Fig 1.
Phenylpropanoid biosynthetic pathway.
(A) C4H catalyzes the conversion of trans-cinnamic acid to p-coumaric acid by adding the hydroxyl group at the 4′ position. (B) A schematic diagram depicting soybean phenylpropanoid pathway highlighting the involment of C4H (red) leading to biosynthesis of specialized metabolites such as isoflavones (daidzein, genistein, glycitein) and other end products (green highlights).
Fig 2.
A map of pESC-leu2D-LjCPR1-GW yeast expression vector for P450 assay.
The bidirectional Gal1/Gal10 promoter of pESC-Leu2d vector drives LjCPR1 and gene cloned within the gateway cassette.
Fig 3.
GmC4H gene structures and motif analysis.
(A) Schematic diagram of gene structure of GmC4Hs. Exon-intron structures of GmC4Hs were compiled from Phyotozome 13 database (https://phytozome-next.jgi.doe.gov/info/Gmax_Wm82_a4_v1) drawn by Gene Structure Display Server 2.0. (B) Multiple sequence alignment of deduced amino acid sequences of candidate GmC4Hs with characterized C4H from other plant species. Identical and similar amino acid residues are indicated by black and gray shades, respectively. Five P450 motifs: the PPGP, the oxygen-binding, the ETLR, the PERF and the heme-binding motifs are indicated by different colored rectangles. Asterisk (*) and red font indicate critical amino acid residues for substrate binding. Accession numbers are as indicated: MsC4H, P37114; CaC4H, O81928; AtC4H, P92994; CsC4H, ASU87408; SbC4H, Q94IP1.
Fig 4.
Phylogenetic analysis of GmC4H proteins.
A maximum likelihood tree was generated by MEGA 10 using the putative amino acid sequences of candidate GmC4Hs and CYP73 candidates from other plant species. Bootstrap replicates of 1000 expressed in percentage are shown next to the branch. Class I and class II CYP73 members are indicated by brown and blue branches of tree, respictively while CYP73 from gymnosperms, mosses, ferns etc are indicated by black branches. Functionally charactersized C4Hs are indicated by black circles and GmC4Hs are shown by red circles. CYP102A1 from Bacillus megaterium (accession no. P14779) was used as an outgroup.
Fig 5.
Molecular docking of GmC4Hs with trans-cinnamic acid.
On the Left, predicted 3D structure of GmC4Hs showing position of trans-cinnamic acid (green) adjacent to heme-group (blue) in protein-ligand complex. Amino acid residues interacting with ligand are shown with red color in protein structure. Middle column depicts magnified view of ligand interacting with heme-group and amino acid residue in the protein-ligand complex. Interaction of ligand with heme group showing with interacting amino acid residues and heme-group is displayed in heme-ligand interaction (right column). Pink dotted line indicates Pi-Sigma interaction, light pink showing Pi-Alkyl interaction, green showing hydrogen bond and light green showing Van der Waals interactions. Conserved alanine residue in GmC4Hs are indicated by solid black arrow.
Fig 6.
Expression analysis of GmC4Hs.
(A) The GmC4H gene expression data across different tissues and developmental stages were obtained from Soybase (https://soybase.org/soyseq/) for the heatmap generation. The color scale below the heat map indicates expression values, blue and red indicating low and high transcript abundance, respectively. (B) Analysis of GmC4H transcript accumulation in soybean embryos during development. cDNA was synthesized from total RNA (1 μg) isolated from soybean embryos (30, 40, 50, 60 and 70 DAP) and qPCR was performed using gene-specific primers. Expression values were normalized against the reference gene CONS4. The error bars represent the SEM of two biological replicates and three technical replicates for each biological replicate. (C) Localization of GmC4H20 in subcellular compartment. A translational fusion of GmC4H20-YFP was transiently expressed in the leaf epidermal cells of N. benthamiana and visualized by confocal microscopy. Co-localization of GmC4H20-YFP fusion with a ER marker fused to CFP was performed for confirmation. A merged signal was obtained by sequentially scanning the two channels. Scale bar: 50 μm.
Fig 7.
Functional characterization of candidate GmC4Hs in vitro.
Microsomal fraction containing candidate GmC4Hs and LjCPR1 was incubated with trans-cinnamic acid and NADPH followed by ethyl acetate extraction. The extract was analyzed by high-performance liquid chromatography (HPLC). (A) standards. (B-E) microsomal proteins + substrate. Spectrum for the substrate and the products are shown on the right.
Table 1.
Characteristics of soybean C4H genes family.