Genetic and molecular analysis of the anthocyanin pigmentation pathway in Epimedium

Introduction Flower color is an ideal trait for studying the molecular basis for phenotypic variations in natural populations of species. Epimedium (Berberidaceae) species exhibit a wide range of flower colors resulting from the varied accumulation of anthocyanins and other pigments in their spur-like petals and petaloid sepals. Methods In this work, the anthocyanidins of eight different Epimedium species with different floral pigmentation phenotypes were analyzed using HPLC. Twelve genes involved in anthocyanin biosynthesis were cloned and sequenced, and their expression was quantified. Results The expression levels of the catalytic enzyme genes DFR and ANS were significantly decreased in four species showing loss of floral pigmentation. Complementation of EsF3’H and EsDFR in corresponding Arabidopsis mutants together with overexpression of EsF3’5’H in wild type Arabidopsis analysis revealed that these genes were functional at the protein level, based on the accumulation of anthocyanin pigments. Discussion These results strongly suggest that transcriptional regulatory changes determine the loss of anthocyanins to be convergent in the floral tissue of Epimedium species.


Introduction
Accumulation of the secondary metabolite anthocyanin is predominantly responsible for red, blue, and purple pigmentation in angiosperms. Pigmentation is a major determinant of a species' pollination syndrome, which refers to the selection of particular floral traits caused by the preference of their pollinators (Fenster et al., 2004). Flower color is intricately regulated by the specific combinations of certain pigment metabolites produced, and is subjected to ecological selection and convergent evolution. Therefore, flower color is an ideal trait for examining ecological and evolutionary selection processes. The anthocyanin biosynthetic pathway (ABP) has been well established in many model species, such as Arabidopsis, petunia (Petunia hybrida E. Vilm.), and snapdragon (Antirrhinum majus L.) (Buer et al., 2010;Pollastri & Tattini, 2011). Most of the knowledge of anthocyanin biosynthesis in Arabidopsis has been obtained from the analysis of transparent testa (tt) mutants, which show loss of seed pigmentation (Lepiniec et al., 2006). In the early steps of the pathway, the key enzymes chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H) condense and convert a phenylpropanoid precursor, p-coumaroyl-CoA, along with three molecules of malonyl CoA, to dihydrokaempferol (Lepiniec et al., 2006). Parallel catalyzation by flavonoid-3'-hydroxylase, flavonoid-3',5'hydroxylase, dihydroflavonol-4-reductase (DFR), and anthocyanidin synthase (ANS) results in the production of various types of anthocyanidin ( Figure 1) (Holton & Cornish, 1995;Boss et al., 1996). The transcriptional regulators controlling flavonoid biosynthetic enzymes have been extensively studied, and include the MYB, the bHLH, and the WD-repeat proteins. Yeastthree-hybrid protein interaction data suggested that a protein complex of the MYB-bHLH-WD40 transcription factors binds the regulatory promoter regions of the flavonoid pathway enzymatic, or structural, genes, to regulate anthocyanin biosynthesis (Gonzalez et al., 2008).
The evolutionary basis for the loss of anthocyanin pigments in floral tissue has been investigated by characterizing major floral pigmentation loci using controlled cross segregating populations (Schwinn, 2006;Whittall et al., 2006;Hoballah et al., 2007;Streisfeld and Rausher, 2009;Smith and Rausher, 2011). Evidence suggests that flower color transition is affected by the transcriptional regulation of several anthocyanin structural genes expression. For example, altered activity of specific transcriptional factors accounts for altered patterns of pigmentation in white Petunia axillaris and some Antirrhinum species (Schwinn, 2006;Hoballah et al., 2007). Cis-regulatory changes in the F3'H gene promoter cause downregulation of F3'H transcription and altered flux in the anthocyanin pathway, resulting in increased production of the red pigment, pelargonidin, instead of blue, in Ipomoea horsfalliae Hook. (Des Marais & Rausher, 2010). Although it has been suggested that mutations in structural genes may incur higher deleterious pleiotropy than those in cis-regulatory elements or transcription factors, the possibility that enzyme coding sequence variation is involved in flower color transition cannot be excluded (Streisfeld and Rausher, 2009).
The Epimedium genus (Berberidaceae), known as "Yinyang Huo" by Chinese druggists, is one of the most popular traditional Chinese medicinal herb genera (Sun et al., 2014;Zhang et al., 2021). A monophyletic group of 50 species of Epimedium is found in western and central China (Huang et al., 2013a;Huang et al., 2013b;Huang et al., 2015;Huang et al., 2016). Epimedium species display a vast range of flower colors; from white and yellow to rose, crimson, and violet ( Figure 2). These color pigments are distributed in petaloid sepals or petals or both. In this study, we studied the phenotypic variation of color in Epimedium species distributed in the Hubei province of China. The expression of genes involved in the anthocyanin biosynthetic pathway (ABP) was also analyzed for the association with the different flower color polymorphisms. Our study focused on answering two questions: (1) Has anthocyanin pigment loss, or variation, in different species resulted from the same mechanism? (2) Which candidate genes are involved in anthocyanin pigmentation in E. sagittatum?

Analysis of pigments and flavonoid intermediates in different Epimedium species
Using HPLC, the major pigments from the floral tissues of anthocyanin species (A+) species were found to comprise delphinidin and cyanidin, whereas no detectable anthocyanins were found in the non-anthocyanin species (A-) flowers ( Figure 3). To further characterize the mechanism responsible for the non-pigmentation of flowers in A-species, E. sagittatum was used as a model for the enzymatic function.

Expression of ABP genes in floral tissues of Epimedium
To determine whether changes in gene expression might be involved in the non-pigmentation phenotype of A-species, the transcript levels of putative anthocyanin biosynthetic enzymes were examined in petal tissue ( Figure 4; Supplementary Figure 1A). Expression of CHS1 not CHS2 and CHS3 was found to be significantly lower in E. wushanese (A-) than in other species. Similarly, down-regulation of CHS2 was observed in E. frachetii (A-), suggesting that loss of anthocyanin may result from low levels of expression of different copies of CHS in E. wushanese and E. franchetii. For CHI and F3H, we found no significant correlation between expression level and the loss of anthocyanins in spur tissues of all A-species. Among the structural genes, ANS was the only ABP locus where all A-species had significantly lower expression levels than that of A+ species. This suggests that the lack of pigmentation production in all A-species could be caused primarily by lower ANS expression. The expression level of DFR was significantly lower in A-species than in A+ species, except for E. lishihchenii. It has been reported that substrate competition between FLS and DFR creates a metabolic flux of the flavonoid biosynthetic pathway in Arabidopsis. In this study, low expression of DFR in the A-species E. franchetii and E. wushanese was correlated with increased accumulation of FLS expression. On the other hand, up-regulation of DFR was positively correlated with FLS expression in E. lishihchenii but E. sagittatum showed no correlation with DFR. In summary, these results suggest that the loss of anthocyanin in E. frachetii, E. wushanese and E. sagittatum  Figure 1B), gene expression was analyzed across five Epimedium species using the same primers. Expression of DFR was lowest in the three A-species and was correlated with ANS expression, suggesting the expression of DFR and ANS could be regulated by a common transcription factor. CHS1 transcripts were not detected in the sepals of E. wushanese, which also had the lowest CHS1 expression in petals. These observations suggest that negative regulation of the DFR and ANS genes together was also correlated with the lowest CHS1 in sepals and petals in E. wushanese.

Complementation analyses
To study the catalytic activity of E. sagittatum ABP gene products, 35S::EsF3'H and 35S::EsDFR genes were individually transferred into their respective Arabidopsis mutants; transparent testa 7 (tt7) lacking flavonoid 3'-hydroxylase, and transparent testa3 (tt3) lacking dihydroflavonol reductase under the control of the cauliflower mosaic virus 35S promoter (Peer et al., 2001). Transgenic and mutant control seedlings were grown under nitrogen stress to determine if the Epimedium genes could rescue the Arabidopsis anthocyanin-null mutant phenotypes. Accumulation of anthocyanins was observed in transgenic seedlings ectopically expressing EsF3'H and EsDFR ( Figure 6). However, the tt7 and tt3 mutant controls did not exhibit anthocyanin accumulation in cotyledons. Thus the E. sagittatum genes showed catalytic activity in Arabidopsis. Given the lack of an Arabidopsis mutant for F3'5'H, in order to determine whether EsF3'5'H can function in vivo, we overexpressed 35S::EsF3'5'H in wild-type Arabidopsis. Under normal conditions on 1/2 MS medium, the seedlings overexpressing EsF3'5'H showed comparable anthocyanin production to wild-type controls ( Figure 6).

Discussion
The four Epimedium A-species (E. sagittatum, E. lishihchenii, E. franchetii and E. wushanense) investigated in this study appeared to exhibit anthocyanin loss at the phenotypic level via reduced activity of the anthocyanin branch of the flavonoid pathway. In all species, this appears to involve reduced transcriptional activity of pathway genes, similar to studies in Mimulus aurantiacus (Streisfeld & Rausher, 2009). Interestingly, one A-species (E. lishihchenii) expressed all ABP loci except for ANS at a high level.
While the data linking conserved gene regulation changes to anthocyanin level changes are purely correlative, we found no evidence for the role of coding-region mutations in determining different anthocyanin levels. In A-specie E. sagittatum, the F3'H and DFR enzymes were shown to rescue anthocyanin production in their corresponding Arabidopsis mutants, suggestive of adequate catalytic function (Huang et al., 2012). Accumulation of anthocyanin in 35S::EsDFR in this study and 35S::EsMYBA1 transformed Arabidopsis indicated functionality of the EsDFR and EsMYBA1 coding region (Huang et al., 2013a). Thus, we concluded that the loss of flower color in E. sagittatum (A-) was due to a tissuespecific regulatory change affecting EsDFR and EsANS transcription and not coding-region mutations of EsDFR, EsANS, or EsMYBA1. We also suggested that the changes responsible for the loss of pigmentation in E. franchetii and E. wushanense flowers were shared with E. sagittatum, based on similar correlative gene expression patterns and anthocyanin production in leaves. Functional assays of the putative cis-elements and trans-regulators involved in DFR or ANS transcription were required to determine the precise regulatory mechanisms resulting in reduced ANS and DFR gene expression in A-species.
Downregulation of CHS was a major cause of white flowers in natural populations of Aquilegia flavellata and Parrya nudicaulis (Whittall et al., 2006;Dick et al., 2011). Although we found an association between the A-phenotype and downregulation of CHS1 in yellow-flowered E. wushanese, DFR, and ANS were also downregulated, which may also have contributed to the A-phenotype. Therefore, the A-phenotype in four Epimedium species was also proposed to be due to alteration at the regulatory level, rather than functional mutations in ABP enzymes. The loci regulating anthocyanin in Epimedium were currently being finemapped and confirmed by transformation assays.

Tissue harvest
Eight Epimedium species (E. acuminatum, E. franchetii, E. leptorrhizum, E. epstenii, E. sagittatum, E. lishihchenii, E. wushanense, and E. zhushanense) grown in the specialized Epimedium nurseries of Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, China (Figure 2). All plants were transplanted from wild populations and growing under the same environmental conditions. Floral tissues including petaloid sepals and spur-like petals were collected in the spring of 2011. The eight species were separated into two groups corresponding to anthocyanin Quantitative expression pattern of ABP structural genes from petal tissue of eight Epimedium species. Colored and empty bars represent A+ and Aspecies, respectively. Le, Zh, Ep, Ac, Sa, Li, Fr and Wu represent E. leptorrhizum, E. zhushanense, E. epstenii, E.acuminatum, E. sagittatum, E. lishihchenii, E. franchetii and E. wushanense. Data presented here are the mean values of three replicates with error bars indicating SE.

HPLC analysis of flavonoid intermediates and anthocyanin
The profiles of anthocyanidins from the samples of A-species and A+ species were analyzed using HPLC. The precursors of anthocyanin pigments were extracted from 100 mg of fresh corolla tissue. For each sample, 20 mL of supernatant was injected into a Shimadzu LC-20 AT liquid chromatograph (Shimadzu Corporation, Japan) and a 250×4.6 mm reverse phase C18 column (Sigma-Aldrich, USA) at a flow rate of 1 ml min -1 . The organic solvent was composed of acetonitrile and 0.1% trifluoroacetic acid, and the polar solvent was 0.1% trifluoroacetic acid in HPLC-grade water. The anthocyanin was measured at 550 nm. The chemical compounds cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin (Poypehenols Laboratories, Norway), were used as anthocyanidin standards.
Transferring ABP candidate genes into other Epimedium species In total, 12 genes from E. sagittatum involved in the ABP were cloned following RT-PCR amplification using degenerate primers or specific primers based on our previous investigation (Zeng et al., 2010;Huang et al., 2013a;Huang et al., 2013b;Huang et al., 2015). These genes were CHS1, CHS2, CHS3, CHI1, CHI2, F3H1, F3H2, F3'H, F3'5'H, FLS, DFR, ANS. In this study, all pairs of primer from E. sagittatum were transferred to other Epimedium species. Analysis of expression profiles of anthocyanin genes in petaloid sepals of five Epimedium species using real-time PCR. The cDNA templates are listed as follows: Le, E. leptorrhizum; Zh, E. zhushanense; Ep, E. epstenii; Wu, E. wushanense and Ac, E. acuminatum. Data presented here are the mean values of three replicates with error bars indicating SE.

Gene expression
Total RNA was extracted from inner sepals and petals at anthesis, at which time the biosynthesis of anthocyanin is completed. First-strand cDNA was synthesized using PrimeScript RT reagent Kit (Takara, Japan) following the manufacturer's instructions. In each 20 mL qRT-PCR reaction, 50 ng of cDNA was amplified using SYBR ® Premix Ex TaqTM II (Takara, Japan) and 100 mM of primers in an ABI7500 Real-Time PCR machine (ABI, USA) as per the manual. Actin was amplified as the control gene. The samples from three tissues were used and three technical replicates were performed for each sample. Data were analyzed by ABI7500 software. In this study, all pairs of primer (CHS1, CHS2, CHS3, CHI1, CHI2, F3H1, F3H2, F3'H, F3'5'H, FLS, DFR, and ANS) from E. sagittatum were transferred in other Epimedium species. All primers used in this manuscript are listed in the supplementary database.
The SalI and SacI digested fragment of each gene was purified and ligated into the pMV plasmid (derived from pBI121) behind the cauliflower 35S promoter. The plasmids were then transformed into Agrobacterium strain EHA105. Arabidopsis wild-type and mutants (tt3 and tt7) were transformed by the floral dip infiltration method (Zhang et al., 2006). Transformants were selected on 1/2 Murashige and Skoog medium supplemented with 50 mg/mL kanamycin. Resistant seedlings were then transferred into the soil to harvest seeds. T1 seedlings were screened on 1/2 MS medium minus nitrogen for observation of anthocyanin accumulation.

Data availability statement
Publicly available datasets were analyzed in this study. This data can be found here: Reference.

Author contributions
WS, HH, and YW conceived and designed the experiments. WS and YM performed the experiments.WS, WH, ZY, XM, and HW analyzed the data. WS wrote the paper. XM and HW revised the paper. YZ provided Figure 2 and collected species. RH and SC supervised this investigation. All authors contributed to the article and approved the submitted version. Phenotypes of the Arabidopsis with overexpression of EsF3'H, EsDFR and F3'5'H(A) Image of anthocyanin in tt7 mutant and rescuing line of EsF3'H in tt7 background, (B) Phenotypes of wild-type, and transgenic Arabidopsis seedling with EsF3'5'H, (C) Phenotypes of tt3 mutant, and transgenic Arabidopsis seedling with EsDFR.

Funding
The authors are grateful for finical support from the Scientific and Technological Innovation Project of the China Academy of Chinese Medical Sciences (CI2021A04806 and CI2021A04008) and National Key Research and Development Program of China (2022YFC3501703).