Genome-wide transcriptome analysis of genes involved in flavonoid biosynthesis between red and white strains of Magnolia sprengeri pamp
© Shi et al.; licensee BioMed Central Ltd. 2014
Received: 22 March 2014
Accepted: 21 August 2014
Published: 23 August 2014
Magnolia sprengeri Pamp is one of the most highly valuable medicinal and ornamental plants of the Magnolia Family. The natural color of M. sprengeri is variable. The complete genome sequence of M. sprengeri is not available; therefore we sequenced the transcriptome of white and red petals of M. sprengeri using Illumina technology. We focused on the identity of structural and regulatory genes encoding the enzymes involved in the determination of flower color.
We sequenced and annotated a reference transcriptome for M. sprengeri, and aimed to capture the transcriptional determinanats of flower color. We sequenced a normalized cDNA library of white and red petals using Illumina technology. The resulting reads were assembled into 77,048 unique sequences, of which 28,243 could be annotated by Gene Ontology (GO) analysis, while 48,805 transcripts lacked GO annotation. The main enzymes involved in the flavonoid biosynthesis, such as phenylalanine ammonia-Lyase, cinnamat-4-Hydroxylase, dihydroflavonol-4-reductase, flavanone 3-hydroxylase, flavonoid-3′-hydroxylase, flavonol synthase, chalcone synthase and anthocyanidin synthase, were identified in the transcriptome. A total of 270 transcription factors were sorted into three families, including MYB, bHLH and WD40 types. Among these transcription factors, eight showed 4-fold or greater changes in transcript abundance in red petals compared with white petals. High-performance liquid chromatography analysis of anthocyanin compositions showed that the main anthocyanin in the petals of M. sprengeri is cyanidin-3-O-glucoside chloride and its content in red petals was 26-fold higher than that in white petals.
This study presents the first next-generation sequencing effort and transcriptome analysis of a non-model plant from the Family Magnoliaceae. Genes encoding key enzymes were identified and the metabolic pathways involved in biosynthesis and catabolism of M. sprengeri flavonoids were reconstructed. Identification of these genes and pathways adds to the current knowledge of the molecular biology and biochemistry of their production in plant. Such insights into the mechanisms supporting metabolic processes could be used to genetically to enhance flower color among members of the Magnoliaceae.
KeywordsTranscriptome Flavonoid biosynthesis Magnolia sprengeri Flower color
Transcriptome analysis of an organism is a particularly effective method for gene discovery, especially in non-model plants for which no reference genome sequences are available . At the same time, it may provide powerful tools to identify differentially expressed genes, and its possible use in modern plant breeding continues to attract the attention of many plant biologists [23–26]. Sequencing technologies have dramatically accelerated genome-wide studies of transcriptomes and have been widely used to explore gene structure and gene expression, even in plants without a genome reference [27–29]. Illumina sequencing technology has been applied recently to transcriptome analyses of plant and animals, and can generate large amounts of sequence data cheaply and quickly [30–33].
Results and discussion
Sequencing and sequence assembly
Summary of sequencing for M. sprengeri
No. of sequences
No. of bases
No. of high-quality reads
No. of bases
Splicing results for M. sprengeri
Total genes (n)
Total isogenes (n)
Total residues (bp)
Average length (bp)
Largest isogene (bp)
Smallest isogene (bp)
In this study, we obtained 35,642,032 sequences and 62,964,028 high-quality sequences in red and white petals of M. sprengeri, respectively (Table 2). By comparison, the assembly of 39,990 M. sprengeri sequences from GenBank (using the GS De novo Assembler) led to only 77,048 unique sequences. The unique sequences derived from GenBank sequences and Illumina sequences were compared by a BLAST search, where matches were defined as having an identity > 90% and an overlap >100 bp. Our Illumina sequencing efforts produced 77,048 unique sequences. Unique sequences that were not present in GenBank were considered as the novel transcripts of M. sprengeri. The large quantity of unique sequences should cover the vast majority of genes from M. sprengeri petals, providing, for the first time, a powerful gene resource for this medicinal and ornamental plant.
Gene ontology (GO) annotation
Clusters of orthologous group (COG) and eukaryote clusters of orthologous groups (KOG) classification
Pathway assignment based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) classification system
The KEGG classification system provides an alternative functional annotation of genes according to their associated biochemical pathways . KEGG annotations for M. sprengeri transcripts were based on sequence similarity searches against the KEGG database, and matches were assigned the corresponding enzyme commission (EC) number. Overall, 12,082 M. sprengeri unique sequences were assigned KEGG annotations, of which only 1,696 unique sequences were assigned to the biosynthesis of secondary metabolites pathways.
Genes encoding enzymes involved in flavonoid biosynthesis in M. sprengeri
Number of transcripts KO no.
Candidate genes encoding enzymes involved in the biosynthesis of flavonoids
Differentially expressed genes related to flavonoid biosynthesis in M. sprengeri red and white
The levels of transcripts encoding the first enzymes in the flavonoids biosynthesis, such as PAL (EC.18.104.22.168), were markedly higher in red petals than in white petals. The transcript abundance of the flavonoids biosynthesis enzymes, including C4H (EC.22.214.171.124), DFR (EC.126.96.36.199), F3H (EC.188.8.131.52), flavonoid-3′-hydroxylase (F3′H), flavonol synthase (FLS, EC.184.108.40.206), CHS (EC.220.127.116.11), and ANS (EC.18.104.22.168), were also higher in red petals.
The Plant Transcription Factor Database was used to search the M. sprengeri transcripts dataset to identify the genes encoding putative TFs or transcriptional regulators . A total of 270 transcripts were predicted to be TFs and were sorted into three families (data not shown). Of these genes, the expression of eight MYB genes showed 4-fold or greater changes in red petals compared with white petals. Further studies are needed to determine whether the changes in transcript abundance of these putative TFs could be related to the regulation of flavonoid metabolism.
Flavonoids are a large group of polyphenolic compounds and are a structurally diverse class of plant secondary metabolites. They are important for defense against pathogens and herbivores, protection from harmful ultraviolet radiation, and flower pigmentation for attracting pollinators [37–39]. In addition to their physiological functions in plants, flavonoids display a wide range of anti-oxidant, anti-microbial, anti-inflammatory, and anti-cancer activities . As a dietary component, flavonoids are considered to have health-promoting and disease-preventing properties. Recently, flavonoids have been intensively investigated as potent pharmaceuticals for treating chronic human pathological conditions [40–44].
Changes in transcript abundance of predicted transcription factors and regulators about flavonoid biosynthesis in M. sprengeri red and white
MYB domain protein 20
R2R3 MYB transcription factor
R2R3-MYB transcription factor MYB9
MYB-related protein 306
MYB-related protein 306 isoform 1
MYB transcription factor
R2R3 Myb24 transcription factor
Based on this comparison, almost all of the candidate genes involved in the flavonoid biosynthesis were present in the transcriptome datasets of M. sprengeri in this study. These results highlight the immense capacity of high-throughput sequencing to discover genes in metabolic pathways.
Illumina next-generation sequencing technology was used for sequencing and transcriptome analysis of the non-model plant M. sprengeri pamp. We identified the genes encoding key enzymes and reconstructed the metabolic pathways involved in biosynthesis and catabolism of flavonoid of M. sprengeri. Our results promote understanding of the mechanisms underlying various metabolic processes, and will enable the genetic manipulation of flower color in M. sprengeri.
The accumulation of flavonoids and the discovery of genes associated with their biosynthesis and metabolism in M. sprengeri are intriguing and worthy of further investigation. The sequences and pathways identified here represent the genetic framework required for further studies. Quantitative transcriptomics in concert with physiological and biochemical analysis in M. sprengeri under conditions that stimulate production and accumulation of flavonoids could help provide insights into the regulation of, and links between, these pathways.
The petals of red and white M. sprengeri were harvested from approximately 50-year-old trees in March 2012 from Wufeng County, Hubei Province, China (Figure 2). We selected 10 trees with red flowers and 10 trees with white flowers for petal collection. Nine petals of each color were selected for RNA-sequencing experiments while three petals (around 0.5 g) of each color were taken for the HPLC experiments. For qPCR, we used additional 5–10 petals to isolate total RNA. After cleaning, the petals were cut into small pieces, immediately frozen in liquid nitrogen, and stored at -80°C until further processing.
The TRIzol® reagent (Invitrogen) was used to extract total RNA from the petals of red and white M. sprengeri according to the manufacturer’s instructions (Invitrogen, USA). The purity of all RNA samples was assessed at an absorbance ratio of OD260/280 and the RNA quality was tested using a 1% ethidium bromide-stained (EtBr-stained) agarose gel. A GeneQuant100 spectrophotometer (GE Healthcare, UK) assessed the RNA concentration before processing.
cDNA synthesis and Illumina sequencing
Clontech’s SMART cDNA synthesis kit (Clontech, USA), was used to produce first-strand cDNA from 5 μg of total RNA extracted from the petals of M. sprengeri, according to the manufacturer’s instructions. The samples were treated with RNase-free DNase I (Takara Biotechnology, China). To construct a cDNA library, oligo (dT) magnetic beads were used to purify poly (A) mRNA from total RNA. The RNA was then fragmented into small pieces by the addition of fragmentation buffer. These short fragments served as templates to synthesize first-strand cDNA using random hexamer primers. Second-strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. A QiaQuick PCR extraction kit purified the short fragments. These fragments were washed with elution buffer for end repair and poly (A) addition and were then ligated to sequencing adapters. Suitable fragments, as judged by agarose gel electrophoresis, were selected for use as templates for PCR amplification. An Illumina HiSeq™2000 sequenced the cDNA library using paired-end technology in a single run.
Transcriptome assembly and annotation
The Solexa GA pipeline 1.6 generated the transcriptome de novo assembly. After the removal of low-quality reads, the Trinity de novo assembler (http://trinityrnaseq.sourceforge.net/) [48, 49] assembled processed reads with an identity value of 95% and a coverage length of 100 bp [48, 49]. First, the overlap information in the short reads was used to construct high-coverage contigs, and then the short reads were assembled into contigs. We then realigned the short reads onto the contigs and estimated the distance and relation of the two contigs using the pair-end linkage and insert size information. Unreliable linkages between the two contigs were filtered and the remaining contigs with compatible connections were linked to each other, and had at least three read-pairs. The last step was to close gaps in the scaffolds. We gathered the paired-end reads with one end mapped to the contigs and another end located in the gaps and performed local assembly with the unmapped end to extend the contig sequence into the small gaps in the scaffolds. CAP3  was used (with default parameters) to reduce redundancy and to combine scaffolds and single-end contigs in the separate assemblies.
To annotate the M. sprengeri transcriptome, we performed a BLAST search against the non-redundant (NR) database in NCBI, SWISS-PROT, KEGG, and COG with a cut-off E-value of ≤10-5. The Blast2GO software (http://www.blast2go.com/b2ghome) obtained the GO annotations and the corresponding EC numbers of the sequences.
Pathway assignment with KEGG
Pathway assignments were mapped according to the KEGG database (http://www.genome.ad.jp/kegg/kegg2.html) (versionKEGG) . EC numbers were assigned to unique sequences that had BLASTX scores with an E value cut-off of 10-5 after searching the KEGG protein databases. The unique sequences were mapped to specific biochemical pathways according to the corresponding EC distribution in the KEGG database.
Quantitative Real-time PCR (qPCR) Analyses
Primers used in quantitative real-time PCR
Primer sequence(forward) 5′-3′
Primer sequence(reverse) 5′-3′
HPLC analysis of anthocyanin
Magnolia sprengeri petals (0.5 g) were ground in 1.5 mL of 70% methanol containing 2% formic acid at 4°C, then centrifuged at 10,000 g for 10 min at 4°C. The supernatant was passed through a 0.22-μm syringe filter before HPLC analysis. Anthocyanins were investigated on an Agilent 1100 HPLC equipped with a diode array detector (Agilent Technology), as described by Zhang et al. . The total anthocyanin concentration was calculated based on a cyanidin-3-O-glucoside standard (Sigma-Aldrich, St. Louis, MO, USA).
This work was supported by the National Forestry Research and Special Public Service Sectors (No. 200904004).
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