Introduction

Epimedium, named as Yin Yang Huo in Chinese, has been extensively utilized in China and East Asia because of its health-promoting prenyl-flavonoids components (Ma et al. 2011), such as icariin, epimedin A, epimedin B, and epimedin C, hereafter designated as bioactive components. Epimedium extracts are also well known to nourish the kidney, reinforce the Yang, regulate bone remodeling (Ming et al. 2013), promote sexual performance, cure cardiovascular diseases, possess anti-cancer (Tong et al. 2011) and anti-aging benefits (Cai et al. 2011). In the last decades, scientists focused on identifying the phenolic components (Zhao et al. 2008; Zhang et al. 2008; Islam et al. 2008) in Epimedium plants. However, little is known about the biosynthesis of prenyl-flavonoids in Epimedium (Fig. 1a). In addition, the wild resource of Epimedium is endangered because of commercial over-exploitation. Therefore, effective methods, including phytohormones or light treatments, to improve the content of bioactive components in Epimedium are alternative (Zeng et al. 2013).

Fig. 1
figure 1

Biosynthesis of bioactive components in Epimedium. a Simplified and predicted biosynthetic pathway of the bioactive components of Epimedium. Enzyme abbreviations: CHS chalcone synthase, CHI chalcone isomerase, F3H flavanone 3 hydroxylase, FLS flavonol synthase, F3′H flavanone 3′ hydroxylase, DFR dihydroflavonol 4-reductase, ANS anthocyanin synthase, ANR anthocyanidin reductase. DHQ dihydroquercetin, DHK dihydrokaempferol. The dotted arrow indicate a predicted step committed by enzyme(s) while black arrows indicate a step committed by a known enzyme. The dashed arrows indicated that the transcription factor(s) putatively regulate corresponding structural genes. Effects of light stress on flavonoid structural genes (b) and regulatory genes (c) in leaves. Mean ± SD determined from three independent samples are shown. Double stars and single star respectively indicate significant differences in the amount of gene transcripts at the level of P < 0.01 and P < 0.05, calculated by Duncan statistical analysis, when compared to CT samples. d Light stress impairs the accumulation of bioactive components in leaves. Mean ± SD determined from three independent samples are shown. Double stars and single star respectively indicate significant differences in the content of bioactive components at the level of P < 0.01 and P < 0.05, calculated by Duncan statistical analysis, when compared to CT samples

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So far, flavonoid biosynthesis has been considered as the well-dissected secondary metabolic pathway (Dixon and Steele 1999). As shown in Fig. 1a, structural genes involved in flavonoid biosynthesis have been isolated and characterized in Arabidopsis and other species. Also, these genes have been isolated and characterized in Epimedium species (Zeng et al. 2010, 2013, submitted). Many transcription factors, including bHLH, R2R3-MYB, and WD-repeat forming a BMW tricomplex to regulate anthocyanin (GL3/PAP1/TTG1) and proanthocyanin (TT8/TT2/TTG1) biosynthesis, are well studied in Arabidopsis (Gonzalez et al. 2008; Baudry et al. 2004). In addition, a flavonol-specific regulator AtMYB12, upregulating AtCHS and AtFLS, was isolated and characterized (Mehrtens et al. 2005). Although the biosynthetic and regulatory genes in model species such as Arabidopsis, tomato, and grape have been well-dissected, those in medicinal plants such as Epimedium remain unclear. So far, thirteen Epimedium MYB members have been isolated and characterized, among which Epimedium MYB7 and MYB9 are homologous to AtTT2 and VvMYB5b, respectively (Huang et al. 2012). Furthermore, transgenic tobacco plant over-expressing MYB9 accumulate higher level of anthocyanin in flowers than in control (Huang et al. 2012).

In the present study, gene expression detected by real-time PCR and the content of bioactive compound revealed by HPLC in leaves responding to light stress were investigated. Finally, the coordinated relationship of gene expression and phytochemical accumulation is discussed.

Materials and methods

Plant materials and light stress treatment

E. pubescens plants were cultivated in Wuhan Botanical Garden, Chinese Academy of Science, P. R. China before growing to florescence stage. E. pubescens plants with approximate 50 % inflorescences flowering were transferred to hydroponic culture and used for light stress treatment. For the light stress treatment, the E. pubescens plants were lighted by incandescent lamp at 63 μmol/m2/s light intensity at room temperature in a light bin and divided into two sets. One set, denoted as control (CT), was lighted under a normal day/night (8 h/16 h) photoperiod. Another set, denoted as light stress (LS), was continuously lighted for 24 h. After treated for 1 day, leathery mature leaves were harvested for investigating gene expression and bioactive components content. Both gene expression and bioactive components content analyses were investigated in three biological specimens and data were presented in a mean value.

RNA isolation and quantitative real-time PCR (qRT-PCR)

Leaves were harvested and powdered in liquid nitrogen. RNA isolation was performed using TRIzol kit (Invitrogen, USA) following the manufacturer’s instructions. For qRT-PCR, total RNA was reverse-transcribed with a PrimeScript RT Reagent Kit with gDNA Eraser (DDR047, TaKaRa, Japan), which digested the residual DNA in the RNA samples and reverse-transcribed in a one-step process. Gene transcripts were amplified with a SYBR Premix Ex TaqTM II (DDR081S, TaKaRa, Japan) and detected by an ABI 7500 Real-Time PCR system. The PCR program was as followed: stage 1: sufficient denaturation at 95 °C for 30 s; stage 2: PCR reaction with 40 cycles at 95 °C for 5 s and 60 °C for 34 s; and stage 3: dissociation at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The qRT-PCR experiments were performed in triplicate. The expression level of Epimedium actin (EsActin) was used to standardize the RNA sample for each qRT-PCR. The expression level of the genes relative to EsActin was presented as a fold change. The primers for the qRT-PCR are listed in Table 1.

Table 1 List of primers used in this study
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Bioactive components determination

To analyze bioactive components content, about 50 mg of dry leaves were powdered with liquid nitrogen, soaked in 5 mL of 70 % ethanol, and ultrasonicated for 30 min. The extract was filtered through 0.45 μm poly filters for high performance liquid chromatography (HPLC) analysis. The HPLC analysis at a 272 nm wavelength was carried out using an Agilent Technologies Series 1100 (Agilent Technologies, Palo Alto, CA, USA) at a flow rate of 1.0 mL/min. The chromatographic column used was a Zorbax SB-C18 (250 × 4.6 mm I.D., 5 μm; Agilent Technologies, Palo Alto, CA, USA) operated at a constant temperature of 25 °C. The detailed HPLC program was performed as previous study (Xu et al. 2013). Epimedin A, epimedin B, epimedin C, and icariin standards were purchased from the ChromaDex Company (Santa Ana, USA). Data analysis was performed using the Agilent ChemStation software, version A.10.02.

Results and discussion

Previous studies demonstrate that transcription factors homologous to Arabidopsis AtTT8, AtGL3, AtTTG1 are involved in anthocyanin biosynthesis in other plants (Spelt et al. 2000; deVetten et al. 1997; Chiu and Li 2012). Furthermore, both tomato SlMYB12 (Ballester et al. 2010; Adato et al. 2009) and grape VvMYBF1 (Czemmel et al. 2009) conserve with Arabidopsis AtMYB12 in regulating flavonol biosynthesis. These studies showed that transcription factors mentioned above are functionally conserved among plants. In this study, several EST fragments, representing Epimedium MYBF, TT8, GL3, and TTG1 homologous to Arabidopsis AtMYB12, AtTT8, AtGL3, and AtTTG1, were retrieved from the Epimedium EST database (Zeng et al. 2010). In addition, flavonoid structural genes had been isolated and characterized in Epimedium (Zeng et al. submitted). Together, all structural genes and several transcription factors involved in flavonoid biosynthesis were used to evaluate their effects on the accumulation of bioactive components.

To investigate the effect of light stress on the gene expression of structural genes and regulatory genes involved in Epimedium flavonoid biosynthesis, the transcripts of these genes were evaluated by real-time PCR. As presented in Fig. 1b, CHS1, CHI1, F3H, FLS, DFR1, DFR2, and ANS expression were significantly up-regulated in LS samples when compared to CT samples. CHS2 and F3′H transcripts were remarkably decreased in LS sample when compared to CT sample. CHI2 transcripts were increased slightly, but not in a significant level. As shown in Fig. 1c, Epimedium MYBF was down-regulated in LS samples, suggesting that CHS1 expression was upregulated by light stress but not MYBF homologous to flavonol-specific transcription factor AtMYB12 regulating the expression of AtCHS and AtFLS (Mehrtens et al. 2005). The transcripts of Epimedium GL3 and TTG1 were also decreased under light stress. In contrast, Epimedium MYB7 and TT8 were up-regulated significantly. Results indicated that expression of Epimedium MYB9 was not affected by light stress under present experimental conditions. To investigate the effect of light stress on the accumulation of bioactive compounds in leaves, the content of bioactive compounds was assayed. As shown in Fig. 1d, the content of epimedin A, B, C, and icariin were significantly decreased in LS sample when compared to CT sample.

The bioactive components content was decreased under light stress, which might be partially attributed to light stress promoting flavonoid degradation (Fahlman et al. 2009) and/or altering the metabolic distribution (Davis et al. 2013). Alternatively, the trade-off of the up-regulated transcripts of DFR1 and DFR2 competing with FLS for the same substrate (dihydrokaempferol) contribute partially to the decreased bioactive components (Fig. 1). In detail, up-regulated Epimedium TT8 and MYB7 promote the high expression of late genes such as DFRs and ANS although the transcripts of Epimedium TTG1 was decreased. In Arabidopsis and other plants, MYB (PAP1), bHLH (TT8 and GL3), and WD40 (TTG1) transcription factors form tricomplex BMW to regulate the expression of structural genes, including DFR and ANS, and further modulate anthocyanin and proanthocyanin biosynthesis (Schaart et al. 2012; Gonzalez et al. 2008; Baudry et al. 2004). Additionally, the increased Epimedium FLS transcripts might be only attributed to light stress induction rather than Epimedium MYBF because Epimedium MYBF, the potential positive regulator of FLS, is decreased (Fig. 1c). Previous study documents that F3′H is possibly regulated dually by WD-dependent (such as BMW model) and WD-independent (such as flavonol-specific regulator AtMYB12) mechanisms, consisting with its function for producing both quercetin-derivative flavonol and cyaniding-derivative anthocyanin (Gonzalez et al. 2008). The decreased F3′H transcripts might be attributed to a large extent to the depressed Epimedium MYBF and TTG1 under light stress even though Epemidium MYB7 and TT8 were up-regulated (Fig. 1c). These results suggested that metabolites might be introduced to a large extent into pelargonidin branch. Consequently, the increase in higher level of the DFRs and ANS transcripts than FLS transcripts resulted in funneling more metabolites into anthocyanin/proanthocyanin branches rather than the bioactive components branch.

In conclusion, the expression of genes involved in flavonoid biosynthesis and the content of bioactive compounds were investigated under light stress. Our results suggested that the decrease of bioactive compounds may be attributed to transcripts of DFRs and ANS increased to a higher level than that of FLS and CHS1, which led to more metabolites introduced into anthocyanin/proanthocyanin branch than bioactive compounds.

Author contribution

Shaohua Zeng designed research, wrote this manuscript and did partial experiment. Yilan Liu did the experiments and Ying Wang designed research.