Phylogenetic analysis of <i>Ga</i>CHS-1 and <i>Ga</i>CHS-2.

<p>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 <i>Ga</i>CHS 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: <i>Grewiaasiatica</i>CHS-1(KX129910), <i>Grewia asiatica</i>CHS-2 (KX129911), <i>Gossypiumhirsutum</i>(AEO96985.1), <i>Gossypiumarboreum</i> CHS-1 (KHG25969.1), <i>Gossypiumraimondii</i>(XP_012454899.1), <i>Theobroma cacao</i> (EOY05368.1), <i>Abelmoschusesculentus</i>(AGW22222.1), <i>Abelmoschusmanihot</i>(ACE60221.1), <i>Gossypiumhirsutum CHS2</i> (AEO96988.1), <i>Gossypiumraimondii</i>CHS1 (XP_012455000.1), <i>Gossypiumarboreum</i>(KHG14899.1), <i>Gossypiumhirsutum</i>CHS1 (ACV72638.1), <i>Gossypiumraimondii</i>CHS2 (XP_012440802.1), <i>Hibiscus cannabinus</i>CHS1 (AIC75908.1), <i>Hibiscus cannabinus</i>CHS2 (AIA22214.1), <i>Mangiferaindica</i>CHS1 (AIY24986.1), <i>Mangiferaindica</i>CHS (AIB06736.1), <i>Mangiferaindica</i>CHS (AIY24987.1), <i>Camellia sinensis</i>(AGI02994.1), <i>Camellia japonica</i> (BAI66465.1), <i>Ziziphus jujube</i> (XP_015887549.1), <i>Populustrichocarpa</i>(EEE78799.1), <i>Pyruscommunis</i> (AAX16494.1), <i>Malus hybridcultivar</i>(ACN25139.1), <i>Malus domestica</i>CHS2 (AFX71920.1), <i>Malus domestica</i>CHS1 (AGE84303.1), <i>Malus domestica</i>PREDICTED:polyketidesynthase5-like (XP_008380608.1), <i>Medicago sativa</i> CHS(AAB41559.1), <i>Silenelittorea</i> CHS1 (AMQ23617.1), <i>Nicotiana tabacum</i> CHS (AAK49457.1), <i>Gypsophila paniculata</i>CHS(AAP74755.1), <i>Polygonum cuspidatum</i> CHS (AFD64563.1), <i>Fagopyrumtataricum</i>CHS(ADG02377.1), <i>Arabidopsis</i>CHS(AAB35812.1). The bar indicates an evolutionary distance of 0.02%.</p

Predicted three-dimensional models and ligand-binding sites of <i>Ga</i>CHSs.

<p>Ribbon model display of the three-dimensional structures of <i>Ga</i>CHS1 and <i>Ga</i>CHS2 with conserved catalytic triad (Cys-His-Asn) shown in the central core of the structures <b>(a and d)</b>;Ribbon model display of the three-dimensional structures of <i>Ga</i>CHS1 <b>(b)</b> and <i>Ga</i>CHS2 <b>(e)</b> 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 <i>Ga</i>CHS1 and Red in <i>Ga</i>CHS2). The ligand binding sites as predicted by 3DLigandSite web server are depicted in the ribbon model <b>(c and f)</b>. The predicted ligand binding sites for <i>Ga</i>CHS1 are Ala²¹¹, Gln²¹², Ala²¹³, Leu²¹⁴, Phe²¹⁵, Ile²⁵⁴, Phe²⁶⁵, Leu²⁶⁷, Lys²⁶⁹, Val²⁷¹, Pro²⁷², Gly³⁰⁵, Gly³⁰⁶, Ala³⁰⁸, Ile³⁰⁹, Ile³³⁶ and for <i>Ga</i>CHS2, 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.</p

Kinetic study of <i>Ga</i>CHSs using different substrates.

<p>The kinetic parameters K_m and V_max were calculated by nonlinear regression analysis in order to determine the relative efficiency of <i>Ga</i>CHS1 and <i>Ga</i>CHS2 against the different substrates including p-coumaroyl-CoA, Acetyl-CoA, Butyryl-CoA, Hexanoyl-CoA and Octanoyl CoA.</p

Primers used in this study.

<p>Primers used in this study.</p

Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in <i>Grewia asiatica</i> L

<div><p>Chalcone synthase constitutes a functionally diverse gene family producing wide range of flavonoids by catalyzing the initial step of the phenylpropanoid pathway. There is a pivotal role of flavonoids in pollen function as they are imperative for pollen maturation and pollen tube growth during sexual reproduction in flowering plants. Here we focused on medicinally important fruit-bearing shrub <i>Grewia asiatica</i>. It is a rich repository of flavonoids. The fruits are highly acclaimed for various putative health benefits. Despite its importance, full commercial exploitation is hampered due to two drawbacks which include short shelf life of its fruits and larger seed volume. To circumvent these constraints, seed abortion is one of the viable options. Molecular interventions tested in a number of economic crops have been to impair male reproductive function by disrupting the chalcone synthase (CHS) gene activity. Against this backdrop the aim of the present study included cloning and characterization of two full-length cDNA clones of <i>Ga</i>CHS isoforms from the CHS multigene family. These included <i>Ga</i>CHS1 (NCBI acc. KX129910) and <i>Ga</i>CHS2 (NCBI acc. KX129911) with an ORF of 1176 and 1170 bp, respectively. <i>Ga</i>CHSs were heterologously expressed and purified in <i>E</i>. <i>coli</i> to validate their functionality. Functionality of CHS isoforms was also characterized via enzyme kinetic studies using five different substrates. We observed differential substrate specificities in terms of their K_m and V_max values. Accumulation of flavonoid constituents naringenin and quercetin were also quantified and their relative concentrations corroborated well with the expression levels of <i>Ga</i>CHSs. Further, our results demonstrate that <i>Ga</i>CHS isoforms show differential expression patterns at different reproductive phenological stages. Transcript levels of <i>Ga</i>CHS2 were more than its isoform <i>Ga</i>CHS1 at the anthesis stage of flower development pointing towards its probable role in male reproductive maturity.</p></div

Multiple sequence alignment of deduced amino acid sequencesof <i>Ga</i>CHS-1 and <i>Ga</i>CHS-2 with related plant CHS sequences using Clustal Omega multiple sequence alignment tool.

<p>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): <i>Grewiaasiatica</i>CHS-1(KX129910), <i>Grewiaasiatica</i>CHS-2 (KX129911),<i>Hibiscus cannabinus</i>CHS1 (AIC75908.1), <i>Bv</i>CHS, <i>Theobroma cacao</i> (EOY05368.1), <i>Gossypiumraimondii</i>(XP_012454899.1), <i>Gossypiumarboreum</i>(KHG14899.1), <i>Abelmoschusesculentus</i>(AGW22222.1), <i>Abelmoschusmanihot</i>(ACE60221.1).</p

Multiple reactions monitoring (MRM) graphs.

<p>MRM chromatograms of standard compounds naringenin and naringenin chalcone eluting at 14.9 and 13.8 min, respectively; <b>(a),</b> MS spectra of naringenin chalcone <b>(b)</b> and naringenin <b>(c)</b>.</p