Abstract
Chalcone synthase (CHS) is a type III polyketide synthase and a key enzyme of the phenylpropanoid pathway that generates precursors for flavonoid biosynthesis. The tree species D. gotadhora is known for having an abundance of rohitukine, which has anti-inflammatory and immune-modulating effects. In this study, we used the leaves of D. gotadhora to clone CHS gene (DbCHS). The 1188-bp open reading frame (ORF) was part of the 1373-bp full-length DbCHS clone. Compared to other parts of the plant, DbCHS is expressed more in the leaves and fruits. This is linked to anti-microbial action against a panel of microbes in these tissues. The leaves and seeds extracts inhibit Bacillus subtilis, Streptococcus pyogenes, Bacillus cereus, and Candida albicans. When a plant is hurt, it leaves its tissues open to attack by microbes. To protect themselves, plants often make chemicals that kill microbes. We found that wounding had a big effect on the production of DbCHS. Based on these tests and the results of phylogenetic analysis and molecular docking, we believe that DbCHS is a wound-inducible enzyme that is needed to make flavonoids, which may give the plant antimicrobial properties.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CHS:
-
Chalcone synthase
- PKS:
-
PolyKetide synthase
- RACE:
-
Rapid amplification of cDNA ends
- QAE:
-
Quercetin equivalent
- ORF:
-
Open reading frame
- UTR:
-
UnTranslated regions
References
Adamczak A, Ozarowski M, Karpinski TM (2019) Antibacterial activity of some flavonoids and organic acids widely distributed in plants. J Clin Med. https://doi.org/10.3390/jcm9010109
Awasthi P, Gupta AP, Bedi YS, Vishwakarma RA, Gandhi SG (2016a) Mannitol stress directs flavonoid metabolism toward synthesis of flavones via differential regulation of two cytochrome P450 monooxygenases in Coleus forskohlii. Front Plant Sci 7:985. https://doi.org/10.3389/fpls.2016.00985
Awasthi P, Jamwal VL, Kapoor N, Rasool S (2016b) Homology modeling and docking study of chalcone synthase gene (CfCHS) from Coleus forskohlii. Cogent Biol 2:1175332. https://doi.org/10.1080/23312025.2016.1175332
Awasthi P, Mahajan V, Jamwal VL, Kapoor N, Rasool S, Bedi YS, Gandhi SG (2016c) Cloning and expression analysis of chalcone synthase gene from Coleus forskohlii. J Genet 95:647–657. https://doi.org/10.1007/s12041-016-0680-8
Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinf 4:29. https://doi.org/10.1186/1471-2105-4-29
Cushnie TP, Lamb AJ (2005) Antimicrobial activity of flavonoids. Int J Antimicrob Agents 26:343–356. https://doi.org/10.1016/j.ijantimicag.2005.09.002
Dao TT, Linthorst HJ, Verpoorte R (2011) Chalcone synthase and its functions in plant resistance. Phytochem Rev 10:397–412. https://doi.org/10.1007/s11101-011-9211-7
Falcone Ferreyra ML, Rius SP, Casati P (2012) Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front Plant Sci 3:222. https://doi.org/10.3389/fpls.2012.00222
Ferrer J-L, Jez JM, Bowman ME, Dixon RA, Noel JP (1999) Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat Struct Biol 6:775–784. https://doi.org/10.1038/11553
Fritze K, Staiger D, Czaja I, Walden R, Schell J, Wing D (1991) Developmental and UV light regulation of the snapdragon chalcone synthase promoter. Plant Cell 3:893–905. https://doi.org/10.1105/tpc.3.9.893
Fukuma K, Neuls ED, Ryberg JM, Suh D-Y, Sankawa U (2007) Mutational analysis of conserved outer sphere arginine residues of chalcone synthase. J Biochem 142:731–739. https://doi.org/10.1093/jb/mvm188
Górniak I, Bartoszewski R, Króliczewski J (2019) Comprehensive review of antimicrobial activities of plant flavonoids. Phytochem Rev 18:241–272. https://doi.org/10.1007/s11101-018-9591-z
Guetsky R et al (2005) Metabolism of the flavonoid epicatechin by laccase of Colletotrichum gloeosporioides and its effect on pathogenicity on avocado fruits. Phytopathology 95:1341–1348. https://doi.org/10.1094/PHYTO-95-1341
Harmon AD, Weiss U, Silverton JV (1979) The structure of rohitukine, the main alkaloid of Amoora rohituka (syn. Aphanamixis polystachya) (Meliaceae). Tetrahedron Lett 20:721–724. https://doi.org/10.1016/S0040-4039(01)93556-7
Jamwal VL et al (2019) Isolation, identification and bioactive potential of bacterial endophytes from Coleus. Indian J Biochem Biophys 56:392–398
Jez JM, Ferrer JL, Bowman ME, Austin MB, Schroder J, Dixon RA, Noel JP (2001) Structure and mechanism of chalcone synthase-like polyketide synthases. J Ind Microbiol Biotechnol 27:393–398. https://doi.org/10.1038/sj.jim.7000188
Kang JN, Lee WH, Won SY, Chang S, Hong JP, Oh TJ, Lee SM, Kang SH (2021) Systemic expression of genes involved in the plant defense response induced by wounding in Senna tora. Int J Mol Sci 22(18):10073. https://doi.org/10.3390/ijms221810073
Khadem S, Marles RJ (2011) Chromone and flavonoid alkaloids: occurrence and bioactivity. Molecules 17:191–206. https://doi.org/10.3390/molecules17010191
Kumar A, Sharma M, Chaubey SN, Kumar A (2020) Homology modeling and molecular dynamics based insights into chalcone synthase and chalcone isomerase in Phyllanthus emblica L. 3 Biotech 10:373. https://doi.org/10.1007/s13205-020-02367-2
Lakshmi V, Khanna AK, Mahdi AA, Agarwal SK (2013) Lipid lowering and antioxidant activity of rohitukine from Dysoxylum binectariferum in hyperlipidemic rats. NPAIJ 9:70–76
Lanz T, Tropf S, Marner FJ, Schroder J, Schroder G (1991) The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways. J Biol Chem 266:9971–9976
Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291. https://doi.org/10.1107/S0021889892009944
Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486. https://doi.org/10.1007/BF00228148
Liu Z, Cheng S, Liu XQ, Kuča K, Hashem A, Al-Arjani AB, Almutairi KF et al (2022) Cloning of a CHS gene of Poncirus trifoliata and its expression in response to soil water deficit and arbuscular mycorrhizal fungi. Front Plant Sci 13:1101212. https://doi.org/10.3389/fpls.2022.1101212
Ma W, Wu Y, Wu M, Ren Z, Zhong Y (2015) Cloning, characterization and expression of chalcone synthase from medicinal plant Rhus chinensis. J Plant Biochem Biotechnol 24:18–24. https://doi.org/10.1007/s13562-013-0231-9
Mahajan V et al (2015) Production of rohitukine in leaves and seeds of Dysoxylum binectariferum: an alternate renewable resource. Pharm Biol 53:446–450. https://doi.org/10.3109/13880209.2014.923006
Mallika V, Sivakumar KC, Soniya EV (2011) Evolutionary implications and physicochemical analyses of selected proteins of type iii polyketide synthase family. Evol Bioinform Online 7:41–53. https://doi.org/10.4137/EBO.S6854
Mishra SK et al (2018) Pharmacological evaluation of the efficacy of Dysoxylum binectariferum stem bark and its active constituent rohitukine in regulation of dyslipidemia in rats. J Nat Med 72:837–845. https://doi.org/10.1007/s11418-014-0830-3
Mohanakumara P et al (2010) Dysoxylum binectariferum Hook.f (Meliaceae), a rich source of rohitukine. Fitoterapia 81:145–148. https://doi.org/10.1016/j.fitote.2009.08.010
Naik RG, Kattige SL, Bhat SV, Alreja B, de Souza NJ, Rupp RH (1988) An antiinflammatory cum immunomodulatory piperidinylbenzopyranone from Dysoxylum binectariferum: isolation, structure and total synthesis. Tetrahedron 44:2081–2086
Pandith SA, Ramazan S, Khan MI, Reshi ZA, Shah MA (2019) Chalcone synthases (CHSs): the symbolic type III polyketide synthases. Planta 251:15. https://doi.org/10.1007/s00425-019-03307-y
Poloni A, Schirawski J (2014) Red card for pathogens: phytoalexins in sorghum and maize. Molecules 19:9114–9133. https://doi.org/10.3390/molecules19079114
Rather IA, Awasthi P, Mahajan V, Bedi YS, Vishwakarma RA, Gandhi SG (2015) Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum. Gene 558:143–151. https://doi.org/10.1016/j.gene.2014.12.055
Richard S, Lapointe G, Rutledge RG, Seguin A (2000) Induction of chalcone synthase expression in white spruce by wounding and jasmonate. Plant Cell Physiol 41:982–987. https://doi.org/10.1093/pcp/pcd017
Romanowski A, Schlaen RG, Perez-Santangelo S, Mancini E, Yanovsky MJ (2020) Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana. Plant J 103:889–902. https://doi.org/10.1111/tpj.14776
Schaffer R, Landgraf J, Accerbi M, Simon V, Larson M, Wisman E (2001) Microarray analysis of diurnal and circadian-regulated genes in Arabidopsis. Plant Cell 13:113–123. https://doi.org/10.1105/tpc.13.1.113
Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc Natl Acad Sci 97:11655–11660. https://doi.org/10.1073/pnas.97.21.11655
Schröder J (1997) A family of plant-specific polyketide synthases: facts and predictions. Trends in Plant Sci 2:373–378. https://doi.org/10.1016/S1360-1385(97)87121-X
Stothard P (2000) The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequencesi. Biotechniques 28:1102–1104. https://doi.org/10.2144/00286ir01
Suh DY, Kagami J, Fukuma K, Sankawa U (2000) Evidence for catalytic cysteine-histidine dyad in chalcone synthase. Biochem Biophys Res Commun 275:725–730. https://doi.org/10.1006/bbrc.2000.3368
Sun Y, Gao M, Kang S, Yang C, Meng H, Yang Y, Zhao X, Gao Z, Xu Y, Jin Y et al (2020) Molecular mechanism underlying mechanical wounding-induced flavonoid accumulation in Dalbergia odorifera T. Chen, an endangered tree that produces chinese rosewood. Genes 11(5):478. https://doi.org/10.3390/genes11050478
Tagousop CN, J-d-D T, Ekom SE, Ngnokam D, Voutquenne-Nazabadioko L (2018) Antimicrobial activities of flavonoid glycosides from Graptophyllum grandulosum and their mechanism of antibacterial action. BMC Complement Altern Med 18:252. https://doi.org/10.1186/s12906-018-2321-7
Tang X, Ren C, Hu J, Chen J, Wang J, Wang R, Wu Q, Liao W, Pei J (2023) Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius. Chin Herb Med 15(2):291–297. https://doi.org/10.1016/j.chmed.2022.12.005
Thain SC, Murtas G, Lynn JR, McGrath RB, Millar AJ (2002) The circadian clock that controls gene expression in Arabidopsis is tissue specific. Plant Physiol 130:102–110. https://doi.org/10.1104/pp.005405
Tropf S, Kärcher B, Schröder G, Schröder J (1995) Reaction mechanisms of homodimeric plant polyketide synthase (stilbenes and chalcone synthase). A single active site for the condensing reaction is sufficient for synthesis of stilbenes, chalcones, and 6′-deoxychalcones. J Biol Chem 270:7922–7928. https://doi.org/10.1074/jbc.270.14.7922
Varshney S et al (2014) Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo. J Lipid Res 55:1019–1032. https://doi.org/10.1194/jlr.M039925
Waheed Janabi AH et al (2020) Flavonoid-rich foods (FRF): a promising nutraceutical approach against lifespan-shortening diseases. Iran J Basic Med Sci 23:140–153. https://doi.org/10.22038/IJBMS.2019.35125.8353
Wang MX, Li WL, Zhang Z, Wei JH, Yang Y, Xu YH, Liang L (2013) Cloning and bioinformatics analysis of chalcone synthase (AsCHS1) gene in Aquilaria sinensis. Zhongguo Zhong Yao Za Zhi 38:149–153
Wang F, Ren G, Li F, Qi S, Xu Y, Wang B, Yang Y, Ye Y, Zhou Q, Chen X (2018) A chalcone synthase gene AeCHS from Abelmoschus esculentus regulates flavonoid accumulation and abiotic stress tolerance in transgenic Arabidopsis. Acta Physiol Plant 40:1–3. https://doi.org/10.1007/s11738-018-2680-1
Waterhouse A et al (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucl Acids Res 46:W296–W303
Wegulo SN, Yang X-B, Martinson CA, Murphy PA (2005) Effects of wounding and inoculation with Sclerotinia sclerotiorum on isoflavone concentrations in soybean. Can J Plant Sci 85:749–760. https://doi.org/10.4141/p04-076
Wen W, Alseekh S, Fernie AR (2020) Conservation and diversification of flavonoid metabolism in the plant kingdom. Curr Opin Plant Biol 55:100–108
Xia F, Li H, Fu C, Yu Z, Xu Y, Zhao D (2011) Cloning, expression and charaterization of chalcone synthase from Saussurea medusa. Chin J Biotechnol 27:1363–1370
Acknowledgements
VM and RC are thankful to CSIR for JRF and SRF fellowships. NK is thankful to ICMR for JRF and SRF fellowships. SGG acknowledges financial support from CSIR Phytopharma Mission: HCP038.
Author information
Authors and Affiliations
Contributions
VM carried out most of the experimental work. NK quantified total flavonoids and determined the antimicrobial activity of extracts. VLJ did the molecular modelling and docking analysis and prepared the figures. RC wrote the manuscript. VLJ assisted in writing. SGG designed the study; supervised VM, NK, VLJ and RC, as well as corrected the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
Authors declare that they do not have any conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Fig. 1
Sequence information of DbCHS from D. gotadhora: The ATG start codon at position 54, the TAA stop codon at position 1241 are highlighted in green and red color, respectively; 5′ and 3′ UTRs are shown in blue boxes; Conserved motifs: MMYQGCF, LFGDG, WIAHPGGPA, GNMSSA and FGFGPGL are encircled; Letters in bold-face indicate strictly conserved amino acids (JPG 5773 KB)
Supplementary Fig. 2
Multiple sequence alignment of DbCHS protein sequence, with the homologous proteins from other species: D. longon (GenBank Acc. No. 346577498), D. albus (GenBank Acc. No. 54311699), L. chinensis (GenBank Acc. No. 283827864) and T. cacao (GenBank Acc. No. 590647992); Conserved amino acid residues: Cys164, His303 and Asn336 of CHN triad are highlighted in a black box (JPG 7965 KB)
Supplementary Fig. 3 a
Antimicrobial activities of extracts of D. gotadhora (at 10 mg/mL) against various microbes – BS - Bacillus subtilis (MTCC 121), BC - Bacillus cereus (IIIM 25), KP- Klebsiella pneumonia (ATCC 75388), SP - Streptococcus pyogenes (MTCC 442), SA - Staphylococcus aureus (MTCC 96), and CA - Candida albicans (ATCC 90028); here 1: Leaf extract, 2: root extract, 3: stem extract, 4: antibiotic control at 0.25 µg/mL (ciprofloxacin for bacteria and clotrimazole for fungi) 5: ripe fruit, 6: unripe fruit b Activities of antibiotic controls (ciprofloxacin for bacteria and clotrimazole for fungi) at different concentrations against microbes; here 1: 0.5 µg/mL, 2: 1 µg/mL, 3: 2 µg/mL (PDF 411 KB)
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Mahajan, V., Chouhan, R., Jamwal, V.L. et al. A wound inducible chalcone synthase gene from Dysoxylum gotadhora (DbCHS) regulates flavonoid biosynthesis. Physiol Mol Biol Plants 29, 959–969 (2023). https://doi.org/10.1007/s12298-023-01344-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12298-023-01344-2