Abstract
The accumulation of total phenylpropanoids and the real time expression of PAL genes in Ocimum tenuiflorum suspension cell cultures were investigated. A comprehensive analysis of the effects of various abiotic elicitors like jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA) and precursor feeder L-phenylalanine (L-phe) was done in O. tenuiflorum suspension cell cultures during elicitation. Ultra performance liquid chromatography based metabolite profiling revealed differential accumulation of three pharmaceutically important phenylpropanoids: caffeic acid, vanillin and ferulic acid. This accumulation corroborated well with real time expression of PAL genes. Biomass analysis during elicitation showed L-phe and JA moderately and MeJA and SA marginally affected the cell growth. Single and repetitive feeding with L-phe induced 22.5 fold accumulations in caffeic acid, ferulic acid and vanillin on day 16 with PAL expression increasing by 9.8–12 fold. SA (1.5 mM) induced 61 fold increase in caffeic acid accumulation with PAL expressions raised by 4.5 fold. MeJA at 25 µM promoted ferulic acid production up to 62 fold having PAL expression raised by seven fold on day 16 of elicitor treatment. Enhanced accumulation of total phenylpropanoids and elevated expression of PAL gene were observed during the course of treatment with elicitors and precursor feeder. This stipulates that PAL may modulate the biosynthesis of phenylpropanoids especially vanillin, caffeic acid and ferulic acid. The present study is of particular significance for using suspension cell culture systems of O. tenuiflorum, a less explored plant, for biotechnological production of pharmaceutically important plant secondary metabolites.
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs40011-017-0858-8/MediaObjects/40011_2017_858_Fig1_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs40011-017-0858-8/MediaObjects/40011_2017_858_Fig2_HTML.jpg)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs40011-017-0858-8/MediaObjects/40011_2017_858_Fig3_HTML.gif)
![](http://media.springernature.com/m312/springer-static/image/art%3A10.1007%2Fs40011-017-0858-8/MediaObjects/40011_2017_858_Fig4_HTML.gif)
Similar content being viewed by others
References
Chase MW, Reveal JL (2009) A phylogenetic classification of the land plants to accompany APG III. Bot J Linn Soc 161:122–127
WHO (2002) Folium Ocimi Sancti. Monogr Sel Med Plants 2:206–216
Simon JE, Morales MR, Phippen WB, Vieira RF, Hao Z (1999) Basil: a source of aroma compounds and a popular culinary and ornamental herb. In: Janick J (ed) Perspectives on new crops and new uses. ASHS Press, Alexandria, pp 499–505
Sarkar B, Kumar D, Sasmal D, Mukhopadhyay K (2014) Antioxidant and DNA damage protective properties of anthocyanin-rich extracts from Hibiscus and Ocimum: a comparative study. Nat Prod Res 28:1393–1398. doi:10.1080/14786419.2014.904309
Graf E (1992) Release of ferulic acid from wheat bran from ferulic acid esterase (FAE-III) from Aspergilus niger esterases. Appl Microbiol Biotechnol 43:1082–1087
Meessen LL, Delattre M, Haon M, Thibault JF, Ceccaldi BC, Brunerie P, Asther M (1996) A two step bioconversion process for vanillin production from ferulic acid combining Aspergilus niger and Pycnoporous cinnabarinus. J Biotechnol 50:107–113
Hansen EH, Møller BL, Kock GR, Bünner CM, Kristensen C, Jensen OR, Okkels FT, Olsen CE, Motawia MS, Hansen J (2009) De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker’s yeast (Saccharomyces cerevisiae). Appl Environ Microbiol 75:2765–2774
Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20. doi:10.1093/mp/ssp106
Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoids metabolism. Ann Rev Plant Physiol Plant Mol Biol 40:347–369
Simlat M, Stobiecki M, Szklarczyk M (2013) Accumulation of selected phenolics and expression of PAL genes in carrots differing in their susceptibility to carrot fly (Psila rosae F.). Euphytica 190:253–266
Wanner LA, Li G, Ware D, Somssich IE, Davis KR (1995) The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol Biol 27:327–338
Lillo C, Lea US, Ruoff P (2008) Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway. Plant Cell Environ 31:587–601
Renu IK, Haque I, Kumar M, Poddar R, Bandopadhyay R, Rai A, Mukhopadhyay K (2014) Characterization and functional analysis of Eugenol O-methyltransferase gene reveal metabolite shifts, chemotype specific differential expression and developmental regulation in Ocimum tenuiflorum L. Mol Biol Rep 41:1857–1870. doi:10.1007/s11033-014-3035-7-7
Ziaei M, Sharifi M, Behmanesh M, Razavik K (2012) Gene expression and activity of phenylalanine ammonia-lyase and essential oil composition of Ocimum basilicum L. at different growth stages. Iran J Biotechnol 10:32–39
Kai GY, Yang S, Zhang Y, Luo XQ, Fu XQ, Zhang A, **ao J (2012) Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus. Mol Biol Rep 39:1721–1729
Zhang W, Curtin C, Franco C (2002) Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures. Enzym Microb Technol 30:688–696. doi:10.1016/S0141-0229(02)00041-8
Murashige T, Skoog FA (1962) Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Planta 15:473–497
Blainski A, Lopes GC, Mello JCP (2013) Application and analysis of folin ciocalteau method for the determination of the total phenolics content from Limonium brasiliense L. Molecules 18:6852–6865
Vyas P, Haque I, Kumar M, Mukhopadhyay K (2014) Photocontrol of differential gene expression and alterations in foliar anthocyanin: a comparative study using red and green forma Ocimum tenuiflorum. Acta Physiol Plant 36:2091–2102
** Q, Yao Y, Cai Y, Lin Y (2013) Molecular cloning and sequence analysis of a phenylalanine ammonia-lyase gene from Dendrobium. PLoS One 8:e62352. doi:10.1371/journal.pone.0062352
Gong Z, Yamazaki Y, Sugiyama M, Tanaka Y, Saito K (1999) Cloning and molecular analysis of structural genes involved in anthocyanin biosynthesis and expressed in a forma-specific manner in Perilla frutescens. Plant Mol Biol 35:915–927
Masoumian M, Arbakariya A, Syahida A, Maziah M (2011) Effect of precursors on flavonoid production by Hydrocotyle bonariensis callus tissues. Afr J Biotechnol 10:6021–6029
Baranek KS, Pietrosiuk A, Naliwajski RM, Kawiak A, Jeziorek M, Wyderska S, Łojkowska E, Chinou I (2012) Effect of L-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst. In Vitro Cell Deve Biol 48:555–564
Pauwels L, Inze D, Goossens A (2009) Jasmonate-inducible gene: what does it mean? Trends Plant Sci 14:87–91
Ram M, Prasad KV, Singh SK, Hada BS, Kumar S (2013) Influence of salicylic acid and methyl jasmonate elicitation on anthocyanin production in callus cultures of Rosa hybrida L. Plant Cell Tissue Organ Cult 113:459–467. doi:10.1007/s11240-013-0287-1
Chaichana N, Dheeranupattana S (2012) Effects of methyl jasmonate and salicylic acid on alkaloid production from in vitro culture of Stemona sp. Int J Biosci Biochem Bioinform 2:146–150
Dong J, Wan G, Liang Z (2010) Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture. J Biotechnol 148:99–104
Sudha G, Ravishankar G (2003) Elicitation of anthocyanin production in callus cultures of Daucus carota and involvement in calcium channel modulators. Curr Sci 84:775–779
Ali MB, Hahn EJ, Paek KY (2007) Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures. Molecules 12:607–621. doi:10.3390/12030607
Guo S, Man S, Gao W, Liu H, Zhang L, **ao P (2013) Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch. Acta Physiol Plant 35:679–686. doi:10.1007/s11738-012-1108-6
Rahimi S, Hasanloo T, Najafi F, Khavari-Nejad RA (2011) Enhancement of silymarin accumulation using precursor feeding in Silybum marianum hairy root cultures. Plant Omics J 4:34–39
Reidel H, Akumo DN, Saw NM, Kütük O, Neubauer P, Smetanska I (2012) Elicitation and precursor feeding influence phenolic acid composition in Vitis vinifera suspension culture. Afr J Biotechnol 11:3000–3008
Waffo TP, Fauconneau B, Deffieux G, Huguet F, Vercauteren J, Merillon JM (1998) Isolation, identification, and antioxidant activity of three stilbene glucosides newly extracted from Vitis vinifera cell cultures. J Nat Prod 61:655–657
Bastide IF, Palama TL, Bory S, Lécolier A, Noirot M, Joët T (2014) Expression profiles of key phenylpropanoid genes during Vanilla planifolia pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis. Plant Physiol Biochem 74:304–314
Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Ann Rev Plant Physiol Plant Mol Biol 48:355–381
Pauwels L, Morreel K, Witte ED, Lammertyn F, Montagu MV, Boerjan W, Inzé D, Goossens A (2008) Map** methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc Nat Acad Sci USA 105:1380–1385
Righetti LM, Franceschetti MF, Annalisa T, Nello B (2007) Resveratrol production in Vitis vinifera cell suspensions treated with several elicitors. Caryologia 60:169–171
Gundlach H, Müller MJ, Kutchan TM, Zenk MH (1992) Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc Natl Acad Sci USA 89:2389–2393
Gadzovska S, Maury S, Delaunay A, Spasenoski M, Joseph C, Hagège D (2007) Jasmonic acid elicitation of Hypericum perforatum L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones. Plant Cell Tissue Organ Cult 89:1–13
Nafie E, Hathout T, Mokadem ASA (2011) Jasmonic acid elicits oxidative defense and detoxification systems in Cucumis melo L. cells. Braz J Plant Physiol 23:161–174
Kikowska M, Budzianowski J, Krawczyk A, Thiem B (2012) Accumulation of rosamarinic, chlorogenic and caffeic acids in in vitro cultures of Eryngium planum L. Acta Physiol Plant 34:2425–2433. doi:10.1007/s11738-012-1011-1
Gurkok T, Turktas M, Parmaksiz I, Unver T (2014) Transcriptome profiling of alkaloid biosynthesis in elicitor induced opium poppy. Plant Mol Bio Rep. doi:10.1007/s11105-014-0772-7
Broeckling CD, Huhman DV, Farag MA, Smith JT, May GD, Mendes P, Dixon RA, Lloyd WS (2005) Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism. J Exp Bot 56:323–336. doi:10.1093/jxb/eri058
Wang JW, Wu JY (2005) Nitric oxide is involved in methyl jasmonate-induced defense responses and secondary metabolism activities of Taxus cells. Plant Cell Physiol 46:923–930
Chen JY, Wen PF, Kong WF, Pan QH, Zhan JC, Li JM, Wan SB, Huang WD (2006) Effect of salicylic acid on phenylpropanoids and phenylalanine ammonia lyase in harvested grape berries. Postharvest Biol Technol 40:64–72
Zhao J, Davis LC, Veerporte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333
Acknowledgements
The work was supported, in part, by University Grants Commission of India [34-275\2008 SR], Ministry of Food Processing Industries, India [47/MFPI/R&D/2006/517] and Infrastructure Development Fund, Department of Agriculture, Govt. of Jharkhand [5/B.K.V/Misc/12/2001]. Drs. I. Haque and I. K. Renu’s excellent technical assistance during UPLC experiments is also acknowledged. Fellowship was provided to PV by BIT-Mesra.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
There is no conflict of interest among the authors for publication of this manuscript.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Vyas, P., Mukhopadhyay, K. Elicitation of Phenylpropanoids and Expression Analysis of PAL Gene in Suspension Cell Culture of Ocimum tenuiflorum L.. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88, 1207–1217 (2018). https://doi.org/10.1007/s40011-017-0858-8
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40011-017-0858-8