title: Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations.
abstract: BACKGROUND
  Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, one of the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides.
  
  
  RESULTS
  In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera. Full length cDNAs of WsCYP98A and WsCYP76A have open reading frames of 1536 and 1545 bp encoding 511 (58.0 kDa) and 515 (58.7 kDa) amino acid residues, respectively. Entire coding sequences of WsCYP98A and WsCYP76A cDNAs were expressed in Escherichia coli BL21 (DE3) using pGEX4T-2 expression vector. Quantitative real-time PCR analysis indicated that both genes express widely in leaves, stalks, roots, flowers and berries with higher expression levels of WsCYP98A in stalks while WsCYP76A transcript levels were more obvious in roots. Further, transcript profiling after methyl jasmonate, salicylic acid, and gibberellic acid elicitations displayed differential transcriptional regulation of WsCYP98A and WsCYP76A. Copious transcript levels of both P450s correlated positively with the higher production of withanolides.
  
  
  CONCLUSIONS
  Two A-types P450 WsCYP98A and WsCYP76A were isolated, sequenced and heterologously expressed in E. coli. Both P450s are spatially regulated at transcript level showing differential tissue specificity. Exogenous elicitors acted as both positive and negative regulators of mRNA transcripts. Methyl jasmonate and salicylic acid resulted in copious expression of WsCYP98A and WsCYP76A. Enhanced mRNA levels also corroborated well with the increased accumulation of withanolides in response to elicitations. The empirical findings suggest that elicitors possibly incite defence or stress responses of the plant by triggering higher accumulation of withanolides.
authors.affiliation: Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. rana27iiim@gmail.com.
authors.forename: Satiander
authors.initials: S
authors.lastname: Rana
authors.affiliation: Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. wajid361@gmail.com.
authors.forename: Wajid Waheed
authors.initials: WW
authors.lastname: Bhat
authors.affiliation: Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. nehasdhar@gmail.com.
authors.forename: Niha
authors.initials: N
authors.lastname: Dhar
authors.affiliation: Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. shahzadiiim@gmail.com.
authors.forename: Shahzad A
authors.initials: SA
authors.lastname: Pandith
authors.affiliation: Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. sumeerbiotech@gmail.com.
authors.forename: Sumeer
authors.initials: S
authors.lastname: Razdan
authors.affiliation: Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. ram@iiim.ac.in.
authors.forename: Ram
authors.initials: R
authors.lastname: Vishwakarma
authors.affiliation: Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, Tawi-180001, India. sklattoo@iiim.ac.in.
authors.forename: Surrinder K
authors.initials: SK
authors.lastname: Lattoo
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005875:Gibberellins / Q000494:pharmacology; D008969:Molecular Sequence Data; D054883:Oxylipins / Q000494:pharmacology; D010802:Phylogeny; D010937:Plant Growth Regulators / Q000494:pharmacology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012459:Salicylates / Q000494:pharmacology; D016415:Sequence Alignment; D032341:Withania / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D054358:Withanolides / Q000378:metabolism
chemical_list: D000085:Acetates; D003517:Cyclopentanes; D005875:Gibberellins; D054883:Oxylipins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D012459:Salicylates; D054358:Withanolides; C072239:methyl jasmonate; D003577:Cytochrome P-450 Enzyme System; C007842:gibberellic acid