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Characterization and expression analysis of chalcone synthase and chalcone isomerase genes in Phyllanthus emblica (L.)

  • Kashmir Singh
  • Avneesh Kumar
  • Monika Kajal
  • Baljinder Singh
Original Article

Abstract

The two decisive enzymes in flavonoid biosynthetic pathway are chalcone synthase (CHS) and chalcone isomerase (CHI), wherein the former carries the first committed step of the pathway and later is involved in isomerization of chalcone. In the present study, full-length cDNA sequence of both the genes (PeCHS and PeCHI) from Phyllanthus emblica (L.) were cloned and sequenced. The 390 and 209 amino acid long polypeptides of PeCHS and PeCHI are coded by 1514 bp nucleotide (nt) (ORF 1173 bp) and 843 bp nt (ORF 630 bp) sequences respectively. Computational analysis revealed the deduced protein of PeCHS contained four CHS protein family specific conserved motifs including residues of active sites and other signature sequences. Four conserved amino acids Thr-48, Tyr-106, Asn-113, and Thr-190 at active sites were identified in PeCHI. Both PeCHI and PeCHS were expressed in E. coli BL21 using pQE-30 UA vector. Expression analysis was carried out in different developmental stages i.e. leaf, flower and fruits. Expression of PeCHS was maximum in mature fruit while PeCHI expression was highest in young leaves. With respect to fruit, PeCHI expression lower down first and then increases with the maturation of fruit whereas PeCHS expression increases gradually with the development of fruit.

Keywords

Chalcone synthase Chalcone isomerase Flavonoid biosynthesis Phyllanthus emblica Gene expression 

Abbreviations

AMF

Arbuscular mycorrhizal fungi

H1N1

Human influenza A

CHS

Chalcone synthase

CHI

Chalcone isomerise

Phyre2

Protein homology/analogy recognition engine V 2.0

Notes

Acknowledgements

Authors are grateful to Department of Science and Technology (DST) Govt. of India for providing research grant. Scholarship provided to Avneesh Kumar by Council of Scientific & Industrial Research (CSIR), is really appreciable. Monika Kajal is thankful to University Grant Commission (UGC), India for providing fellowship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13562_2018_467_MOESM1_ESM.ppt (1.2 mb)
Supplementary material 1 (PPT 1246 kb)

References

  1. Antunes PM, Rajcan I, Goss MJ (2006) Specific flavonoids as interconnecting signals in the tripartite symbiosis formed by arbuscular mycorrhizal fungi, Bradyrhizobium japonicum (Kirchner) Jordan and soybean (Glycine max (L.) Merr.). Soil Biol Biochem 38(3):533–543CrossRefGoogle Scholar
  2. Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N (2010) ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 38(suppl_2):W529–W533CrossRefPubMedPubMedCentralGoogle Scholar
  3. Baker ME (1992) Evolution of regulation of steroid-mediated intercellular communication in vertebrates: insights from flavonoids, signals that mediate plant-rhizobia symbiosis. J Steroid Biochem Mol Biol 41(3–8):301–308CrossRefPubMedGoogle Scholar
  4. Baliga MS, Dsouza JJ (2011) Amla (Emblica officinalis Gaertn), a wonder berry in the treatment and prevention of cancer. Eur J Cancer Prev 20(3):225–239CrossRefPubMedGoogle Scholar
  5. Cheng H, Li L, Cheng S, Cao F, Wang Y, Yuan H (2011) Molecular cloning and function assay of a chalcone isomerase gene (GbCHI) from Ginkgo biloba. Plant Cell Rep 30(1):49–62CrossRefPubMedGoogle Scholar
  6. Feng C et al (2012) Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNA-Seq. BMC Genom 13(1):19CrossRefGoogle Scholar
  7. Ghosal S, Tripathi VK, Chauhan S (1996) Active constituents of Emblica officinalis, Part I, the chemistry and antioxidant effects of two new hydrolysable tannins, emblicanin A and B. Indian J Chem 35:941Google Scholar
  8. Harris N, Luczo J, Robinson SP, Walker A (2013) Transcriptional regulation of the three grapevine chalcone synthase genes and their role in flavonoid synthesis in Shiraz. Aust J Grape Wine Res 19(2):221–229CrossRefGoogle Scholar
  9. He F et al (2010) Biosynthesis of anthocyanins and their regulation in colored grapes. Molecules 15(12):9057–9091CrossRefPubMedGoogle Scholar
  10. Jez JM, Noel JP (2002) Reaction mechanism of chalcone isomerase pH dependence, diffusion control, and product binding differences. J Biol Chem 277(2):1361–1369CrossRefPubMedGoogle Scholar
  11. Jez JM, Bowman ME, Dixon RA, Noel JP (2000) Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nat Struct Mol Biol 7(9):786CrossRefGoogle Scholar
  12. Karppinen K, Hirvelä E, Nevala T, Sipari N, Suokas M, Jaakola L (2013) Changes in the abscisic acid levels and related gene expression during fruit development and ripening in bilberry (Vaccinium myrtillus L.). Phytochemistry 95:127–134CrossRefPubMedGoogle Scholar
  13. Khan KH (2009) Roles of Emblica officinalis in medicine-A review. Bot Res Int 2(4):218–228Google Scholar
  14. Kumar A, Singh K (2012) Isolation of high quality RNA from Phyllanthus emblica and its evaluation by downstream applications. Mol Biotechnol 52(3):269–275CrossRefPubMedGoogle Scholar
  15. Kumaran A, Karunakaran RJ (2006) Nitric oxide radical scavenging active components from Phyllanthus emblica L. Plant Foods Hum Nutr 61(1):1CrossRefPubMedGoogle Scholar
  16. Kusirisin W et al (2009) Antioxidative activity, polyphenolic content and anti-glycation effect of some Thai medicinal plants traditionally used in diabetic patients. Med Chem 5(2):139–147CrossRefPubMedGoogle Scholar
  17. Loake GJ, Choudhary AD, Harrison MJ, Mavandad M, Lamb CJ, Dixon RA (1991) Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter. Plant Cell 3(8):829–840CrossRefPubMedPubMedCentralGoogle Scholar
  18. Markham KR, Gould KS, Ryan KG (2001) Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration. Phytochemistry 58(3):403–413CrossRefPubMedGoogle Scholar
  19. Martin C, Prescott A, Mackay S, Bartlett J, Vrijlandt E (1991) Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J 1(1):37–49CrossRefPubMedGoogle Scholar
  20. Mishio T, Honma T, Iwashina T (2006) Yellow flavonoids in Centaurea ruthenica as flower pigments. Biochem Syst Ecol 34(2):180–184CrossRefGoogle Scholar
  21. Moilanen E (1997) Anti-inflammatory activity of extracts lirom leaves of Phyllanthus emblica. Planta Med 63:518–524CrossRefPubMedGoogle Scholar
  22. Muir SR et al (2001) Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nat Biotechnol 19(5):470CrossRefPubMedGoogle Scholar
  23. Nakatsuka T et al (2008) Flower color modification of gentian plants by RNAi-mediated gene silencing. Plant Biotechnol 25(1):61–68CrossRefGoogle Scholar
  24. Ngamkitidechakul C, Jaijoy K, Hansakul P, Soonthornchareonnon N, Sireeratawong S (2010) Antitumour effects of Phyllanthus emblica L.: induction of cancer cell apoptosis and inhibition of in vivo tumour promotion and in vitro invasion of human cancer cells. Phytother Res 24(9):1405–1413CrossRefPubMedGoogle Scholar
  25. Niu S-S et al (2010) Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor. Planta 231(4):887–899CrossRefPubMedGoogle Scholar
  26. Peterson J, Dwyer J (1998) Flavonoids: dietary occurrence and biochemical activity. Nutr Res 18(12):1995–2018CrossRefGoogle Scholar
  27. Poltanov EA et al (2009) Chemical and antioxidant evaluation of Indian gooseberry (Emblica officinalis Gaertn., syn. Phyllanthus emblica L.) supplements. Phytother Res 23(9):1309–1315CrossRefPubMedGoogle Scholar
  28. Roschek B Jr, Fink RC, McMichael MD, Li D, Alberte RS (2009) Elderberry flavonoids bind to and prevent H1N1 infection in vitro. Phytochemistry 70(10):1255–1261CrossRefPubMedGoogle Scholar
  29. Singh B, Sharma RA (2015) Antioxidant and antimicrobial activities of callus culture and fruits of phyllanthus emblica L. J Herbs Spices Med Plants 21(3):230–242CrossRefGoogle Scholar
  30. Singh I, Soyal D, Goyal PK (2006) Emblica officinalis (Linn.) fruit extract provides protection against radiation-induced hematological and biochemical alterations in mice. J Environ Pathol Toxicol Oncol 25(4):643–654CrossRefPubMedGoogle Scholar
  31. Singh MK, Yadav SS, Gupta V, Khattri S (2013) Immunomodulatory role of Emblica officinalis in arsenic induced oxidative damage and apoptosis in thymocytes of mice. BMC Complement Altern Med 13(1):193CrossRefPubMedPubMedCentralGoogle Scholar
  32. Srirama R et al (2012) Hepatoprotective activity of Indian Phyllanthus. Pharm Biol 50(8):948–953CrossRefPubMedGoogle Scholar
  33. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739CrossRefPubMedPubMedCentralGoogle Scholar
  34. Tarwadi K, Agte V (2007) Antioxidant and micronutrient potential of common fruits available in the Indian subcontinent. Int J Food Sci Nutr 58(5):341–349CrossRefPubMedGoogle Scholar
  35. Teixeira S et al (2005) Structure–property studies on the antioxidant activity of flavonoids present in diet. Free Radic Biol Med 39(8):1099–1108CrossRefPubMedGoogle Scholar
  36. Wang Y, Li J, Xia R (2010) Expression of chalcone synthase and chalcone isomerase genes and accumulation of corresponding flavonoids during fruit maturation of Guoqing No. 4 satsuma mandarin (Citrus unshiu Marcow). Sci Hortic 125(2):110–116CrossRefGoogle Scholar
  37. Wang W et al (2012) Chalcone isomerase in grape vine: gene expression and localization in the developing fruit. Biol Plant 56(3):545–550CrossRefGoogle Scholar
  38. Warrier PK, Nambiar V (1993) Indian medicinal plants: a compendium of 500 species, vol 5. Orient Blackswan, ChennaiGoogle Scholar
  39. Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5(3):218–223CrossRefPubMedGoogle Scholar
  40. Wollenweber E, Dietz VH (1981) Occurrence and distribution of free flavonoid aglycones in plants. Phytochemistry 20(5):869–932CrossRefGoogle Scholar
  41. Yuan Y, Ma X, Shi Y, Tang D (2013) Isolation and expression analysis of six putative structural genes involved in anthocyanin biosynthesis in Tulipa fosteriana. Sci Hortic 153:93–102CrossRefGoogle Scholar
  42. Zhang Y-J, Tanaka T, Iwamoto Y, Yang C-R, Kouno I (2000) Novel norsesquiterpenoids from the roots of Phyllanthus emblica. J Nat Prod 63(11):1507–1510CrossRefPubMedGoogle Scholar
  43. Zhang L-Z, Zhao W-H, Guo Y-J, Tu G-Z, Lin S, Xin L (2003) Studies on chemical constituents in fruits of Tibetan medicine Phyllanthus emblica. Zhongguo Zhong yao za zhi Zhongguo zhongyao zazhi China J Chin Mater Med 28(10):940–943Google Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2018

Authors and Affiliations

  • Kashmir Singh
    • 1
  • Avneesh Kumar
    • 1
    • 2
  • Monika Kajal
    • 1
  • Baljinder Singh
    • 1
  1. 1.Department of BiotechnologyPanjab UniversityChandigarhIndia
  2. 2.Department of BotanyAkal UniversityTalwandi SaboIndia

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