davies KM (2009) Light-induced vegetative anthocyanin pigmentation in petunia. J Exp Bot 60:2191–2202View ArticlePubMedPubMed CentralGoogle Scholar
- Bai Y, Pattanaik S, Patra B, Werkman JR, Xie CH, Yuan L (2011) Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active. Planta 234:363–375View ArticlePubMedGoogle Scholar
- Bovy A, de Vos R, Kemper M, Schijlen E, Almenar Pertejo M, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, Santos-Buelga C, van Tunen A (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell 14:2509–2526View ArticlePubMedPubMed CentralGoogle Scholar
- Bradley JM, Davies KM, Deroles SC, Bloor SJ, Lewis DH (1998) The maize Lc gene up regulates the flavonoids biosynthetic pathway in petunia. Plant J 13:381–392View ArticleGoogle Scholar
- Campanella JJ, Smalley JV, Dempse ME (2014) A phylogenetic examination of the primary anthocyanin production pathway of the Plantae. Bot Stud 55:1–10View ArticleGoogle Scholar
- Fan X, Fan B, Wang Y, Yang W (2016) Anthocyanin accumulation enhanced in Lc-transgenic cotton under light and increased resistance to bollworm. Plant Biotechnol Rep 10:1–11View ArticlePubMedGoogle Scholar
- Forkmann G, Ruhnau B (1987) Distinct substrate specificity of dihydroflavonol 4-reductase from flowers of Petunia hybrida. Z Naturforsch 42C:1146–1148Google Scholar
- Franken P, Schrell S, Peterson PA, Saedler H, Wienand U (1994) Molecular analysis of protein domain function encoded by the myb-homologous maize genes C1, Zm1 and Zm38. Plant J 6:21–30View ArticlePubMedGoogle Scholar
- Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780View ArticlePubMedGoogle Scholar
- Halbwirth H (2010) The creation and physiological relevance of divergent hydroxylation patterns in the flavonoid pathway. Int J Mol Sci 11:595–621View ArticlePubMedPubMed CentralGoogle Scholar
- Han YJ, Kim YM, Lee JY, Kim SJ, Cho KC, Chandrasekhar T, Song PS, Woo YM, Kim JI (2009) Production of purple-colored creeping bentgrass using maize transcription factor genes Pl and Lc through Agrobacterium-mediated transformation. Plant Cell Rep 28(3):397–406View ArticlePubMedGoogle Scholar
- Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083View ArticlePubMedPubMed CentralGoogle Scholar
- Huang ZA, Zhao T, Fan HJ, Wang N, Zheng SS, Ling HQ (2012) The upregulation of NtAN2 expression at low temperature is required for anthocyanin accumulation in juvenile leaves of Lc-transgenic tobacco (Nicotiana tabacum L.). J Genet Genom 39:149–156View ArticleGoogle Scholar
- Li SJ, Deng XM, Mao HZ, Hong Y (2005) Enhanced anthocyanin synthesis in foliage plant Caladium bicolour. Plant Cell Rep 23:716–720View ArticlePubMedGoogle Scholar
- Lloyd AM, Walbot V, Davis RW (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258:1773–1775View ArticlePubMedGoogle Scholar
- Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86:7092–7096View ArticlePubMedPubMed CentralGoogle Scholar
- Merken HM, Beecher GR (2000) Liquid chromatographic method for the separation and quantification of prominent flavonoid aglycones. J Chromatogr A 897:177–184View ArticlePubMedGoogle Scholar
- Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:677–678View ArticlePubMedGoogle Scholar
- Mol J, Grotewold E, Koes R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217View ArticleGoogle Scholar
- Nishihara M, Nakatsuka T (2011) Genetic engineering of flavonoid pigments to modify flower colour in floricultural plants. Biotechnol Lett 33:433–441View ArticlePubMedGoogle Scholar
- Nishihara M, Nakatsuka T, Yamamura S (2005) Flavonoid components and flower colour change in transgenic tobacco plants by suppression of chalcone isomerase gene. FEBS Lett 579:6074–6078View ArticlePubMedGoogle Scholar
- Pattanaik S, Kong Q, Zaitlin D, Werkman JR, Xie CH, Patra B, Yuan L (2010) Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231:1061–1076View ArticlePubMedGoogle Scholar
- Qiu J, Sun S, Luo S, Zhang J, Xiao X, Zhang L, Wang F, Liu S (2014) Arabidopsis AtPAP1 transcription factor induces anthocyanin production in transgenic Taraxacum brevicorniculatum. Plant Cell Rep 33:669–680View ArticlePubMedGoogle Scholar
- Quattrocchio F, Wing JF, Leppen HTC, Mol JNM, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5:1497–1512View ArticlePubMedPubMed CentralGoogle Scholar
- Sambrook J, Maccallum P, Russel D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Springs Harbour Press, Cold Spring Harbor. ISBN 0-87969-577-3Google Scholar
- Song YE, Wang X, Shen ZW, Xu Y, Li JY (2013) Expressing the maize anthocyanin regulatory gene Lc increased flavonoid content in the seed of white pericarp rice and purple pericarp rice. Genetika 49:1292–1299Google Scholar
- Wang CK, Chen PY, Wang HM, To KY (2006) Cosuppression of tobacco chalcone synthase using petunia chalcone synthase construct results in white flowers. Bot Stud 47:71–82Google Scholar
- Wang H, Yang J, Zhang M, Fan W, Firon N, Pattanaik S, Yuan L, Zhang P (2016) Altered phenylpropanoid metabolism in the maize Lc-expressed sweet potato (Ipomoea batatas) affects storage root development. Sci Rep 6:18645–18659View ArticlePubMedPubMed CentralGoogle Scholar
- Xie Y, Tan H, Ma Z, Huang J (2016) DELLA proteins promote anthocyanin biosynthesis via sequestering MYBL2 and JAZ suppressors of the MYB/bHLH/WD40 complex in Arabidopsis thaliana. Mol Plant 9:711–721View ArticlePubMedGoogle Scholar
- Yang CC, Yu JJ, Zhao Q, Zhu DY, Ao GM (2007) Influence of the maize regulatory gene on flower colour of transgenic tobacco and petunia. J Agric Biotech 15:85–89 (in Chinese) Google Scholar