Cloning and Characterization of a Tryptophan–Aspartic Acid Repeat Gene Associated with the Regulation of Anthocyanin Biosynthesis in Oriental Hybrid Lily

in Journal of the American Society for Horticultural Science

Anthocyanins are major pigments responsible for the color of lily (Lilium sp.) flowers. Anthocyanin synthesis is part of the flavonoid metabolic pathway. Numerous transcription factors, including R2R3-MYBs, basic helix-loop-helix (bHLH), and tryptophan–aspartic acid repeat (also known as WD40 or WD repeat) proteins, known to regulate flavonoid biosynthesis have been identified in various plant species. However, there is limited information available on WD repeat proteins in lilies. In this study, we identified a WD repeat gene in the Oriental hybrid lily ‘Sorbonne’ (Lilium hybrid WD repeat, LhWDR). LhWDR contains no introns, and has a 1100–base pair open reading frame, encoding a putative protein of 370 amino acids. LhWDR was found to be localized in the cytoplasm of transgenic Arabidopsis thaliana root cells. Expression patterns of LhWDR in different organs and at different periods of lily tepal growth revealed that the expression levels of this gene are closely associated with anthocyanin accumulation. A yeast two-hybrid assay demonstrated that full-length LhWDR interacts with the 420 N-terminal amino acids of Lilium hybrid bHLH2. Interestingly, overexpression of LhWDR in A. thaliana led to an upregulation of the dihydroflavonol 4-reductase gene, which is an important structural gene downstream of the anthocyanin pathway. These results indicate that the WD repeat protein LhWDR might interact with a bHLH transcription factor to regulate anthocyanin biosynthesis.

Abstract

Anthocyanins are major pigments responsible for the color of lily (Lilium sp.) flowers. Anthocyanin synthesis is part of the flavonoid metabolic pathway. Numerous transcription factors, including R2R3-MYBs, basic helix-loop-helix (bHLH), and tryptophan–aspartic acid repeat (also known as WD40 or WD repeat) proteins, known to regulate flavonoid biosynthesis have been identified in various plant species. However, there is limited information available on WD repeat proteins in lilies. In this study, we identified a WD repeat gene in the Oriental hybrid lily ‘Sorbonne’ (Lilium hybrid WD repeat, LhWDR). LhWDR contains no introns, and has a 1100–base pair open reading frame, encoding a putative protein of 370 amino acids. LhWDR was found to be localized in the cytoplasm of transgenic Arabidopsis thaliana root cells. Expression patterns of LhWDR in different organs and at different periods of lily tepal growth revealed that the expression levels of this gene are closely associated with anthocyanin accumulation. A yeast two-hybrid assay demonstrated that full-length LhWDR interacts with the 420 N-terminal amino acids of Lilium hybrid bHLH2. Interestingly, overexpression of LhWDR in A. thaliana led to an upregulation of the dihydroflavonol 4-reductase gene, which is an important structural gene downstream of the anthocyanin pathway. These results indicate that the WD repeat protein LhWDR might interact with a bHLH transcription factor to regulate anthocyanin biosynthesis.

Lilies are popular ornamental plants globally, owing to their range of tepal colors and floral scents. Lilium (Liliaceae) consists of more than 100 species belonging to several sections (Fatihah et al., 2019; Lim et al., 2008). Hybridization within each section is easy, and the hybrids are divided into several groups: longiflorum, trumpet, Oriental, and Asiatic. Oriental hybrid lilies are popular in the cut flower industry because of the aesthetic flower shape, fragrance, and color (Yamagishi and Akagi, 2013). Oriental hybrid lily flowers are mostly white and pink, and anthocyanins are the major pigments that accumulate in the tepals (Wang and Yamagishi, 2019). Anthocyanins are found in a broad range of plant species, in which they contribute to the vivid colors of flowers, including red, pink, purple, and blue. The colors conferred by anthocyanins to plants are used to attract pollinators and seed-dispersing animals (Winkel-Shirley, 2001) and play important roles in fertility (Grotewold, 2006) and oxidation resistance (Gould et al., 2002).

Anthocyanin biosynthesis is a branch of the flavonoid metabolic pathway, and a range of enzymes are responsible for the synthesis of anthocyanin (Nie et al., 2015). The major enzymes include chalcone synthase (CHS), chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) (Nie et al., 2015). CHS and CHI are early enzymes of anthocyanin biosynthesis, and DFR and ANS are late enzymes. The activity of enzymes involved in anthocyanin biosynthesis is mainly regulated by MYB-bHLH-WD repeat (MBW) ternary complexes composed of R2R3-MYB transcription factors (TFs), a bHLH protein, and WD repeat proteins (Albert et al., 2014; Baudry et al., 2004; Lai et al., 2013; Xu et al., 2015). In plants, R2R3-MYB proteins are among the most abundant transcription factor subfamilies involved in regulating the development of phenotypes, such as plant color (Yamagishi, 2018). The bHLH proteins contain a basic helix-loop-helix region, which plays an important role in DNA binding (Nakatsuka et al., 2009). The WD repeat proteins are characterized by WD repeat motif, which is defined as an ≈40-amino acid structure ending with tryptophan–aspartic acid dipeptide (Lloyd et al., 2017). WD repeat proteins generally serve as a protein–protein interaction platform for the formation of complexes and as mediators of transient connections between other proteins, thereby stabilizing complexes by binding MYBs or interacting with bHLHs (Lloyd et al., 2017). The mechanisms underlying the regulatory role of the MBW protein complex in anthocyanin biosynthesis have been studied in some plant species (de Vetten et al., 1997; Gonzalez et al., 2008; Morita et al., 2006; Quattrocchio et al., 1999; Schwinn et al., 2006; Spelt et al., 2000; Walker et al., 1999; Zhang et al., 2003). In petunia (Petunia ×hybrida), ANTHOCYANIN 1 (AN1) and ANTHOCYANIN 2 (AN2) encode bHLH and MYB TFs, respectively (Mol et al., 1996), whereas ANTHOCYANIN 11 (AN11) encodes a WD repeat protein (de Vetten et al., 1997). AN1, AN2, and AN11 form a regulatory network to determine the timing, level, and patterning of anthocyanin accumulation (Albert et al., 2011). In Arabidopsis thaliana, MBW complexes regulate the expression of late anthocyanin synthesis genes to promote anthocyanin production and pigmentation in the seedcoat (Baudry et al., 2004; Gonzalez et al., 2008; Matus et al., 2010).

In lilies, some of the TFs that regulate anthocyanin biosynthesis have been previously described; among which, R2R3-MYBs are the most extensively studied. Lilium has several R2R3-MYB TFs with different expression profiles, which contribute to a diverse range of color patterns in the flower tepals (Yamagishi et al., 2018). Lilium MYB12 (LhMYB12) generally regulates the synthesis of anthocyanins in the whole tepals in Asiatic hybrids (Yamagishi et al., 2010, 2012) and Oriental hybrids (Yamagishi, 2011). LhMYB12-Lat regulates the development of splatter-type spots in the Tango Series cultivars of Asiatic hybrid lilies (Yamagishi et al., 2014b), whereas Lilium regale MYB15 (Lr MYB15) is related to the bud-blush pigmentation (Yamagishi, 2016), and LhMYB18 is associated with the big-spot pigmentation in Asiatic hybrid lilies (Yamagishi et al., 2018). Two bHLH-type TFs have been found in lily, and LhbHLH2 is directly related to anthocyanin synthesis (Nakatsuka et al., 2009). It has been confirmed that in Lilium flowers, several R2R3-MYBs control the formation of anthocyanin in conjunction with LhbHLH2 (Nakatsuka et al., 2009; Yamagishi, 2016; Yamagishi et al., 2010, 2014a). So far, the overall molecular mechanisms that underlie tepal pigmentation remain limited. Little is known about the regulation of WD repeat protein in the process of anthocyanin synthesis in lilies.

WD repeat proteins exist in all eukaryotes and participate in a range of biological processes, such as signal transduction, pre–messenger RNA (mRNA) splicing, transcriptional regulation, cytoskeletal assembly, and vesicular trafficking (Neer et al., 1994). WD repeat proteins typically have several conserved regions of 40 to 60 amino acids, the sequences of which start with an N-terminal glycine-histidine (GH) dipeptide and end with a C-terminal WD dipeptide (Kamran et al., 2014; Lai et al., 2012; Neer et al., 1994; Smith et al., 1999). Although WD repeat domains are essential for protein–protein interactions (Yang and Sale, 1998), they lack DNA-binding regions (del March Naval et al., 2016). Recently, the regulation of anthocyanin synthesis by WD repeat protein has been reported in many plants. In petunia, AN11 is a conserved cytoplasmic component of the signal transduction cascade that regulates AN2 function, and thus activates cellular signaling and transcriptional activation (de Vetten et al., 1997). AN11 is capable of intercellular movement in the petal epidermis, which may facilitate anthocyanin pigment pattern formation (Albert et al., 2014). In A. thaliana, TRANSPARENT TESTA GLABRA 1 (TTG1, a WD repeat protein) interacts with R2R3-MYB and bHLH proteins to regulate the production of proanthocyanidin, anthocyanidin, seedcoat mucilage, trichomes, and root hairs (Albert et al., 2014; Feller et al., 2011; Liu et al., 2017). A ttg1 mutant has been reported to be defective in anthocyanin function and the production of seedcoats and trichomes (Walker et al., 1999). Overexpression of Fagopyrum tataricum WD40 (FtWD40) in tobacco (Nicotiana tabacum) has been found to induce upregulated expression of the DFR and ANS genes and increase the accumulation of anthocyanins (Yao et al., 2017). However, little is currently known regarding the regulation of WD repeat proteins during the process of anthocyanin synthesis in lilies. To date, based on an analysis of RNA-sequencing (RNA-seq) data, Suzuki et al. (2016) found a WD40 gene, which was related to the synthesis of anthocyanin lilies, namely LhWD40a. LhWD40a is a unigene that was identified by blasting the amino acid sequence of TTG1 against the Lollypop sequence database. However, the authors of this study obtained only a part of the coding region of the LhWD40a gene, and did not conduct further research on it. Therefore, more WD repeat genes need to be researched in lilium, which is of great significance for studying the regulation of MBW complex in the anthocyanin synthesis process.

In this study, a novel WD repeat gene, LhWDR, was isolated from the Oriental hybrid lily ‘Sorbonne’. The relationships between LhWDR and other proteins involved in regulating the anthocyanin biosynthetic pathway were also investigated to determine the function of LhWDR in the regulatory network of anthocyanin biosynthesis.

Materials and Methods

Plant materials.

The Oriental hybrid lily ‘Sorbonne’ was used for gene cloning and gene expression assays. Bulbs of ‘Sorbonne’ were purchased from Beijing Plant Horticulture Co. (Beijing, China) and grown in a greenhouse (natural conditions without heating) of the Beijing Radiation Center (Beijing, China). To analyze the gene expression patterns in different organs, we collected the roots, leaves, stems, bulbs, tepals, and anthers of ‘Sorbonne’. A. thaliana ecotype Columbia (Col) was grown in soil at 22 °C under a 16/8-h (light/dark) photoperiod.

Isolation of the LhWDR gene.

Total RNA was extracted from tepals of ‘Sorbonne’ at stage 4 (S4) or stage 5 (S5) of growth using an RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan). First-strand DNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Degenerate primers were designed based on the sequences of TTG1, AN11, Perilla frutescens WD repeat gene (PFWD), Vitis vinifera WD repeat gene (VvWDR1), and PALE ALEURONE COLOR1 (PAC1) WD repeat gene from Zea mays. A partial complementary DNA (cDNA) fragment of the LhWDR gene was amplified using the primers LhWDR-C-5′ and LhWDR-C-3′ (Table 1). The fragment was then inserted into a pEASY-T1 cloning vector using the TA cloning method (TransGen Biotech, Beijing, China), and the recombinant vector was sequenced. The 5′ and 3′ ends of the cDNA sequences were amplified using a SMARTer Rapid Amplification of cDNA ends (RACE) Kit (Clontech, Mountain View, CA). The primers used in this study are listed in Table 1. Full-length cDNA was amplified with rTaq (TaKaRa, Tokyo, Japan) using the primers LhWDR-F and LhWDR-R (Table 1). The DNA fragment thus obtained was subsequently sequenced. Genomic DNA was isolated from the young leaves of ‘Sorbonne’ using the DNA Plant System Kit (Tiangen, Beijing, China). Sequencing and primer synthesis were performed by the Majorbio Biology Technology Co. (Beijing, China).

Table 1.

Primer names, sequences, and the amplified fragments used for polymerase chain reaction amplification.

Table 1.

Sequence analysis.

Multiple WD repeat sequences were aligned using DNAMAN 8 (Lynnon Biosoft, San Ramon, CA). A phylogenetic tree was constructed using the neighbor-joining method (Saitou and Nei, 1987) and was visualized using MEGA version 6. Tree nodes were evaluated for 1000 replicates via bootstrap analysis. Publicly available sequences were obtained from GenBank via the National Center for Biotechnology Information (Bethesda, MD). The amino acid sequence was analyzed using the MyHits website of the Swiss Institute of Bioinformatics (University of Lausanne, Lausanne, Switzerland).

Determination of total anthocyanin content.

The tepals of ‘Sorbonne’ flowers were ground in liquid nitrogen, and the total anthocyanin content was determined according to the method of Rabino and Mancinelli (1986). We added 5 mL of acidic methanol solution (1% HCl, v/v) per gram of tepals material and shook at 4 °C for 48 h. Absorbances of the filtered extracts were subsequently measured at wavelengths of 530 and 657 nm. The anthocyanin contents in the preparations were determined using the formula: (A530 – 0.25 A657)/M, in which A530, and A657 are the absorptions at 530 and 657 nm, respectively, and M is the weight of tepals (grams). For each sample, we performed determinations for three replicates.

Gene expression analysis.

The roots, stems, bulbs, leaves, anthers, and tepals of ‘Sorbonne’ were harvested immediately after flowering (S5). Total RNA was obtained as previously described for isolating the LhWDR gene. Approximately 2 μg of total RNA was used to synthesize first-strand cDNA using a Transcriptor First Strand cDNA Synthesis Kit and an anchored-oligo (dT)18 primer. The reaction product thus obtained was then directly used as a template for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR amplification.

RT-PCR was performed using 20 μL reaction solution containing 1 μL of first-strand cDNA and 2 × TransTaq-T PCR SuperMix (TransGen Biotech). Lilium Actin gene (LhActin) cDNA was used as an internal control to normalize the amounts of cDNA. The primers LhActin-RT1 and LhActin-RT2 (Table 1) were used to amplify the LhActin cDNA fragment. The primers LhWDR-RT1 and LhWDR-RT2 (Table 1) were used to amplify the LhWDR cDNA fragment. Assessments were carried out as described by Yang et al. (2009). The expression levels of LhWDR, LhbHLH2, LhMYB12, LhCHS, LhCHI, LhDFR, and LhANS were determined using 20-μL reaction solutions containing 2 μL of 10× diluted first-strand cDNA and a LightCycler 480 SYBR Green I master mix (Roche, Basel, Switzerland). PCR amplification was performed in a real-time PCR instrument (480, Roche) using the following program: 95 °C for 10 mins, followed by 40 cycles of 95 °C for 30 s and 60 °C for 1 min.

LhActin levels were quantified as an internal control for lily gene expression to normalize the RNA levels. The primers used to amplify the genes LhWDR, LhbHLH2, LhMYB12, LhCHS, LhCHI, LhDFR, and LhANS are listed in Table 1. The relative mRNA levels of the reaction products were calculated using the comparative Ct method (Liu et al., 2007). All PCRs were performed in triplicate and repeated twice.

The gene names and accession numbers are as follows: LhWDR (KY706354), LhActin (AB438963) LhbHLH2 (BAE20058), LhMYB12 (BAJ05398), LhCHS (HQ161731), LhCHI (KJ784468), LhDFR (BAM28975), and LhANS (BAM28976).

Determination of protein subcellular localization.

The LhWDR open reading frame (ORF) lacking stop codon was amplified with rTaq using the primers LhWDR-CDS-F and LhWDR-CDS-R (Table 1). After verification by sequencing, the LhWDR coding sequence (CDS) fragment was cloned in front of the GREEN FLUORESCENT PROTEIN (GFP) sequence in a modified pCAMBIA1300 vector containing the Cauliflower mosaic virus 35S promoter and a GFP-coding region, resulting in the 35S:LhWDR-GFP-NOS construct. This construct was initially transformed into Agrobacterium tumefaciens (strain GV3101) and then into A. thaliana wild-type Col plants via Agrobacterium-mediated flower bud transformation (Bechtold and Pelletier, 1998). The transformed plants were selected using 20 mg·L−1 hygromycin. The localization of the LhWDR-GFP fusion protein in the roots of transgenic plants was observed using a confocal microscope LSM510 (Carl Zeiss, Oberkochen, Germany).

Ectopic expression of LhWDR in A. thaliana.

The 35S:LhWDR-GFP-NOS transgenic plants were used to analyze the effects of related genes in A. thaliana with wild-type plants being used as a control. We selected four T2 lines homozygous for the insertion with strong fluorescence for detection, which were named OE1 to OE4 with wild-type plants being used as a control. The method used for the determination of A. thaliana CHS (AtCHS), A. thaliana (AtCHI), DFR, ANS, A. thaliana PURPLE ACID PHOSPHATASE 1 (AtPAP1), and GLABRA 3 (GL3) gene expression levels was as described previously. The 18S ribosomal RNA (18S rRNA) level was used as internal control to normalize the RNA levels. The primers used to amplify the A. thaliana genes are listed in Table 1. The gene names and accession numbers are as follows: AtCHS (At5g13930), AtCHI (At3g55120), DFR (At5g42800), ANS (At4g22880), AtPAP1 (At1g13750), and GL3 (At5g41315). Three independent plants of each homozygous T2 insertion line were selected for biological replication.

Yeast two-hybrid assays.

The Gal4 vector system was used for yeast two-hybrid (Y2H) assays (Clontech, Mountain View, CA). The CDS of LhWDR was cloned into the pGADT7 vector. The full-length CDS and a CDS fragment of LhbHLH were cloned into the pGBKT7 vector. These constructs were cotransformed into the yeast strain Y187 using the PEG/LiAC method, as described by Causier and Davies (2002). The transformed Y187 cells were adjusted to an OD600 of 0.3–0.4 and then cultured on synthetic medium plates lacking tryptophan and leucine acid (SD/-Trp/-Leu) and synthetic medium plates lacking tryptophan, leucine, histidine, and adenine acid (SD/-Trp/-Leu/-His/-Ade) plates for 2 to 6 d at 30 °C. The primers used in this experiment are shown in Table 1.

Results

Cloning of the LhWDR gene from the tepals of Oriental hybrid lily.

Our primary aim was to isolate a WD repeat gene that may be involved in anthocyanin biosynthesis in Lilium. Using homology cloning and RACE technology, full-length cDNA of the LhWDR gene was isolated from the tepals of Oriental hybrid lily ‘Sorbonne’, including 15 base pairs (bp) of the 5′ untranslated region (UTR), 1110 bp of CDS, and 244 bp of 3′ UTR. Comparisons of the genomic DNA and cDNA sequences indicated that LhWDR lacks introns, as has previously been observed for TTG1 and AN11. The sequence of LhWDR has been deposited in GenBank under the accession number KY706354.

Sequence and phylogenetic analyses.

Sequence analysis revealed that the full-length cDNA of LhWDR consists of a 1110-bp ORF that encodes a predicted protein of 370 amino acids (Fig. 1A). The results of a basic local alignment search tool revealed that LhWDR contains five WD repeat sequences, and the deduced amino acid sequence indicated sequence similarity with other members of the WD protein family. The amino acid sequence was highly conserved with respect to WD repeats 2–5 compared with those of WD repeat proteins from A. thaliana, Z. mays, V. vinifera, and Gossypium hirsutum (Fig. 1A). Most of the homology was observed within the WD repeat motifs. The core length of the WD repeat motifs was ≈40 amino acids, and these motifs were characterized by GH and WD doublet residues. Multiple sequence alignment of the deduced amino acid sequences revealed that LhWDR shared 83% amino acid identity with the V. vinifera WD repeat 2 (VvWDR2) protein, 81% with A. thaliana AN11 (AtAN11), 80% with G. hirsutum TRANSPARENT TESTA GLABRA 4 (GhTTG4), 65% with Z. mays MP1 and Sorghum bicolor WD repeat (SbWDR) (Fig. 1A). WD repeat proteins associated with plant flavonoids are classified in the 4WD-repeat subfamily, which is divided into the PAC1 and MP1 clades (Carey et al., 2004). The phylogenetic tree we constructed using LhWDR and homologous proteins from fourteen other species (Fig. 1B) revealed that LhWDR clustered in the MP1 clade, which included VvWDR2, MP1, and AtAN11.

Fig. 1.
Fig. 1.

Protein sequence alignment and phylogenetic tree of Lilium hybrid WD repeat (LhWDR) and selected tryptophan–aspartic acid (WD) repeat domains. (A) The deduced protein sequence of LhWDR was compared with the sequences of Gossypium hirsutum TRANSPARENT TESTA GLABRA 2 (GhTTG2), G. hirsutum TRANSPARENT TESTA GLABRA 4 (GhTTG4), Zea mays MP1, Sorghum bicolor WD repeat (SbWDR), Vitis vinifera WD repeat 2 (VvWDR2), and Arabidopsis thaliana ANTHOCYANIN 11 (AtAN11). Predicted WD repeats are labeled (WD-1 to WD-5). (B) The tree was constructed using the neighbor-joining method and bootstrapped with 1000 replicates. The scale bar indicates the branch length that corresponds to the number of substitutions per amino acid position. The names and GenBank accession numbers are as follows: GhTTG2, AAM95644; GhTTG4, AAM95646; Morus notabilis WD repeat (MnLWD1), XP_010090944; Populus trichocarpa WD repeat (PtWDR), XP_002301564; Arachis ipaensis WD repeat 1 (AiLWD1), XP_016162111; AtAN11, AAC18912; Glycine max WD repeat (GmWDR), XP_003553993; VvWDR2, XP_010660592; Ipomoea batatas WD repeat (IpWDR), AHM25607; Z. mays PALE ALEURONE COLOR1 (PAC1), AAM76742; Populus euphratica WD repeat 1 (PeLWD1), XP_011003601; LhWDR, KY706354; SbWDR, XP_002453914; MP1, AY339884; Oryza sativa Japonica group WD repeat 1 (OsLWD1), XP_015627230; G. hirsutum TRANSPARENT TESTA GLABRA 3 (GhTTG3), AAM95645; Perilla frutescens WD repeat gene (PFWD), BAB58883; G. hirsutum TRANSPARENT TESTA GLABRA 1 (GhTTG1), AAM95641; A. thaliana TRANSPARENT TESTA GLABRA 1 (TTG1), CAB45372; Petunia ×hybrida ANTHOCYANIN 11(PhAN11), AAC18914; and V. vinifera WD repeat 1 (VvWDR1), ABF66625. The proteins in parentheses indicated that they were clustered in PALE ALEURONE COLOR1 or MP1 clade.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 2; 10.21273/JASHS04791-19

Expression patterns of LhWDR and anthocyanin-related genes.

The expression pattern of the LhWDR gene was observed during plant development and inflorescence. RT-PCR and quantitative real-time PCR were performed to evaluate the expression levels of LhWDR in different plant tissues. The results revealed that LhWDR is expressed in all the tissues we examined (Fig. 2A), which is similar to the expression patterns of the constitutively expressed genes AN11 and PFWD (de Vetten et al., 1997; Sompornpailin et al., 2002). However, the expression levels of LhWDR varied in different tissues, with the pink tepals and light-pink bulbs exhibiting higher transcript levels than other tissues [roots, leaves, stems, and anthers (Fig. 2A and B)], suggesting that the expression of LhWDR may be associated with pigmentation.

Fig. 2.
Fig. 2.

Expression patterns of Lilium hybrid WD repeat (LhWDR). (A) Reverse-transcription polymerase chain reaction (RT-PCR) assay to determine LhWDR mRNA levels in roots, stems, bulbs, leaves, tepals, and anthers. (B) Quantitative real-time PCR assays to determine the relative expression of LhWDR in roots, stems, bulbs, leaves, tepals, and anthers. (C and D) Quantitative real-time PCR assays to determine the relative expression levels of LhCHS, LhCHI, LhDFR, LhANS, LhWDR, LhMYB12, and LhbHLH2 in stage 2 (S2) to stage 5 (S5) tepals. (E) The process of ‘Sorbonne’ inner tepal coloring. (F) Quantification of total anthocyanins in ‘Sorbonne’ inner tepals at S2 to S5 of development. Each value represents the mean of three replicates, and error bars indicate standard deviations (±SD); white bar = 1 cm.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 2; 10.21273/JASHS04791-19

The transcription profiles of the genes during flower development from S2 to S5 were also investigated using quantitative real-time PCR. Purple spots began to appear on the tepals of ‘Sorbonne’ from S2 and the tepals became pale pink at S3, and had turned dark pink by S5 (Fig. 2E). During the period of development from S2 to S5, we detected a gradual increase in the total content of anthocyanins (Fig. 2F). Quantitative real-time PCR results showed that there was an accumulation of LhWDR transcripts from S2 to S5, with transcription peaking at S4 (Fig. 2C). Furthermore, the transcription profiles of LhMYB12 and LhbHLH2 were found to be similar to the transcription profile of LhWDR, in that transcription peaked at S4. However, the increase in LhMYB12 transcript levels was higher than that in the transcript levels of LhWDR and LhbHLH2. Subsequent analysis of four key structural genes from lilies predicted to be involved in flavonoid and anthocyanin biosynthesis, namely, LhCHS, LhCHI, LhDFR, and LhANS, revealed that the transcription of these genes was low or undetectable at S2 and highest at S4 or S5. Moreover, expression of the LhDFR and LhANS genes was observed to be similar to that of LhMYB12, LhbHLH2, and LhWDR (Fig. 2D). The similarity of these expression patterns accordingly indicated that LhWDR may be associated with the anthocyanin metabolic pathway.

Subcellular localization of the LhWDR protein.

To investigate the subcellular localization of LhWDR, the p35S:LhWDR-GFP construct was transformed into wild-type A. thaliana and a total of 47 transgenic plants were subsequently obtained. Observation of the root tips of T1 transgenic plants revealed that 31 plants showed green fluorescence, and PCR analysis indicated that all of these plants had the transgene insertion. The GFP signal was observed in transformed root cells by comparison with cells stained with propidium iodide. The LhWDR-GFP fusion proteins were observed under fluorescent light as green signals localized in the cytoplasm (Fig. 3A), whereas the signals obtained with the control vector 35S:GFP were distributed throughout the cells (Fig. 3B). These results are consistent with those obtained for AN11 and PFWD, and the proteins encoded by these genes were also detected in the cytoplasm (de Vetten et al., 1997; Sompornpailin et al., 2002).

Fig. 3.
Fig. 3.

Subcellular localization of Lilium hybrid WD repeat–green fluorescent protein (LhWDR-GFP) in Arabidopsis thaliana root cells. (A) The GFP signals of LhWDR-GFP were located in the cytoplasm of A. thaliana root cells. (B) Signals of 35S-GFP (positive control). Propidium iodide (PI) was used to stain the cell profile and the signal was merged with the GFP signal in the right hand column. Scale bar = 1 μm.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 2; 10.21273/JASHS04791-19

Ectopic expression of LhWDR promotes DFR gene expression in A. thaliana.

Observations of T1 plants harboring the p35S:LhWDR-GFP construct throughout the growth cycle revealed no significant phenotypic changes. To characterize the function of LhWDR in anthocyanin biosynthesis, four T1 plants with strong GFP fluorescence (OE1–OE4) were selected to obtain homozygous T2 generation plants by selfing. The homozygous transgenic insertion was selected for gene detection and we found that the LhWDR gene was overexpressed in the T2 OE1–OE4 lines (Fig. 4A and B). Quantitative real-time PCR analysis of the expression levels of the anthocyanin biosynthesis genes ATCHS, ATCHI, DFR, and ANS; the regulatory MYB TF gene ATPAP1; and the bHLH gene GL3 revealed that the average expression level of DFR in the transgenic plants was twice that in the control plants (Fig. 4C), whereas no clear changes were observed in the expression of the other genes (Fig. 4C). Our findings indicated that overexpression of the LhWDR gene upregulates DFR, a key gene in late anthocyanin synthesis in A. thaliana, and we thus speculate that LhWDR may participate in the anthocyanin synthesis pathway by regulating the transcription of DFR.

Fig. 4.
Fig. 4.

Expression levels of related genes in Arabidopsis thaliana overexpressing Lilium hybrid WD repeat (LhWDR). Reverse-transcription polymerase chain reaction (RT-PCR) (A) and quantitative real-time PCR (B) assays to determine LhWDR mRNA levels in wild-type plant (WT) and LhWDR-overexpressing plants (OE1–OE4). (C) Expression levels of AtCHS, AtCHI, DFR, ANS, AtPAP1, and GL3 in WT and OE1–OE4 plants. **Significant difference at the 1% level (t-test).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 2; 10.21273/JASHS04791-19

The LhWDR protein interacts with LhbHLH2 in yeast.

In A. thaliana, TTG1 has been shown to interact with the bHLH TFs GL3 and ENHANCER OF GLABRA3 (EGL3) to regulate the synthesis of procyanidins and the formation of epidermal hairs (Walker et al., 1999; Zhang et al., 2003). To date, two bHLH TFs have been studied in Asiatic hybrid lilies (LhbHLH1 and LhbHLH2); however, only LhbHLH2 has been reported to regulate anthocyanin metabolism, via regulating the expression of the downstream gene LhDFR (Nakatsuka et al., 2009). To examine the relationship between LhWDR and LhbHLH2, we used a Y2H system. LhbHLH2 is a self-activating protein (Fig. 5) owing to the bHLH structure, which is 420 to 600 amino acids long. Therefore, the sequence of LhbHLH2 was divided into two fragments when constructing the vectors. The N-terminal 420 amino acids were cloned into pGBKT7 to construct the LhbHLH2-420-BD vector, whereas the C-terminal 260 amino acids were cloned into pGBKT7 to construct the LhbHLH2-260-BD vector. The results showed that the fragment comprising the N-terminal 420 amino acids did not exhibit self-activation, whereas the C-terminal 260 amino acids fragment was self-activated (Fig. 5). The full-length LhWDR was cloned into pGADT7 to construct the LhWDR-AD vector and the Y2H assay revealed that this full-length LhWDR could interact with the N-terminal 420 amino acids of the LhbHLH2 protein (Fig. 5).

Fig. 5.
Fig. 5.

Yeast two-hybrid analysis of protein-protein interactions. The cotransformed yeast cells were grown on synthetic medium plates lacking tryptophan and leucine acid (SD/-Trp/-Leu) at 30 °C for 2 d and synthetic medium plates lacking tryptophan, leucine, histidine, and adenine acid (SD/-Trp/-Leu/-His/-Ade) at 30 °C for 3–6 d. The yeast cell carrying AD/BD vectors were used as a negative control. The yeast cell carrying AD/LhbHLH2 and AD/LhbHLH2–260 grew on SD/-Trp/-Leu/-His/-Ade suggesting that the full-length amino acids of LhbHLH2 and the C-terminal 260 amino acids fragment of LhbHLH2 were self-activated. The yeast cell carrying AD/LhbHLH2–420 and LhWDR/BD could not grow on SD/-Trp/-Leu/-His/-Ade, suggesting that the N-terminal 420 amino acids fragment of LhbHLH2 and the full-length amino acids of LhWDR were not self-activated. The yeast cell carrying LhWDR/ LhbHLH2–420 grew on SD/-Trp/-Leu/-His/-Ade and the 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) was blue, indicating that the full-length LhWDR could interact with the N-terminal 420 amino acids of the LhbHLH2 protein. AD = pGADT7 vector, BD = pGBKT7 vector.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 145, 2; 10.21273/JASHS04791-19

Discussion

WDR anthocyanin regulatory proteins are highly conserved.

WD repeat motifs are typically 40 to 60 amino acids in length, and the 40- to 60-residue sequence generally contains a GH dipeptide in the N-terminal region and a WD dipeptide in the C-terminal region (Kamran et al., 2014; Smith et al., 1999). Studies have reported that the motif generally consists of 4 to 10 tandemly repeated units that form a beta-propeller structure (van Nocker and Ludwig, 2003). In the present study, we cloned an anthocyanin biosynthesis regulatory gene (LhWDR) belonging to the WD repeat gene family, which is characterized by five WD repeat sequences. In each WD repeat motif of the LhWDR protein, the core length between the GH and WD sequences is ≈40 amino acids. We found that WD repeats 2 to 5 have >80% identity with the corresponding repeats in WD regions of homologous proteins. Plant flavonoid-related WD repeat proteins have been grouped into two clades, namely, the PAC1 and MP1 clades (Matus et al., 2010), and the phylogenetic tree we constructed revealed that LhWDR clustered in the MP1 clade. It is predicted that the members of both PAC1 and MP1 clades function in DNA and protein binding, and based on sequence alignment, it has been found that the two clades differ with respect to the number and position of WD repeat domains. Previous analysis has revealed that MP1 clade sequences lack nuclear localization signals (Yao et al., 2017), and we have found that neither PAC1 nor MP1 exhibit typical nuclear localization signals. Both AN11 and PFWD belong to the PAC1 clade, and the proteins encoded by these genes have been detected in the cytoplasm (de Vetten et al., 1997; Sompornpailin et al., 2002). Based on subcellular localization experiments, we confirmed that LhWDR is localized in the cytoplasm rather than in the nucleus.

To date, there have been numerous studies that have focused on the genes of WD repeat proteins in the PAC1 cluster, among which, TTG1 is essential for the accumulation of purple anthocyanins in leaves and stems, as well as the development of hairs and root hairs (Walker et al., 1999). The fragment of LhWD40a gene obtained by Suzuki et al. (2016) through analyzing RNA-seq also belongs to the PAC1 clade. Overexpression of homologous genes from the PAC1 cluster proteins, such as VvWDR1, GhTTG1/3, and Setaria italica TTG1, in A. thaliana ttg1 mutant plants has been found to restore many of the mutant phenotypes (Humphries et al., 2005; Liu et al., 2017; Matus et al., 2010). In contrast, overexpression of the genes VvWDR2, G. hirsutum TRANSPARENT TESTA GLABRA 2 (GhTTG2), and GhTTG4, which are clustered in the MP1 clade, was found to be ineffective in restoring ttg1 phenotypes (Humphries et al., 2005; Matus et al., 2010). Although LhWDR, which is also an MP1 clade member, has been observed to increase the expression level of DFR when ectopically expressed in Arabidopsis, LhWDR cannot complement anthocyanin and trichome defects in the ttg1 mutant (data not shown). These results thus indicate that, although both MP1 and PAC1 contain WD40 family proteins related to anthocyanin synthesis, the two clades are not functionally redundant. The fragment of LhWD40a gene obtained by Suzuki et al. (2016) through analyzing RNA-seq also belongs to the PAC1 clade.

Regulation of LhWDR in anthocyanin synthesis.

Anthocyanin biosynthesis is generally regulated by the MBW complex, and the ratios and amounts of R2R3-MYB, bHLH, and WDR transcripts determine the amount of anthocyanin produced (De Majnik et al., 1998). In Asiatic hybrid lilies, changes in the expression levels of LhMYB12 have been found to be consistent with changes in the anthocyanin contents in tepals, filaments, and styles (Yamagishi et al., 2010). Moreover, the transcription of LhbHLH2 has been detected in most tissues and found to be positively correlated with anthocyanin accumulation (Nakatsuka et al., 2009). In the present study, we observed that LhWDR is transcribed in most organs of ‘Sorbonne’, and high transcript levels were observed in bulb scales and tepals, which are the two pigmented organs in ‘Sorbonne’. Anthocyanins are the main visible pigments in Oriental hybrid lily flowers (Yamagishi, 2011), and the observed correlation between the expression levels of LhWDR and pigmentation suggest that LhWDR might be associated with anthocyanin synthesis. In lilies, LhCHS, LhCHI, LhDFR, and LhANS are the major enzymes involved in the synthesis of anthocyanin pigments, with the highest expression levels of these genes being detected in the S4 or S5 tepals. LhMYB12 has been found to directly activate the LhCHSa and LhDFR genes (Lai et al., 2012; Yamagishi, 2011), and LhDFR transcription has been demonstrated to be associated with the activity of LhbHLH2 (Nakatsuka et al., 2009). In the present study, we found that LhWDR, LhMYB12, LhbHLH2, LhDFR, and LhANS showed similar patterns of expression, which might indicate interactions or regulatory relationships among these genes. When we overexpressed LhWDR in A. thaliana, although we detected no epidermal or anthocyanin-related abnormalities in the transgenic plants, the expression level of the DFR gene increased with overexpression of LhWDR, and the expression of GL3 was also slightly upregulated. DFR is a late gene of flavanol biosyntheses and is necessary for anthocyanin biosynthesis, and ANS is located downstream of DFR and directly controls the synthesis of anthocyanins. The expression of ANS is directly related to the amounts of anthocyanins, and in the present study, we found that whereas the overexpression of LhWDR in A. thaliana increased the expression of the DFR gene, it did not significantly affect the expression of ANS, nor did it alter the content of anthocyanin. We accordingly suspect that LhWDR might participate in anthocyanin biosynthesis pathways by regulating the DFR gene.

Interaction between LhWDR and LhbHLH2.

In Arabidopsis, both GL3 and EGL3 encode bHLH proteins, which interact with TTG1 (Zhang et al., 2003). The gl3 egl3 double mutant has been found to exhibit phenotypes similar to those of the ttg1 mutant, such as defects in anthocyanin production, seedcoat mucilage production, and root hair growth (Zhang et al., 2003). MYB proteins in A. thaliana, such as GL1, PAP1, PAP 2, CAPRICE, and TRIPTYCHON, form heterodimers with GL3, and in the MBW model, which is dependent on TTG1, there is more than one member of the MYB and bHLH protein families involved and functional redundancy within the same family (Zhang et al., 2003). Although both LhbHLH1 and LhbHLH2 are bHLH-type TFs in lilies, only LhbHLH2 is involved in mediating anthocyanin metabolism (Nakatsuka et al., 2009). Similarly, LhMYB6 and LhMYB12 are MYB proteins that mediate anthocyanin metabolism in lilies, and Y2H analysis has revealed that both these proteins interact with LhbHLH2 (Yamagishi et al., 2010).

In the present study, we used the Y2H assay to examine the relationship between LhWDR and LhbHLH2. We accordingly found that that the C-terminal 240 amino acids of LhbHLH2 exhibited self-activation, and therefore used the remaining 420-amino acid sequence as bait, which was observed to interact with the full-length LhWDR. In addition, we examined the interaction between the WD repeat and MYB TFs and found that LhWDR interacted with neither LhMYB6 nor LhMYB12 in the yeast system (data not shown). TFs and coactivators must enter the nucleus to exert regulatory control (Matus et al., 2010), and LhbHLH2 has been shown to be nuclear-localized (Li et al., 2014; Nakatsuka et al., 2009). In contrast, we found that LhWDR is localized in the cytoplasm. Thus, LhWDR would need to enter the nucleus to interact with LhbHLH2 and regulate the expression of downstream genes. This process should be further investigated. Anthocyanin biosynthesis in different tissues is considered to be determined by combinations of the R2R3-MYB, bHLH, and WDR factors and the interactions of these factors, which control the expression of a set of structural genes (Morita et al., 2006). Further examination of the MBW complex will be valuable for elucidating the role of this complex in anthocyanin biosynthesis.

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Contributor Notes

This work was supported by the Beijing Academy of Science and Technology Municipal Financial Project (PXM2019_178217_000001), the 12th Five Years Key Programs for Science and Technology Development of China (2013BAD01B0706), and the BJAST Young Researcher Training Plan (201524).We thank Kezhen Yang for providing Arabidopsis thaliana seeds and transformation vectors and Lei Zhu for her assistance with yeast two-hybrid experiments.J.B. is the corresponding author. E-mail: bjr301@126.com
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    Protein sequence alignment and phylogenetic tree of Lilium hybrid WD repeat (LhWDR) and selected tryptophan–aspartic acid (WD) repeat domains. (A) The deduced protein sequence of LhWDR was compared with the sequences of Gossypium hirsutum TRANSPARENT TESTA GLABRA 2 (GhTTG2), G. hirsutum TRANSPARENT TESTA GLABRA 4 (GhTTG4), Zea mays MP1, Sorghum bicolor WD repeat (SbWDR), Vitis vinifera WD repeat 2 (VvWDR2), and Arabidopsis thaliana ANTHOCYANIN 11 (AtAN11). Predicted WD repeats are labeled (WD-1 to WD-5). (B) The tree was constructed using the neighbor-joining method and bootstrapped with 1000 replicates. The scale bar indicates the branch length that corresponds to the number of substitutions per amino acid position. The names and GenBank accession numbers are as follows: GhTTG2, AAM95644; GhTTG4, AAM95646; Morus notabilis WD repeat (MnLWD1), XP_010090944; Populus trichocarpa WD repeat (PtWDR), XP_002301564; Arachis ipaensis WD repeat 1 (AiLWD1), XP_016162111; AtAN11, AAC18912; Glycine max WD repeat (GmWDR), XP_003553993; VvWDR2, XP_010660592; Ipomoea batatas WD repeat (IpWDR), AHM25607; Z. mays PALE ALEURONE COLOR1 (PAC1), AAM76742; Populus euphratica WD repeat 1 (PeLWD1), XP_011003601; LhWDR, KY706354; SbWDR, XP_002453914; MP1, AY339884; Oryza sativa Japonica group WD repeat 1 (OsLWD1), XP_015627230; G. hirsutum TRANSPARENT TESTA GLABRA 3 (GhTTG3), AAM95645; Perilla frutescens WD repeat gene (PFWD), BAB58883; G. hirsutum TRANSPARENT TESTA GLABRA 1 (GhTTG1), AAM95641; A. thaliana TRANSPARENT TESTA GLABRA 1 (TTG1), CAB45372; Petunia ×hybrida ANTHOCYANIN 11(PhAN11), AAC18914; and V. vinifera WD repeat 1 (VvWDR1), ABF66625. The proteins in parentheses indicated that they were clustered in PALE ALEURONE COLOR1 or MP1 clade.

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    Expression patterns of Lilium hybrid WD repeat (LhWDR). (A) Reverse-transcription polymerase chain reaction (RT-PCR) assay to determine LhWDR mRNA levels in roots, stems, bulbs, leaves, tepals, and anthers. (B) Quantitative real-time PCR assays to determine the relative expression of LhWDR in roots, stems, bulbs, leaves, tepals, and anthers. (C and D) Quantitative real-time PCR assays to determine the relative expression levels of LhCHS, LhCHI, LhDFR, LhANS, LhWDR, LhMYB12, and LhbHLH2 in stage 2 (S2) to stage 5 (S5) tepals. (E) The process of ‘Sorbonne’ inner tepal coloring. (F) Quantification of total anthocyanins in ‘Sorbonne’ inner tepals at S2 to S5 of development. Each value represents the mean of three replicates, and error bars indicate standard deviations (±SD); white bar = 1 cm.

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    Subcellular localization of Lilium hybrid WD repeat–green fluorescent protein (LhWDR-GFP) in Arabidopsis thaliana root cells. (A) The GFP signals of LhWDR-GFP were located in the cytoplasm of A. thaliana root cells. (B) Signals of 35S-GFP (positive control). Propidium iodide (PI) was used to stain the cell profile and the signal was merged with the GFP signal in the right hand column. Scale bar = 1 μm.

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    Expression levels of related genes in Arabidopsis thaliana overexpressing Lilium hybrid WD repeat (LhWDR). Reverse-transcription polymerase chain reaction (RT-PCR) (A) and quantitative real-time PCR (B) assays to determine LhWDR mRNA levels in wild-type plant (WT) and LhWDR-overexpressing plants (OE1–OE4). (C) Expression levels of AtCHS, AtCHI, DFR, ANS, AtPAP1, and GL3 in WT and OE1–OE4 plants. **Significant difference at the 1% level (t-test).

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    Yeast two-hybrid analysis of protein-protein interactions. The cotransformed yeast cells were grown on synthetic medium plates lacking tryptophan and leucine acid (SD/-Trp/-Leu) at 30 °C for 2 d and synthetic medium plates lacking tryptophan, leucine, histidine, and adenine acid (SD/-Trp/-Leu/-His/-Ade) at 30 °C for 3–6 d. The yeast cell carrying AD/BD vectors were used as a negative control. The yeast cell carrying AD/LhbHLH2 and AD/LhbHLH2–260 grew on SD/-Trp/-Leu/-His/-Ade suggesting that the full-length amino acids of LhbHLH2 and the C-terminal 260 amino acids fragment of LhbHLH2 were self-activated. The yeast cell carrying AD/LhbHLH2–420 and LhWDR/BD could not grow on SD/-Trp/-Leu/-His/-Ade, suggesting that the N-terminal 420 amino acids fragment of LhbHLH2 and the full-length amino acids of LhWDR were not self-activated. The yeast cell carrying LhWDR/ LhbHLH2–420 grew on SD/-Trp/-Leu/-His/-Ade and the 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) was blue, indicating that the full-length LhWDR could interact with the N-terminal 420 amino acids of the LhbHLH2 protein. AD = pGADT7 vector, BD = pGBKT7 vector.

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