Proteins differentially expressed in elicited cell suspension culture of Podophyllum hexandrum with enhanced podophyllotoxin content

Abstract

Background: Podophyllotoxin (PTOX), the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to methyl jasmonate (MeJA) elicitation. High-resolution two-dimensional gel electrophoresis (2-DE) followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell's proteome.

Result: The HPLC analysis showed approximately 7-8 fold change in accumulation of PTOX, in the 12day old cell suspension culture (i.e. after 9days of elicitation) elicited with 100 μM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defense related protein, transcription and DNA replication and signaling are also discussed.

Conclusions: Elicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level.

Figure 1

A flowering twig of Podophyllum hexandrum.

Figure 2

Schematic representation of podophyllotoxin biosynthetic pathway. Dashed arrows represent unknown multiple reactions. Enzyme abbreviations: PAL– Phenylalanine ammonia lyase, C4H– Cinnamate 4-hydroxylyase, C3H– p-coumarate 3-hydroxylyase, CAOMT– caffeic acid–O-methyltransferase, 4CL– 4coumarate: CoA ligase, CCOMT– caffeoyl CoA o-methyltransferase, CCR– cinnamoyl CoA reductase, CAD- cinnamyl alcohol dehydrogenase, DP oxidase– dirigent protein oxidase, PLR– pinoresinol lariciresinol reductase, SDG– Secoisolariciresinol dehydrogenase.

Figure 3

(a) Callus of P. hexandrum used for the initiation of cell suspension culture. (b) Estimation of podophyllotoxin in control and MeJA elicited cell suspension cultures of P. hexandrum using HPLC analysis. Data are the mean ± SD for three individual experiments (n = 3).

Figure 4

Representative two-dimensional gel electrophoresis of total soluble proteins extracted from (a) control and (b) MeJA-elicited cell suspension cultures of P. hexandrum.

Figure 5

Annotated gel image representing predicted functions of proteins. Master gel combining spots of control and MeJA-elicited cell culture protein extracts. Some of the representative spots of major categories are annotated corresponding to their respective SSPs as listed in Additional file 2.

Figure 6

Putative functional classifications of differentially accumulated proteins in MeJA elicited cell suspension cultures of P. hexandrum .