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An inducible NADPH–cytochrome P450 reductase from Picrorhiza kurrooa — an imperative redox partner of cytochrome P450 enzymes

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Abstract

Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH–cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8 ± 0.05 μmol min−1 mg−1, 8.1 ± 0.12 μmol min−1 mg−1 and 7.8 μM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa.

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Abbreviations

IPTG:

Isopropyl-β-d-thiogalactopyranoside

ORF:

Open reading frame

CPR:

Cytochrome P450 reductase

RACE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse transcription-polymerase chain reaction

MeJA:

Methyl jasmonate

SA:

Salicylic acid

2,4-D:

2,4-Dicholorophenoxy acetic acid

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TSS:

Transcriptional start site

UTR:

Untranslated region

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Acknowledgments

This work was supported by a grant from the Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, under Network Project BSC0108. The authors are thankful to Dr. R.K. Khajuria, Instrumentation Division, Indian Institute of Integrative Medicine Jammu, India, for facilitating HPLC analysis. W.W. Bhat, S. Razdan, S. Rana and N. Dhar are highly thankful to Council of Scientific and Industrial Research (CSIR), Government of India, New Delhi, for Senior Research Fellowship (CSIR-SRF). S. A. Pandith is grateful to University Grants Commission, Government of India, New Delhi, for Senior Research Fellowship (UGC-SRF). This manuscript represents institutional communication number IIIM/1606/2013.

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Authors and Affiliations

  1. Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, India

    Wajid Waheed Bhat, Satiander Rana, Niha Dhar, Sumeer Razdan, Shahzad A. Pandith & Surrinder K. Lattoo

  2. Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi-180001, India

    Ram Vishwakarma

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  1. Wajid Waheed Bhat
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Fig. S1

Nucleotide and the deduced amino acid sequence of PkCPR. 5′ UTR and 3′ UTR are shown in italics. The dash marks the translation termination codon (PPTX 1401 kb)

Fig. S2

Transmembrane domain prediction of PkCPR using Phobious (a) and TMHMM (b) web server (PPTX 79 kb)

Fig. S3

Phylogenetic tree of PkCPR and other plant CPR genes constructed using the ClustalW2 program and MEGA 5.2 software based on the neighbour-joining method. A total of 15 protein sequences used for analysis were from following plant species: Picrorhiza kurrooa (AEW43314), Perilla frutescens (ADC94831), Salvia miltiorrhiza (AGL46979), Catharanthus roseus (Q05001), Petroselinum crispum (AAB97737; AAB97736), Withania somnifera (ADI49691; ADG29353), Pisum sativum (AAC09468), Arabidopsis thaliana (CAA46814; NP_194750), Gossypium hirsutum (ACN54323; ACN54324) and Petunia × hybrida (AAZ39648; AAZ39649) (PPTX 71 kb)

Fig. S4

HPLC chromatogram of standard (picroside I and picroside II mix) at 270 nm (a) and standard (apocynin, androsin and feruloylcatalpol) at 283 nm (b). HPLC chromatogram of the Dhanwas sample of Picrorhiza kurrooa showing five marker compounds viz. picroside I, picroside II, apocynin, androsin and feruloylcatalpol (c and d) (PPTX 387 kb)

Fig. S5

Nucleotide sequences of the PkCPR gene promoter. Numbering starts from the predicted transcription start site. The putative core promoter consensus sequences and the motifs with significant similarity to the previously identified cis-acting elements are boxed and the names are given (PPTX 629 kb)

Fig. S6

Ramachandran plot of PkCPR 3D model of Picrorhiza using PROCHECK server. Most favoured regions are coloured red (A, B, L), additional allowed (a, b, l, p), generously allowed (~a, ~b, ~l, ~p), and disallowed regions are indicated as yellow, light yellow and white fields, respectively (PPTX 2437 kb)

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Bhat, W.W., Rana, S., Dhar, N. et al. An inducible NADPH–cytochrome P450 reductase from Picrorhiza kurrooa — an imperative redox partner of cytochrome P450 enzymes. Funct Integr Genomics 14, 381–399 (2014). https://doi.org/10.1007/s10142-014-0362-7

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