Research ArticlePLANT BIOCHEMISTRY

A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis

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Science Advances  08 Apr 2016:
Vol. 2, no. 4, e1501780
DOI: 10.1126/sciadv.1501780
  • Fig. 1 Specialized flavones found in S. baicalensis Georgi plant.

    (A) S. baicalensis Georgi plant. (B) The dried roots of S. baicalensis Georgi used in traditional Chinese medicine. (C) Structures of its major flavones. (D) The proposed pathway responsible for biosynthesis of 4′-deoxyflavones in S. baicalensis.

  • Fig. 2 Phylogenetic and RNA interference (RNAi) silencing of SbFNSII-1 and SbFNSII-2 in hairy root cultures of S. baicalensis.

    (A) Bootstrap consensus tree of the CYP93B subfamily. Maximum likelihood (ML) was used to construct this tree with 1000 replicate bootstrap support. The tree was rooted with Sorghum bicolor CYP93G. GenBank ID of the proteins used in the tree: CYP93B6, BAB59004.1; CYP93B23, AGF30365.1; CYP93B3, BAA84071.1; CYP93B17, BAF49323.1; CYP93B2, AAD39549.1; CYP93B5, AAF04115.1; CYP93B14, ACB56919.1; CYP93B12, ABC59104.2; CYP93B20P, KHN21998.1; CYP93B16, ACV65037.1; CYP93B19, NP_001241129.1; CYP93G3, XP_002461286.1. (B) Relative levels of SbFNSII-1 and SbFNSII-2 transcripts compared to β-actin were determined by qRT-PCR analyses performed on total RNA extracted from different organs. R, roots; S, stem; L, leaves; F, flowers. (C) Relative expression of SbFNSII-1 and SbFNSII-2 subjected to MeJA treatment for 24 hours. The expression levels were normalized to corresponding values from mock treatments. (D) Silencing of SbFNSII-1 was measured by monitoring relative transcript levels by qRT-PCR. The expression levels were measured relative to those obtained with empty vector as a control. (E) Measurements of RSFs from the SbFNSII-1 RNAi lines used for transcript analysis. (F) Silencing of SbFNSII-2 was measured by monitoring relative transcript levels by qRT-PCR. (G) Measurements of RSFs from the SbFNSII-2 RNAi lines used for transcript analysis. Bin, baicalin; Wde, wogonoside; Bein, baicalein; Win, wogonin. SEs were calculated from three biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test).

  • Fig. 3 Overexpression of SbFNSII-2 in Arabidopsis.

    (A) Transcript levels of SbFNSII-2 relative to Arabidopsis UBI in two empty vector (EV) control lines and five transgenic lines determined by qRT-PCR. (B) Measurements of pinocembrin (pin) and chrysin (chr) from two empty vector lines and five transgenic lines grown on MS supplemented with pinocembrin. SEs were calculated from three biological replicates.

  • Fig. 4 Phylogenetic tree of CLLs and qRT-PCR analysis of Sb4CLs genes.

    (A) Phylogenetic analysis of CLLs. ML method was used to construct this tree with 1000 replicates bootstrap support. TAIR (The Arabidopsis Information Resource) ID of the proteins used in the tree: At4CL1, AT1G51680; At4CL2, AT3G21240; At4CL3, AT1G65060; At4CLL3, AT1G20490; At4CL4, AT1G20500; At4CL5, AT3G21230; At4CLL6, AT4G19010; At4CLL7, AT4G05160; AT4CL8, AT5G38120; At4CLL9, AT5G63380; At4CLL10, AT3G48990; AtCNL, AT1G65880. (B) Relative SbCLL-1, (C) SbCLL-5, and (D) SbCLL-7 transcript levels to β-actin were determined by qRT-PCR analyses performed on total RNA extracted from different organs. (E) Relative expression of the three genes subjected to MeJA treatment for 24 hours. The expression levels were measured relative to those obtained from mock treatment as a control. SEs were calculated from three biological replicates. *P < 0.05, and **P < 0.01 (Student’s t test).

  • Fig. 5 RNAi of SbCLL-7 in hairy root cultures of S. baicalensis.

    (A) Silencing of SbCLL-7 was measured by monitoring relative transcript levels by qRT-PCR. The expression levels were measured relative to those obtained from an empty vector line as a control. (B) Measurements of RSFs from SbCLL-7 RNAi lines used for transcript analysis. SEs were calculated from three biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test).

  • Fig. 6 Phylogenetic analysis of Scutellaria CHS isoforms and the expression patterns of their genes.

    (A) Phylogenetic tree of CHS proteins. ML was used to construct this tree with 1000 replicate bootstrap support. The tree was rooted with Physcomitrella patens CHS. GenBank ID of the proteins used in the tree: AmCHS, CAA27338.1; SiCHS, XP_011091402.1 ; PfCHS, O04111.1; ArCHS, CAA27338.1; PcCHS, AJO53275.1; SvCHS, ACC68839.1; CcCHS, P48385.2; GhCHS, CAA86220.1; PtCHS, XP_002303821.2; MtCHS1, XP_003601647.1; GmCHS1a, AAB01004.1; PpCHS, ABB84527.1. (B) Relative levels of SbCHS-1 and SbCHS-2 transcripts compared to β-actin were determined by qRT-PCR analyses performed on total RNA extracted from different Scutellaria organs. (C) Relative expression of SbCHS-1 and SbCHS-2 subjected to MeJA treatment for 24 hours. The expression levels were measured relative to those obtained from mock treatment as a control. SEs were calculated from three biological replicates. *P < 0.05 and **P < 0.01 (Student′s t test).

  • Fig. 7 Proposed pathways for synthesis of flavones in S. baicalensis.

    The proteins labeled in red are those for which genes encoding specific isoforms are reported in this study.

  • Table 1 Kinetic parameters of FNSIIs toward pinocembrin and naringenin.

    Each data set represents the mean ± SE from triplicate measurements.

    Protein and substrateKm (μM)Vmax (pkat mg protein−1 )Vmax/Km
    FNSII-1
      Pinocembrin0.24 ± 0.0727.65 ± 3.07114.73
      Naringenin0.28 ± 0.0660.93 ± 0.67215.31
    FNSII-2
      Pinocembrin0.46 ± 0.069.02 ± 0.2219.60
      Naringenin
  • Table 2 Kinetic parameters of CLLs toward different substrates.

    Each data set represents the mean ± SE from triplicate measurements.

    EnzymeSubstrateKm (μM)Vmax (nkat mg protein−1)Vmax /Km
    SbCLL-1Cinnamic acid5300 ± 712180 ± 160.03
    4-Coumaric acid45 ± 3536 ± 811.88
    Caffeic acid18 ± 2190 ± 910.58
    SbCLL-5Cinnamic acid3060 ± 331279 ± 230.09
    4-Coumaric acid31 ± 3687 ± 3522.15
    Caffeic acid260 ± 15600 ± 102.31
    SbCLL-7Cinnamic acid172 ± 17168 ± 70.97
    4-Coumaric acid
    Caffeic acid
  • Table 3 Tobacco leaves infiltrated with different combinations of flavone biosynthetic genes.
    Scutellaria genes usedCinnamic acid (supplemented)Pinocembrin (detected)Chrysin (detected)
    GFPNoNo
    +NoNo
    CLL-7NoNo
    +NoNo
    CLL-7, CHS-2YesNo
    +YesNo
    CLL-7, CHS-2, CHIYesNo
    +YesNo
    CLL-7, CHS-2, CHI, FNSII-2NoYes
    +NoYes
    CLL-7, CHS-2, FNSII-2NoYes
    +NoYes

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/4/e1501780/DC1

    Fig. S1. Multiple alignment of CPY93B6, CPY93B24, and CPY93B25.

    Fig. S2. Flavone accumulation patterns in S. baicalensis.

    Fig. S3. RNAi of SbFNSII-2 in hairy root cultures of S. baicalensis.

    Fig. S4. In vitro assay of SbFNSII-1 and SbFNSII-2 and in vivo assay of SbFNSII-2.

    Fig. S5. HPLC metabolite profiles of Arabidopsis plants carrying empty vector or a representative SbFNSII-2 line, grown on MS with or without supplementation of pinocembrin.

    Fig. S6. Western blot analysis of the recombinant SbCLLs and SPB domain analysis of At4CLs and SbCLL-7.

    Fig. S7. Metabolite profiles by HPLC from empty vector line and a representative SbCLL-7 RNAi line.

    Fig. S8. Metabolite profiles of HPLC analysis of infiltrated N. benthamiana leaves.

    Fig. S9. Transcript levels of SbC4H relative to actin.

    Table S1. Primers used in this study. Underlined sequences mean recombination cites for Gateway cloning.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Multiple alignment of CPY93B6, CPY93B24, and CPY93B25.
    • Fig. S2. Flavone accumulation patterns in S. baicalensis.
    • Fig. S3. RNAi of SbFNSII-2 in hairy root cultures of S. baicalensis.
    • Fig. S4. In vitro assay of SbFNSII-1 and SbFNSII-2 and in vivo assay of SbFNSII-2.
    • Fig. S5. HPLC metabolite profiles of Arabidopsis plants carrying empty vector or a representative SbFNSII-2 line, grown on MS with or without supplementation of pinocembrin.
    • Fig. S6. Western blot analysis of the recombinant SbCLLs and SPB domain analysis of At4CLs and SbCLL-7.
    • Fig. S7. Metabolite profiles by HPLC from empty vector line and a representative SbCLL-7 RNAi line.
    • Fig. S8. Metabolite profiles of HPLC analysis of infiltrated N. benthamiana leaves.
    • Fig. S9. Transcript levels of SbC4H relative to actin.
    • Table S1. Primers used in this study. Underlined sequences mean recombination cites for Gateway cloning.

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