The mechanism of action of ptox is based on inhibiting the polymerisation of tubulin and arresting of the cell cycle in the metaphase (Ayres and Loike, 1990; Buss and Waigh, 1995). Glycosidic derivatives of ptox, namely, etoposide, etopophos, and teniposide inhibit the cell cycle by inhibiting the DNA topo isomerase II (Fleming et al., 1989; Von Wartburg and Stahelin, 1993). Previous studies have also shown that ptox and its derivatives exhibit biological activity as strong antiviral agents and as antineoplastic drugs and its glycosidic derivatives, etoposide, etopophos (etoposide phosphate), teniposide are thus widely used for the treatment for various types of malignancy (Stahelin and von Wartburg, 1991; Canela et al., 2000). Ptox, is a polyphenolic substance, and formed by the combination of two phenylpropane units. Ptox is one of the most frequently used cytotoxic lignan isolated from podophyllin, which is an ethanolic extract of an endangered medicinal plant, P. hexandrum (Stahelin and von Wartburg, 1991; Gordaliza et al., 2004). This medicinal herb is generally grown at high altitude of the Himalayas and belongs to the Berberidaceae family. In one recent study, the Podophyllum Germination Network (PGN) has been constructed in reference to Arabidopsis thaliana to explore the underlying potential of seed germination mechanisms (Dogra et al., 2015). Previous studies reported that the chemical synthesis of cyclolignan like ptox is yet to be feasible. Thus, various biotechnological techniques like biotransformations, etc. (Kutney, 1999), especially transgenic hairy roots produced by infection of plants with Agrobacterium rhizogenes and cell culture based production of cyclolignan like ptox have been studied extensively (Kadkade, 1981; van Uden et al., 1989; Heyenga et al., 1990; Seidel et al., 2002; Chattopadhyay et al., 2003). It was also reported that MeJA treatment induce the production of lariciresinol, another lignan, in hairy root cultures of Isatis indigotica (Chen et al., 2015).

Previously, the cell suspension cultures of Linum spp. was noted with higher yield of ptox using MeJA and salicylic acid (SA) as elicitors (van Furden et al., 2005; Yousefzadi et al., 2010). MeJA treatment also induced the other secondary metabolie production like paclitaxel in the cell culture of Taxus cuspidate (Lenka et al., 2015). Treatment of the cell culture of L. nodiflorum with coronalon, indanoyl-isoleucine and MeJA induced the accumulation of 6-methoxypodophyllotoxin, a lignan related to ptox (Berim et al., 2005). In our previous studies, we have also reported the 8- to 10-fold higher yield of ptox in MeJA treated old cell culture of P. hexandrum (Bhattacharyya et al., 2012).

Jasmonic acid (JA) and its methyl derivative, MeJA, are well known as plant signaling compound that involves in stress management and development. Wang and Wu (2005) reported that exogenous treatment of JA and MeJA, actually mimic the effects of wounding and these elicitors bring the relative response like production of secondary metabolite as well as ROS. It has also been reported that MeJA induced ROS alter the mitochondrial dynamics that resulted in photosynthetic dysfunction and cell death (Zhang and Xing, 2008). According to Wang and Wu (2005) MeJA induces the ROS, which includes H₂O₂ and NO, in cell culture of Taxus sp. and the introduction of ROS inhibitor to culture reduce the MeJA induced taxol production. Similar to the changes in taxol from Taxus sp., concentration of other secondary metabolites like phytoalexin were also increased by ROS via cyclopentenone isoprostanes in tomato (Thoma et al., 2003). MeJA induced production of H₂O₂ also reported in rice leaves. According to Orozco-Cárdenas et al. (2001) H₂O₂ acts as a second messenger upon treatment with MeJA and induced systemin production to the tomato plant.

Previously, we also did a whole transcriptome analysis of control and MeJA treated cell culture of P. hexandrum and identified ptox specific CAD isoforms namely, PhCAD3 and PhCAD4 (Bhattacharyya et al., 2016, 2013). However, the mechanism through which MeJA regulates ptox biosynthetic pathway genes is yet to be understood. This study revealed the molecular mechanism of MeJA altered ptox accumulation by regulating ptox biosynthetic pathway genes at transcript level. This has shown that MeJA induced ROS alters the mRNA stability of selected pathway genes to regulate their expression at transcript level. In addition to that, it was also noted that MeJA up regulates other ptox biosynthetic pathway genes which were not regulated by ROS. These ROS non-responsive genes may be regulated by the down regulation of particular MeJA responsive miRNAs, which are interfering with ptox pathway genes. Together, this study suggests two possible molecular mechanisms of MeJA induced ptox accumulation in P. hexandrum cell culture.

It is well established that MeJA is a signal molecule in plant defense responses and an effective inducer of secondary metabolite accumulation in plant cell cultures by means of ROS as a secondary signaling molecule. According to You and Chan (2015) during stress condition ROS plays a vital role in the regulation of complex signaling network of plant stress response. It was also reported by Wang and Wu, 2005 that MeJA also induced the production of H₂O₂, intracellular malondialdehyde content and lipoxygenase. ROS also regulates the gene expression, translation, metabolism, and turnover (Singh et al., 2016). Exogenous application of MeJA also alters the stomatal closure by ROS where ROS inhibitor DPI prohibited the stomatal closure (Suhita et al., 2004). In this study, we explore the mechanism through which MeJA affects the increased accumulation of ptox. Previously, we have reported that MeJA significantly induces accumulation of ptox by up regulating the genes in the ptox biosynthetic pathway (Bhattacharyya et al., 2013, 2016). However, the molecular mechanism of MeJA-induced expression of ptox biosynthetic pathway genes is still to be elucidated. In this study ROS has been detected by the FACS and confocal microscopy in MeJA treated P. hexandrum cell culture which was simultaneous to the enhanced ptox accumulation (Figures 1A–C). Results also indicate the enhanced ptox accumulation in CCCP and H₂O₂ treated P. hexandrum cell cultures which may be attributed to the enhanced ROS production (Figures 1A–C). To further confirm the involvement of ROS in ptox accumulation, in both MeJA and CCCP treated cultures, ROS was inhibited by DPI and DETC and that led to the lower degree of ptox accumulation. This observation confirmed the involvement of MeJA induced ROS in increased accumulation of ptox.

It was reported earlier that ROS plays a significant role as a signaling molecule in plant development, abiotic and biotic stress management, photorespiration and photosynthesis, etc. (Noctor and Foyer, 1998; Apel and Hirt, 2004; Mittler et al., 2004).

To understand how MeJA induced ROS affect the ptox biosynthetic pathway genes consequently up accumulating ptox biosynthesis, the expression pattern of 19 ptox biosynthetic pathway genes was checked at transcript level after the MeJA treatment. Results revealed that all these 19 genes were up regulated after the MeJA treatment. Interestingly, when MeJA induced ROS was inhibited by DPI and DETC treatment, consequently, the up regulation of three genes, namely PhCAD3, PhCAD4, and NAC3 was decreased significantly, whereas other 13 genes were noted with the almost same rate of up regulation as obtained after MeJA treatment without ROS scavenging (Figure 2A). Hence, it can be assumed that MeJA induced ROS affect the regulation of PhCAD3, PhCAD4, and NAC3. To confirm the ROS-responsiveness of these three genes, we further treated the culture with CCCP, which induced the ROS generation (Chaudhari et al., 2007; Ji et al., 2016) as well as with H₂O_2. Results indicated that only three genes, namely, PhCAD3, PhCAD4, and NAC3, were significantly up regulated after the treatment amongst all 19 genes (Figures 2B,C). Furthermore, when ROS generated in CCCP treated culture was inhibited by DPI and DETC treatment, the up regulation of these three genes were deceased significantly. Together, it can be deduced that MeJA induced ROS is the responsible factor for up regulation of these three ROS-responsive genes namely PhCAD3, PhCAD4, and NAC3. In our previous study, we have demonstrated that these two ROS-sensitive isoforms of PhCAD, viz. PhCAD3 and PhCAD4, were specifically responsible for ptox biosynthesis (Bhattacharyya et al., 2016).

We further investigated the underlying mechanism through which ROS-responsive genes of the ptox biosynthetic pathway were regulated by ROS. It is well known that the regulation of mRNA decay is a major checkpoint for controlling the expression level of different genes (Guhaniyogi and Brewer, 2001; Shim and Karin, 2002). According to Chung et al. (2008) the mRNA stability of a particular gene can be altered by ROS. They showed that H₂O₂ treatment increases the mRNA stability of the SOS1 gene in A. thaliana. In our study, the mRNA stability of PhCAD3, PhCAD4, and NAC3 were increased after independent MeJA, CCCP, and H₂O₂ treatment. Further, when ROS generated in MeJA and CCCP treated culture, was inhibited by DPI and DETC, the mRNA stability of these three genes was decreased (Figure 3). That observation indicates that MeJA induced ROS up regulates PhCAD3, PhCAD4, and NAC3 by increasing their mRNA stability.

We also proposed the possible mechanism of MeJA mediated up regulation of upstream and downstream genes of PhCADs which are not regulated by MeJA produced ROS. According to Carlborg and Haley, (2004) the differential expression of genes can result from cis- and/or trans-regulatory changes, leading to epistatic effects. miRNA plays a vital role as a trans-acting regulator of gene expression. It was previously reported that miR163 plays important role in plant secondary metabolism (Ng et al., 2011). Robert et al. (2011) also reported that miR163 modulates the secondary metabolite pathway from camalexin toward glucosinolates. It was suggested earlier that biosynthesis of benzylisoquinoline alkaloids were might be regulated by pso-miR13, pso-miR2161, and pso-miR408 in Papaver somniferum (Boke et al., 2015).

In our study, among eight selected miRNAs, which are targeted to the phenylpropanoid pathway genes, only five miRNAs namely miR172i, miR035, miR1438, miR2275, miR8291 were significantly down regulated after MeJA treatment. Heovere, H₂O₂ as well as CCCP induced ROS, did not show any effect on these eight miRNAs (Figure 4). Hence, these can be concluded that these five down regulated miRNAs were not regulated by the MeJA induced ROS. These five miRNAs are targeted to four coumarate ligage [4CL], Cytochrome P450, Caffeoyl–CoA methyltransferase, Cytochrome P450, and C. synthase as identified by KEGG analysis in our previous report (Bhattacharyya et al., 2016; Biswas et al., 2016). So the down regulation of these ROS non-responsive miRNA in trun bring about the up regulation ptox biosynthetic pathway genes. As we noted here, almost all genes located upstream and downstream of PhCADs, were ROS non-responsive whereas, PhCADs are ROS-responsive. Again, five above mentioned ROS non-responsive miRNAs were also targeted to the genes up- and downstream of PhCADs. These five miRNAs interfere with the target genes expression and the decline in the level of these microRNAs may bring about an up-regulation of the ptox biosynthetic pathway genes. Hence it can be concluded that may be miRNA is just one of the another way to regulate the other sixteen ROS non-responsive genes of the ptox biosynthetic pathway.

Taken together, it can be concluded that MeJA can up regulate the ptox biosynthetic genes by two possible ways, one through increasing the mRNA stability of PhCAD3, PhCAD4, and NAC3 by MeJA induced ROS and another through the down regulating some miRNAs which are targeted to other important genes of the ptox biosynthetic pathway.

SH carried out the experimental work, analyzed the data, and draft the manuscript. DB helps to analyze additional data and performed initial experiments. SC conceived and designed the experiments and prepared the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.