An investigation on podophyllotoxin biosynthetic pathway in Podophyllum hexandrum

Hazra, Saptarshi (2017) An investigation on podophyllotoxin biosynthetic pathway in Podophyllum hexandrum. PhD thesis, J U.

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    Supervisors

    SupervisorsEmail
    Chattopadhyay, Sharmila

    Abstract

    Ptox is primarily obtained from a critically endangered medicinal plant P. hexandrum. It is used for synthesis semi-synthetic anti cancer drug etoposide (VP-16-213), teniposide (VM-26) and etopophos which are well know against testicular and lung cancer treatment (Stahelin and von Wartburg, 1991). Due to the comparatively lesser content of ptox in P. hexandrum and the limited knowledge on the molecular mechanism of ptox biosynthesis, an in depth investigation on ptox biosynthetic pathway in P. hexandrum is the need of the hour with a view to obtain enhanced the ptox content using modern cutting-edge techniques. It was well established that MeJA treatment to the cell culture of P. hexandrum increased ptox content maximum after 12 days of culture (Bhattacharyya et al., 2012). A comparative whole transcriptome analysis of control and 12 days MeJA treated culture was reported. It was revealed that along with all ptox biosynthetic pathway genes and other related transcription factors, CAD was most significantly up regulated (Bhattacharyya et al., 2013, 2016). Considering this, further downstream studies were performed with the CAD, one of the important rate limiting enzymes of ptox biosynthetic pathway. CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from CAld and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. It has been revealed that out of four CAD isoforms viz. PhCAD1, PhCAD2, PhCAD3 and PhCAD4, isolated from P. hexandrum, PhCAD3 and PhCAD4 were characterized as ptox specific. Furthermore, four transgenic cell cultures and calli of P. hexandrum overexpressing CAD isoforms independently, were raised and the ptox content was noted significantly enhanced by HPLC analysis in ptox specific CAD overexpressing lines. However, the molecular mechanism of MeJA induced ptox accumulation is yet to be explored. Here, it has been demonstrated that MeJA induced ROS production stimulated ptox accumulation significantly. MeJA induced ROS increased the mRNA stability of three ROSresponsive genes namely, PhCAD3, PhCAD4, NAC3 resulting enhanced ptox content. It has also been noted that, MeJA up regulated other ptox biosynthetic pathway genes, which are not affected by the MeJA induced ROS, however, by the down regulation of five secondary metabolites biosynthesis specific miRNAs viz. miR172i, miR035, miR1438, miR2275 and miR8291. To summarize, among the four isolated PhCAD isoforms only PhCAD3 and PhCAD4 favour ptox specifically than lignin, whereas PhCAD1 favours the lignin and ptox almost equally. MeJAenhanced the ptox content in P. hexandrum by up regulating the ptox specific ROS-responsive PhCAD isoforms namely PhCAD3 and PhCAD4 via increasing the mRNA stability. MeJA also regulates other ROS non-responsive ptox biosynthetic genes by down regulation of ROS nonresponsive miRNAs.

    Item Type: Thesis (PhD)
    URI: http://www.eprints.iicb.res.in/id/eprint/2627
    Subjects: Chemistry
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Ms Sutapa Ganguly
    Date Deposited: 28 Apr 2017 10:51
    Last Modified: 28 Apr 2017 10:51
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