A search was made for proteins differentially accumulated in cell suspension cultures of P. hexandrum elicited with MeJA resulting enhanced accumulation of PTOX. The ability to perform proteomic analyses of medicinal plants without a fully sequenced and annotated genome will provide a useful platform and form a baseline for exploring and investigating the complex physiological pathways related to metabolism, defence and signaling of these medicinal plants. In this investigation 100 μM MeJA was noted as optimum for enhanced accumulation of PTOX which can be corroborated with previous report where 2 to 10- fold enhancement of PTOX was noted in cell suspension culture of L. album
. The role of MeJA in triggering phenyl propanoid biosynthetic pathway and elicitation of defense related proteins are the major effects described previously. Thus, our analysis is focused on proteins closely related to these effects. However, proteins related to other categories have also been discussed in brief.
Proteins related to secondary metabolism
The major category of the identified protein was related to secondary metabolism comprising of 13% of the total proteins identified. This is in consistence with previous reports of elicited cell cultures in which several secondary metabolic enzymes were identified . However, the proteomes of a few medicinal plants with rich but incomplete sequence information have been explored, including Taxus cuspidate
, Catharanthus roseus
, Papaver somniferum
[28, 34], ginseng  and Chelidonium majus
. For proteomic analysis of such organisms, one approach utilizes sequence homology to proteins already in a database. This approach allows identification of highly conserved proteins such as primary metabolic pathways proteins but not necessarily enzymes involved in specific secondary metabolic pathways like PTOX biosynthesis which are specific to individual plant species. Elicitor-mediated accumulation of phenylpropanoid compounds in plant cell cultures has been studied in greatest detail in parsley, chickpea, soybean and alfalfa cells [37–40]. In our study also several proteins related to phenylpropanoid biosynthetic pathways were identified, which is consistent with previous studies, for example- Chalcone synthase, it plays an essential role in the biosynthesis of plant phenylpropanoids and catalyzes the first step in flavonoid biosynthesis . Likewise, plant O-methyltransferases (OMTs) constitutes a large family of enzymes that methylate the oxygen atom of a variety of secondary metabolites including phenylpropanoids, flavonoids, and alkaloids. O-Methylation plays a key role in lignin biosynthesis, stress tolerance, and disease resistance in plants . Monolignol pathway enzymes like Caffeoyl CoA 3-O-methyltransferase (CCOMT), a class A O-methyltransferase was found to be up-accumulated in elicited culture  and is essential for the biosynthesis of coniferyl and sinapyl alcohols, the precursors of lignins and lignans [44, 45]. Likewise S-adenosyl-L-methionine-dependent methyltransferases (SAM-methyltransferases) and caffeic acid-O-methyl transferase (CAOMT) identified as up-accumulated in the present study are the key enzymes in phenylpropanoid, flavonoid and monolignol pathway [46, 47]. The identification of these methyltransferases, suggests the probability of the presence of such homologous proteins in P. hexandrum, which may have a role in PTOX biosynthetic pathway.
The biosynthetic pathway of PTOX is yet to be revealed to its full extent, however the identification of several monolignol as well as phenylpropanoid pathway related enzymes, after MeJA-elicitation substantially adds to the available information. This approach may be used to identify and trace the poorly categorized pathway of PTOX. Similarly differential expression of proteins related to other secondary metabolic pathways were also observed indicating the enhancement of homologous proteins related to PTOX biosynthetic pathways following the MeJA elicitation. Proteins such as polyphenol oxidase was identified as well, these have diverse roles related to secondary metabolism as well as defense, induction of these proteins following wounding and elicitations have been reported vividly . Proteins related to other secondary metabolic pathways like anthocyanin pathway such as chalcone-flavone isomerase and resveratrol synthase, involved in synthesis of resveratrol (phytoalexin) were also observed which may be due to MeJA elicitation as reported previously . Likewise dioxygenases and tyrosine decarboxylase were also identified, these are important in the biosynthesis of plant signaling compounds such as abscisic acid, gibberellins, and ethylene and also of secondary metabolites, notably flavonoids and alkaloids [50, 51].
Stress and defense related protein
Jasmonates are known as defense gene regulators in virtually all plant systems. The MeJA elicited culture also showed an up-accumulation of defense/stress-related enzyme, the second major category of the identified protein, which forms 15% of the total proteins identified. Pathogenesis related proteins Pr-1 like protein and intracellular PR-104 proteins were up-accumulated in the elicited cells, which can be correlated with previous reports [52, 53]. In general, pathogenesis related proteins are induced by pathogen challenge and exhibit antibacterial and antifungal activities . Although PR-10 are classified as defense response proteins, however, the down-regulation of PR-10 was observed in present study, which suggests that they may be involved in functions different from defense, for example some have ribonuclease activity and ligand binding capacity  as well, but its precise function is still unclear. Similarly a downregulation of alcohol dehydrogenase was observed. Likewise the identification of other proteins like glutathione transferase, benzothiadiazole-induced S-adenosyl L-methionine salicylic acid carboxyl methyl transferase, NBS/LRR resistance protein and LRR kinase protein as a defense response following elicitation is also a well known fact and may be correlated with previous studies [49, 56]. The ability of jasmonate to induce variety of defence related proteins reported previously can be corroborated with our investigation [57–59].
Most proteins identified as signal transduction components belong to calcium signaling pathways and include a calmodulin, which increased in abundance in response to elicitor treatment. Calcium flux is important for the elicitor-induced accumulation of secondary metabolites  and calmodulin is a ubiquitous calcium sensor. Calmodulin has been implicated in the salicylic acid-independent defense response in tobacco . Phosphoinositide specific phospholipase C family protein was found to be down-regulated. Binding of TFs to DNA can be regulated via protein phosphorylation and dephosphorylation which in turn regulate expression of many target genes, including TF genes . Therefore, regulation of TF biosynthesis, activation, and inactivation provides a flexible network for regulation of plant secondary metabolites.
Transcription and DNA replication
It is believed that all signal transduction pathways finally converge on transcription factors (TFs), and almost all genes for secondary metabolite biosynthesis are regulated by specific TFs. Identification of TFs like Apetala 1 can be corroborated with previous reports where rapid accumulation of AP2/ERF-domain TFs in response to fungal elicitor or MeJA treatment has been shown . Many genes in the phenylpropanoid pathway possess the same TF-binding cis-elements and are regulated by the same TFs. Similarly role of cis acting elements such as MYB TFs in binding to specific phenylpropanoid pathway genes has also been reported previously . Myb and bHLH, essential for regulating phenylpropanoid and flavonoid biosynthesis pathway, can physically interact with each other . Role of R2R3 Myb transcription factor in MeJA signal transduction and induction of phenylpropanoid biosynthetic pathway genes is also reported  indicating the essential roles of TFs in regulation of plant polyphenolics like lignans. Down-regulation of certain transcription factors like F-box proteins has been observed. Role of these proteins has been observed in various developmental processes in plants, for example photomorphogenesis, circadian clock regulation, self incompatibility, and floral meristem and floral organ identity determination, involves F-box proteins [67–69].
Proteins related to other metabolic pathways
The P. hexandrum cell suspension cultures were induced from leaves therefore spots corresponding to proteins related to various categories like electron transfer chain, carbohydrate pathways, glycolysis, pyruvate pathways, tricarboxylic acid pathway and other carbon metabolism enzymes were also identified. Significant down-regulation of some of these proteins like galactonolactone dehydrogenase, quinone oxidoreductase and inositol polyphosphate 5 phosphatase were observed. Proteins related to other biological functions like protein translocation, folding, modification, and degradation were also identified. Previous work of cell proteome elicited with fungal elicitors has also shown similar proteins [27, 28], which can be correlated with our study.
Proteins related to other categories like cell wall biosynthesis, lipid metabolism, nucleotide metabolism, amino acid metabolism was observed. Down regulation of some of these proteins like aspartate aminotransferase was noted. Interestingly cyclin A like protein involved in cell cycle was observed to be down regulated which may be correlated with previous study of significant decrease in cell growth following MeJA elicitation [70, 71]. Hormone biosynthetic enzymes like 12-oxophytodienoic acid reductase, involved in the biosynthesis of jasmonic acid, a signaling molecule commonly induced in response to stress, wound, and pathogenic and herbivore attacks and was identified in our study and in a proteomics survey of a C. roseus cell culture producing monoterpenoid indole alkaloids. Jasmonic acid is a critical signaling molecule in plant defense responses . Rapid accumulation of endogenous jasmonic acid in response to elicitation has been noted in many plant cell cultures of diverse taxonomic origins, and it has been suggested to mediate the induction of many defense-responsive natural products. In general, identification of proteins related to diverse metabolic pathways following MeJA elicitation can be corroborated with previous reports . However, less is known about how primary and secondary metabolic pathways are coordinated to support increased secondary metabolite production. Proteins related to transposon and unknown biological processes were also observed (see Additional file 2). The sequence information on P. hexandrum is unavailable and the number of functionally annotated ESTs is low at this time. As this number increases, the proteome data will be proven even more useful. In the existing databases, the proteins of unknown function are denoted as unknown protein, hypothetical protein or putative protein. The hypothetical proteins are peptides generated by computer assisted theoretical translation of nucleotide sequences but have never been identified as protein products.
Understanding the temporal, spatial, developmental, and environmental expression of proteins is useful for further analysis of its function in any associated condition. The use of protein profiling, identification of protein classes and their expression profile at subcellular level define the functions involved in metabolic pathways, which ultimately dictate the cellular physiology. Thus, assigning a functional role to each of the yet unidentified protein will be challenging and quite valuable in order to know their physiological significance in P. hexandrum.