Fig 1.
Flavonoid biosynthetic pathway.
A schematic representation of precursors used in the biosynthesis of flavonoids, flavones and flavonols with some important enzymes involved in flavonoid biosynthetic pathway. Abbreviations: ACC = Acetyl-CoA carboxylase;PAL = phenylalanine ammonia lyase; CHS = chalcone synthase and CHI = chalcone isomerase.
Table 1.
Primers used in this study.
Fig 2.
Predicted three-dimensional models and ligand-binding sites of GaCHSs.
Ribbon model display of the three-dimensional structures of GaCHS1 and GaCHS2 with conserved catalytic triad (Cys-His-Asn) shown in the central core of the structures (a and d);Ribbon model display of the three-dimensional structures of GaCHS1 (b) and GaCHS2 (e) as predicted by I-TASSER web server, showing geometry of active site a malonyl-CoA binding motif shown as mesh structures and gatekeepers Phe215and Phe265 (Pink in GaCHS1 and Red in GaCHS2). The ligand binding sites as predicted by 3DLigandSite web server are depicted in the ribbon model (c and f). The predicted ligand binding sites for GaCHS1 are Ala211, Gln212, Ala213, Leu214, Phe215, Ile254, Phe265, Leu267, Lys269, Val271, Pro272, Gly305, Gly306, Ala308, Ile309, Ile336 and for GaCHS2, the predicted ligand binding sites were Lys55, Phe56, Asp57, Leu58, Ser59, Ala60, Val62, Thr63, Ile64, Leu164, Leu206, Asp207, Leu209, Val210, Gly211, Leu214, Phe215, Ile254, Phe265, Leu267, Lys269, Val271, Pro272, Gly305, Gly306, Ala308, Asn336.
Fig 3.
Multiple sequence alignment of deduced amino acid sequencesof GaCHS-1 and GaCHS-2 with related plant CHS sequences using Clustal Omega multiple sequence alignment tool.
Functionally important conserved residues are highlighted with a coloured background: Dark yellow, the four catalytic residues (Cys-His-Asp triad + F) that are conserved in all chalcone synthases; red, the 13 residues that shape the geometry of the active site; pink, the malonyl-CoA binding motif; and green, the highly conserved CHS signature sequence, N-myristoylation motif. (GenBank accession numbers): GrewiaasiaticaCHS-1(KX129910), GrewiaasiaticaCHS-2 (KX129911),Hibiscus cannabinusCHS1 (AIC75908.1), BvCHS, Theobroma cacao (EOY05368.1), Gossypiumraimondii(XP_012454899.1), Gossypiumarboreum(KHG14899.1), Abelmoschusesculentus(AGW22222.1), Abelmoschusmanihot(ACE60221.1).
Fig 4.
Phylogenetic analysis of GaCHS-1 and GaCHS-2.
The phylogenetic analysis was performed based on Maximum Likelihood methodwith 1000 bootstrap replicates using the MUSCLE program and MEGA- 5 software. The analysis involved alignment of 34 amino acid sequences which were chosen by BLAST search of GaCHS genes from NCBI data-base. The desired sequences were selected based on the complete cds information available. The evolutionary distances were computed using the Poisson correction method. The database accession numbers of the CHS sequences used are as follows: GrewiaasiaticaCHS-1(KX129910), Grewia asiaticaCHS-2 (KX129911), Gossypiumhirsutum(AEO96985.1), Gossypiumarboreum CHS-1 (KHG25969.1), Gossypiumraimondii(XP_012454899.1), Theobroma cacao (EOY05368.1), Abelmoschusesculentus(AGW22222.1), Abelmoschusmanihot(ACE60221.1), Gossypiumhirsutum CHS2 (AEO96988.1), GossypiumraimondiiCHS1 (XP_012455000.1), Gossypiumarboreum(KHG14899.1), GossypiumhirsutumCHS1 (ACV72638.1), GossypiumraimondiiCHS2 (XP_012440802.1), Hibiscus cannabinusCHS1 (AIC75908.1), Hibiscus cannabinusCHS2 (AIA22214.1), MangiferaindicaCHS1 (AIY24986.1), MangiferaindicaCHS (AIB06736.1), MangiferaindicaCHS (AIY24987.1), Camellia sinensis(AGI02994.1), Camellia japonica (BAI66465.1), Ziziphus jujube (XP_015887549.1), Populustrichocarpa(EEE78799.1), Pyruscommunis (AAX16494.1), Malus hybridcultivar(ACN25139.1), Malus domesticaCHS2 (AFX71920.1), Malus domesticaCHS1 (AGE84303.1), Malus domesticaPREDICTED:polyketidesynthase5-like (XP_008380608.1), Medicago sativa CHS(AAB41559.1), Silenelittorea CHS1 (AMQ23617.1), Nicotiana tabacum CHS (AAK49457.1), Gypsophila paniculataCHS(AAP74755.1), Polygonum cuspidatum CHS (AFD64563.1), FagopyrumtataricumCHS(ADG02377.1), ArabidopsisCHS(AAB35812.1). The bar indicates an evolutionary distance of 0.02%.
Fig 5.
Multiple reactions monitoring (MRM) graphs.
MRM chromatograms of standard compounds naringenin and naringenin chalcone eluting at 14.9 and 13.8 min, respectively; (a), MS spectra of naringenin chalcone (b) and naringenin (c).
Fig 6.
Kinetic study of GaCHSs using different substrates.
The kinetic parameters Km and Vmax were calculated by nonlinear regression analysis in order to determine the relative efficiency of GaCHS1 and GaCHS2 against the different substrates including p-coumaroyl-CoA, Acetyl-CoA, Butyryl-CoA, Hexanoyl-CoA and Octanoyl CoA.
Fig 7.
Real-Time expression analysis of GaCHS1 and GaCHS2and estimation of total flavonoid content in Grewiaasiatica.
Quantitative estimation of the relative expression of GaCHS1 and GaCHS2 in leaf, stem and root tissues of G.asiatica(a). Relative accumulation of flavonoids in leaf, stem and root tissues of G.asiatica(b). Quantitative estimation of the relative expression of GaCHS1 and GaCHS2 in different floral phenological stages represented by (A) bud initiation, (B) bud growth, (C) pre-anthesis, (D) anthesis, (E) senescence and (F) fruit initiation (c) Corresponding relative accumulation of flavonoids in above mentioned floral phenological stages (d) Quantitative estimation of the relative expression of GaCHS1 and GaCHS2 in different floral tissues including sepals, petals, stamens, carpel and ovary (e) Relative accumulation of flavonoids in different floral tissues including sepals, petals, stamens, carpel and ovary (f). The data were compared and analyzed with one-way analysis of variance (ANOVA) using GraphPad Prism 6 software. Values are expressed as mean ± standard deviation, with standard errors indicated by bars representing at least three replicates. The statistical significance was considered at P < 0.001.