Abstract
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.
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Abbreviations
- MeJA:
-
Methyl jasmonate
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
- SA:
-
Salicylic acid
- ABA:
-
Abscisic acid
- 4CL:
-
4-Coumarate:coenzyme A Ligase
- UV:
-
Ultraviolet
- PAL:
-
Phenylalanine ammonia-lyase
- C4H:
-
Cinnamate 4-hydroxylase
- qPCR:
-
Quantitative real-time RT-PCR
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Acknowledgements
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).
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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.
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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)
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)
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)
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)
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)
Supplementary Table 1
Nucleotide sequence of primers used in the study (DOCX 16 kb)
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Awasthi, P., Mahajan, V., Jamwal, V.L. et al. Characterization of the gene encoding 4-coumarate:CoA ligase in Coleus forskohlii. J. Plant Biochem. Biotechnol. 28, 203–210 (2019). https://doi.org/10.1007/s13562-018-0468-4
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DOI: https://doi.org/10.1007/s13562-018-0468-4