Characterization of the gene encoding 4-coumarate:CoA ligase in Coleus forskohlii

  • Praveen Awasthi
  • Vidushi Mahajan
  • Vijay Lakshmi Jamwal
  • Rekha Chouhan
  • Nitika Kapoor
  • Yashbir S. Bedi
  • Sumit G. GandhiEmail author
Original Article


4-coumarate:coenzyme A ligase (4CL) converts 4-coumaric acid and its hydroxylated derivatives into the CoA thiol esters, directing carbon flux into various end-products of phenylpropanoid metabolism, such as flavonoids and lignins. In this study, full-length cDNA showing homology with plant 4CL genes was cloned from Coleus forskohlii and was designated as Cf4CL (Accession No. KF643242). Cf4CL was found to contain an ORF of 1626 bps. The computational translation of Cf4CL encoded a protein of 541 amino acids. Theoretical isoelectric point and molecular weight of Cf4CL were calculated to be 5.55 and 58.65 kDa, respectively. Phylogenetic tree clustered Cf4CL with the class I 4CLs (involved in lignin biosynthesis). Spatial distribution of Cf4CL in different tissues and its expression in response to various stresses was carried out through qPCR. Abscisic acid (ABA) treatment strongly induced the expression of Cf4CL. Homology modeling and docking studies further ascertain the role of Cf4CL gene in lignin biosynthesis. In silico prediction suggested that Cf4CL may be post-transcriptionally regulated by microRNAs. Decreased expression of miR1886 in response to ABA treatment was associated with an increase in Cf4CL transcripts and lignin content, thus suggesting a possible role of miR1886 in regulating lignin biosynthesis in C. forskohlii.


4CL Docking studies MicroRNAs Phenylpropanoid pathway qPCR 



Methyl jasmonate


2,4-Dichlorophenoxyacetic acid


Salicylic acid


Abscisic acid


4-Coumarate:coenzyme A Ligase




Phenylalanine ammonia-lyase


Cinnamate 4-hydroxylase


Quantitative real-time RT-PCR



PA and VM were supported by CSIR-Senior Research Fellowship. NK was supported by ICMR Junior Research Fellowship. RC was supported by CSIR Junior Research Fellowship. SGG acknowledges the financial support for this work from CSIR 12th FYP Project ‘PMSI’ (BSC0117) and (BSC0106) of Council of Scientific and Industrial Research (CSIR).

Author contributions

PA did the expression profiling, homology modeling and docking studies and lignin quantification. VM carried out cloning work. PA and VM wrote the manuscript. VLJ, RC and NK assisted PA in carrying out lignin quantification and in preparation of manuscript. YSB provided critical inputs for the study as well as during preparation of the manuscript. SGG designed the study, analyzed the results and edited the manuscript and figures.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13562_2018_468_MOESM1_ESM.tif (140 kb)
Supplementary Fig. 1 Nucleotide and the deduced amino acid sequence of Cf4CL from C. forskohlii. The ATG start codon at position 70, the TAA stop codon at position 1696 and conserved regions are highlighted. Conserved amino acid motifs- SSGTTGLPKGV and GEICIRG are also shown (TIFF 140 kb)
13562_2018_468_MOESM2_ESM.tif (4.9 mb)
Supplementary Fig. 2 Sequence similarity of Cf4CL and its homologs in other plant species. (a): Multiple sequence alignment of the Cf4CL protein sequence, with the homologous proteins from other species S. baicalensis (GenBank Acc. No. BAD90937.1), M. officinalis (GenBank Acc. No. CBJ23825.1), P. fortunei (GenBank Acc. No. ACL31667.1) and S. miltiorrhiza (GenBank Acc. No. AAP68991.1). (b): Represents percentage similarity and identity among the related 4CL proteins of different plant species calculated using MatGAT tool (TIFF 4989 kb)
13562_2018_468_MOESM3_ESM.tif (3 mb)
Supplementary Fig. 3 Phylogenetic tree of Cf4CL. (a) Clustering of sequences from different taxonomic groups. Cf4CL clusters with related sequences from dicotyledonous plants. (b) Clustering of Cf4CL with respect to the two known classes of 4CL: class 1 and class II. Cf4CL falls in class I category (TIFF 3063 kb)
13562_2018_468_MOESM4_ESM.tif (7.6 mb)
Supplementary Fig. 4 Protein 3D structure and Ramachandran plot analysis of Cf4CL protein. (a) Protein model of Cf4CL (b) Ramachandran plot analysis of Cf4CL (TIFF 7804 kb)
13562_2018_468_MOESM5_ESM.tif (187 kb)
Supplementary Fig. 5 Computational prediction of miRNAs targeting Cf4CL. The picture shows conservation of target sites of predicted miRNAs in Cf4CL and related homologs from other plant species, and their possible modes of action (cleavage or inhibition of translation) (TIFF 187 kb)
13562_2018_468_MOESM6_ESM.docx (17 kb)
Supplementary Table 1 Nucleotide sequence of primers used in the study (DOCX 16 kb)


  1. Ahmed B, Vishwakarma RA (1988) Coleoside, a monoterpene glycoside from Coleus forskohlii. Phytochemistry 27:3309–3310. CrossRefGoogle Scholar
  2. Alasbahi RH, Melzig MF (2010a) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology—part 2. Planta Med 76:653–661. CrossRefPubMedGoogle Scholar
  3. Alasbahi RH, Melzig MF (2010b) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology—part 1. Planta Med 76:653–661. CrossRefPubMedGoogle Scholar
  4. Allina SM, Pri-Hadash A, Theilmann DA et al (1998) 4-Coumarate:coenzyme A ligase in hybrid poplar. Properties of native enzymes, cDNA cloning, and analysis of recombinant enzymes. Plant Physiol 116:743–754CrossRefPubMedPubMedCentralGoogle Scholar
  5. Awasthi P, Mahajan V, Rather IA et al (2015) Plant Omics: isolation, identification, and expression analysis of cytochrome P450 gene sequences from Coleus forskohlii. Omi A J Integr Biol 19:782–792. CrossRefGoogle Scholar
  6. Awasthi P, Gupta AP, Bedi YS et al (2016a) Mannitol stress directs flavonoid metabolism toward synthesis of flavones via differential regulation of two cytochrome P450 monooxygenases in Coleus forskohlii. Front Plant Sci 7:1–13. CrossRefGoogle Scholar
  7. Awasthi P, Lakshmi Jamwal V, Kapoor N et al (2016b) Homology modeling and docking study of chalcone synthase gene (CfCHS) from Coleus forskohlii. Cogent Biol 2:1175332. CrossRefGoogle Scholar
  8. Awasthi P, Mahajan V, Jamwal VL (2016c) Cloning and expression analysis of chalcone synthase gene from Coleus forskohlii. J Genet 95:647–657. CrossRefPubMedGoogle Scholar
  9. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  10. Bjellqvist B, Hughes GJ, Pasquali C et al (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14:1023–1031CrossRefPubMedGoogle Scholar
  11. Bjellqvist B, Basse B, Olsen E, Celis JE (1994) Reference points for comparisons of two-dimensional maps of proteins from different human cell types defined in a pH scale where isoelectric points correlate with polypeptide compositions. Electrophoresis 15:529–539CrossRefPubMedGoogle Scholar
  12. Camera L, Gouzerh G, Dhondt S et al (2004) Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways. Immunol Rev 198:267–284CrossRefPubMedGoogle Scholar
  13. Cao Y, Hu S, Huang S et al (2012) Molecular cloning, expression pattern, and putative cis-acting elements of a 4-coumarate:CoA ligase gene in bamboo (Neosinocalamus affinis). Electron J Biotechnol 15:1–13CrossRefGoogle Scholar
  14. Chowdhury MEK, Choi B, Cho B-K et al (2013) Regulation of 4CL, encoding 4-coumarate:coenzyme A ligase, expression in Kenaf under diverse stress conditions. Plant Omics 6:254–262Google Scholar
  15. Cukovic D, Ehlting J, VanZiffle JA, Douglas CJ (2001) Structure and evolution of 4-coumarate:coenzyme A ligase (4CL) gene families. Biol Chem 382:645–654. PubMedCrossRefGoogle Scholar
  16. Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol 61:651–679. CrossRefPubMedGoogle Scholar
  17. de Oliveira DM, Finger-Teixeira A, Rodrigues Mota T et al (2015) Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis. Plant Biotechnol J 13:1224–1232. CrossRefPubMedGoogle Scholar
  18. Douglas CJ, Ellard M, Hauffe KD, Molitor E, de Sá MM, Reinold S, Subramaniam R, Williams F (1992) General phenylpropanoid metabolism: regulation by environmental and developmental signals. In: Stafford HA, Ibrahim RK (eds) Phenolic metabolism in plants. Springer, Boston, pp 63–90CrossRefGoogle Scholar
  19. Egan JF, Maxwell BD, Mortensen DA et al (2011) 2,4-Dichlorophenoxyacetic acid (2,4-D)-resistant crops and the potential for evolution of 2,4-D-resistant weeds. Proc Natl Acad Sci U S A 108:E37. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Ehlting J, Buttner D, Wang Q et al (1999) Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant J 19:9–20. CrossRefPubMedGoogle Scholar
  21. Gallego-Giraldo L, Escamilla-Trevino L, Jackson LA, Dixon RA (2011) Salicylic acid mediates the reduced growth of lignin down-regulated plants. Proc Natl Acad Sci U S A 108:20814–20819. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Gandotra N (2013) Plant signaling: understanding the molecular crosstalk. Springer, BerlinGoogle Scholar
  23. Goujon T, Sibout R, Eudesa A et al (2003) Genes involved in the biosynthesis of lignin precursors in Arabidopsis thaliana. Plant Physiol Biochem 41:677–687. CrossRefGoogle Scholar
  24. Guo H, Kan Y, Liu W (2011) Differential expression of miRNAs in response to topping in flue-cured tobacco (Nicotiana tabacum) roots. PLoS ONE 6:e28565. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369. CrossRefGoogle Scholar
  26. Hamberger B, Hahlbrock K (2004) The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc Natl Acad Sci U S A 101:2209–2214. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hu Y, Gai Y, Yin L et al (2010) Crystal structures of a Populus tomentosa 4-coumarate:CoA ligase shed light on its enzymatic mechanisms. Plant Cell 22:3093–3104. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Jia C, Zhao H, Wang H et al (2004) Obtaining the transgenic poplars with low lignin content through down-regulation of4CL. Chin Sci Bull 49:905–909. CrossRefGoogle Scholar
  29. Kasirajan L, Charles S, Aruchamy K (2017) Cloning and expression analysis of 4-coumarate CoA ligase (4CL) gene in sugarcane clones varying in lignin content. Proc Natl Acad Sci India Sect B Biol Sci. CrossRefGoogle Scholar
  30. Kavitha C, Rajamani K, Vadivel E (2010) Coleus forskohlii: a comprehensive review on morphology, phytochemistry and pharmacological aspects. J Med Plants 4:278–285Google Scholar
  31. Kumar A, Ellis BE (2003) 4-coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and expression. Plant Mol Biol 51:327–340CrossRefPubMedGoogle Scholar
  32. Lee D, Ellard M, Wanner LA et al (1995) The Arabidopsis thaliana 4-coumarate:CoA ligase (4CL) gene: stress and developmentally regulated expression and nucleotide sequence of its cDNA. Plant Mol Biol 28:871–884CrossRefPubMedGoogle Scholar
  33. Li Y, Kim JI, Pysh L, Chapple C (2015) Four isoforms of Arabidopsis 4-coumarate:CoA ligase have overlapping yet distinct roles in phenylpropanoid metabolism. Plant Physiol 169:2409. PubMedPubMedCentralCrossRefGoogle Scholar
  34. Lindermayr C, Möllers B, Fliegmann J et al (2002) Divergent members of a soybean (Glycine max L.) 4-coumarate:coenzyme A ligase gene family. Eur J Biochem 269:1304–1315. CrossRefPubMedGoogle Scholar
  35. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. CrossRefPubMedGoogle Scholar
  36. Miedes E, Vanholme R, Boerjan W, Molina A (2014) The role of the secondary cell wall in plant resistance to pathogens. Front Plant Sci 5:358. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Morris GM, Huey R, Lindstrom W et al (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Paul M, Radha A, Kumar DS (2013) On the high value medicinal plant, Coleus forskohlii Briq. Hygeia 5:69–78Google Scholar
  39. Porth I, Hamberger B, White R, Ritland K (2011) Defense mechanisms against herbivory in Picea: sequence evolution and expression regulation of gene family members in the phenylpropanoid pathway. BMC Genom 12:608. CrossRefGoogle Scholar
  40. Rather IA, Awasthi P, Mahajan V et al (2015) Molecular cloning and functional characterization of an antifungal PR-5 protein from Ocimum basilicum. Gene 558:143–151. CrossRefPubMedGoogle Scholar
  41. Rogers LA, Dubos C, Surman C et al (2005) Comparison of lignin deposition in three ectopic lignification mutants. New Phytol 168:123–140. CrossRefPubMedGoogle Scholar
  42. Saballos A, Sattler SE, Sanchez E et al (2012) Brown midrib2 (Bmr2) encodes the major 4-coumarate:coenzyme A ligase involved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench). Plant J 70:818–830. CrossRefPubMedGoogle Scholar
  43. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  44. Schmelz S, Naismith JH (2009) Adenylate-forming enzymes. Curr Opin Struct Biol 19:666–671. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Shann JR, Blum U (1987) The utilization of exogenously supplied ferulic acid in lignin biosynthesis. Phytochemistry 26:2977–2982. CrossRefGoogle Scholar
  46. Song Y (2014) Insight into the mode of action of 2,4-dichlorophenoxyacetic acid (2,4-D) as an herbicide. J Integr Plant Biol 56:106–113. CrossRefPubMedGoogle Scholar
  47. Stuible H, Büttner D, Ehlting J et al (2000) Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes. FEBS Lett 467:117–122CrossRefPubMedGoogle Scholar
  48. Sunkar R, Chinnusamy V, Zhu J, Zhu J-K (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309. CrossRefPubMedGoogle Scholar
  49. Tamura K, Peterson D, Peterson N et al (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Tuteja N (2007) Abscisic acid and abiotic stress signaling. Plant Signal Behav 2:135–138CrossRefPubMedPubMedCentralGoogle Scholar
  51. Tuteja N, Sopory SK (2008) Chemical signaling under abiotic stress environment in plants. Plant Signal Behav 3:525–536CrossRefPubMedPubMedCentralGoogle Scholar
  52. Voelker SL, Lachenbruch B, Meinzer FC et al (2010) Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiol 154:874–886. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Vogt T (2010) Phenylpropanoid biosynthesis. Mol Plant 3:2–20. CrossRefPubMedGoogle Scholar
  54. Voo KS, Whetten RW, O’Malley DM, Sederoff RR (1995) 4-coumarate:coenzyme a ligase from loblolly pine xylem. Isolation, characterization, and complementary DNA cloning. Plant Physiol 108:85–97CrossRefPubMedPubMedCentralGoogle Scholar
  55. Wei H-Y, Rao G-D, Wang Y-K et al (2013) Cloning and analysis of a new 4CL-like gene in Populus tomentosa. For Sci Pract 15:98–104. CrossRefGoogle Scholar
  56. Xu Z, Zhang D, Hu J et al (2009) Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom. BMC Bioinform 10:S3. CrossRefGoogle Scholar
  57. Yang J, Yan R, Roy A et al (2014) The I-TASSER Suite: protein structure and function prediction. Nat Methods 12:7–8. CrossRefGoogle Scholar
  58. Yoon J, Choi H, An G (2015) Roles of lignin biosynthesis and regulatory genes in plant development. J Integr Plant Biol 57:902–912. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Zhang X-H, Chiang VL (1997) Molecular cloning of 4-coumarate:coenzyme A ligase in Loblollv Pine and the roles of this enzyme in the biosynthesis of lignin in compression wood. Plant Physiol 113:65–74CrossRefPubMedPubMedCentralGoogle Scholar
  60. Zhao S-J, Hu Z-B, Liu D, Leung FCC (2006) Two divergent members of 4-coumarate:coenzyme A ligase from Salvia miltiorrhiza Bunge: cDNA cloning and functional study. J Integr Plant Biol 48:1355–1364. CrossRefGoogle Scholar
  61. Zhu J-K (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2018

Authors and Affiliations

  • Praveen Awasthi
    • 1
  • Vidushi Mahajan
    • 1
    • 2
  • Vijay Lakshmi Jamwal
    • 1
  • Rekha Chouhan
    • 1
  • Nitika Kapoor
    • 1
  • Yashbir S. Bedi
    • 1
    • 2
  • Sumit G. Gandhi
    • 1
    • 2
    Email author
  1. 1.Plant Biotechnology DivisionIndian Institute of Integrative Medicine (CSIR-IIIM)JammuIndia
  2. 2.Division of Biological Science, Faculty of ScienceAcademy of Scientific and Innovative ResearchKolkataIndia

Personalised recommendations