Comparative Genomics of Spatholobus suberectus and Insight Into Flavonoid Biosynthesis

Abstract

Spatholobus suberectus Dunn (S. suberectus), has been widely used in traditional medicines plant source of the Leguminosae family. Its vine stem of which plays an important role in the prevention and treatment of various diseases because it contains various flavonoids. Comparative genome analysis suggested well-conserved genomic components and genetic collinearity between the genome of S. suberectus and other genera of Leguminosae such as Glycine max. We discovered two whole genome duplications (WGD) events in S. suberectus and G. max lineage underwent a WGD after speciation from S. suberectus. The determination of expansion and contractions of orthologous gene families revealed 1,001 expanded gene families and 3,649 contracted gene families in the S. suberectus lineage. Comparing to the model plants, many novel flavonoid biosynthesis-related genes were predicted in the genome of S. suberectus, and the expression patterns of these genes in the roots are similar to those in the stems [such as the isoflavone synthase (IFS) genes]. The expansion of IFS from a single copy in the Leguminosae ancestor to four copies in S. suberectus, will accelerate the biosynthesis of flavonoids. MYB genes are widely involved in plant flavonoid biosynthesis and the most abundant member of the TF family in S. suberectus. Activated retrotransponson positive regulates the accumulation of flavonoid in S. suberectus by introducing the cis-elements of tissue-specific expressed MYBs. Our study not only provides significant insight into the evolution of specific flavonoid biosynthetic pathways in S. suberectus, but also would facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding for alleviating resource shortage of S. suberectus.

Keywords: Spatholobus suberectus Dunn; comparative genome analysis; flavonoid biosynthesis; isoflavone synthase; transcription factors.

Figure 1

The dry stem of Spatholobus suberectus.

Figure 2

Comparative analyses of Spatholobus suberectus with other plants. (A) The gene number in four clusters of eight plant species. (B) Shared and unique gene families.

Figure 3

Whole-genome duplication and gene family expansion analysis. (A) Whole genome duplication (WGD) events detected in genome of Spatholobus suberectus, Glycine max, and Glycyrrhiza uralensis. 4dTv distribution of transversion substitutions at fourfold degenerate sites. (B) Circular diagram showing genetic collinearity between S. suberectus and G. max. Circles from inside to outside are as followed: a, the genome collinear blocks of between S. suberectus and G. max, which connected by curved lines and set as same color; b, gene density (green). All distributions are drawn in a window size of 300 kb, chromosomes_units = 500,000. (C) Gene family expansions and contractions in S. suberectus and seven other plants.

Figure 4

The metabolic profiles and detailed biosynthetic pathways of flavonoid of various tissues in Spatholobus suberectus. (A) The percentage of total flavonoid content. (B) The content of formononetin. (C) The content of isoliquiritigenin. (D) The content of genistein. (E) The content of catechin. (F) Detailed biosynthetic pathways of flavonoid in S. suberectus. The abbreviated name of enzyme in each catalytic step is showed in blue font. Gene expression levels [log₁₀ (RPKM+1)] in five tissues are represented by color gradation. Gene expression with RPKM≤ 1 was set to 0 after log10 transformation. Genes with more than one homology are represented by equal colored horizontal stripe and are termed from top to bottom. The names of enzymes are listed as followed: PAL, C4H, 4CL, CHS, CHI, IFS, HID, F3H, F3’H, DFR, FLS, LAR, OMT, CHR. Each plant tissue for gene expression had three biological repetitions.

Figure 5

The co-expression networks of transcripts involved in the biosynthesis and metabolism of flavonoid, formononetin, isoliquiritigenin, genistein, and catechin. (A) Hierarchical cluster tree and color bands indicating the 14 modules identified by weighted gene co-expression network analysis (WGCNA). (B) The analysis of module–trait correlations. Each row represents a module and each column represents a specific chemical compound. Each cell at the row–column intersection is color-coded by correlation according to the color legend. (C) The expression abundance and cluster of flavonoid, formononetin, isoliquiritigenin, genistein, and catechin in roots, steams, leaves, flowers, and fruits. (D) Gene ontology (GO) enrichment analysis of genes belonging to the related cell of flavonoid, formononetin, isoliquiritigenin, genistein, and catechin.

Figure 6

Gene family analysis showing expansion in isoflavonoid biosynthesis genes involved in the biosynthesis of genistein and formononetin compounds. (A) Two gene families involving in the biosynthesis of flavonoid were expanded in Spatholobus suberectus, including the anthocyanidin synthase (ANS) and isoflavone synthase (IFS) family. (B) IFS is the key enzyme for biosynthesis of isoflavones, which catalyzes 5,7,4’-trihydroxyflavanone (naringenin) to genistein. (C) Phylogeny of the IFS genes in S. suberectus, Glycine max, Glycyrrhiza uralensis, Cicer arietinum, and Lotus japonicus showing four copies of S. suberectus IFS genes. Numbers correspond to branching posterior probabilities. IFS genes are upregulated in the stem and root of S. suberectus. The heatmap of IFS genes expression is corresponding to the order of IFS genes in the tree. (D) The four genomic regions in chromosome five containing the S. suberectus. IFS genes show clear synteny with the G. max genome, while the four IFS genes occur in tandem. This suggests the involvement of both whole-genome duplication (WGD) and tandem duplication events in IFS family expansion. Lines linking the two bars indicate regions with >70% similarity and coverage length >=100. (E) The arrangement of cis-elements on the promoter of IFS (Chr5.1660), the percentage of genes with different cis-elements in their promoter regions and the expression profiles of MYBs related to the biosynthesis of flavonoid.

Figure 7

Regulate networks of the MYBs. (A) Expression profiles of MYBs, DFRs, LARs, and IFSs in the roots, stems, leaves, flowers, and fruits. (B) Promoter fragments of DFR, LAR, and IFSs were connected to pLacZi and transformed into YM4271 strain harboring GAL4-AD-MYBs. The β-galactosidase activity was validated using X-gal staining. All bars represent means± s.d, and three biological replicates in the experiment. Significant differences (Student’s t-test) at P < 0.01 (**).