ABSTRACT
Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.
Subject(s)
Alcohol Oxidoreductases/metabolism , Podophyllotoxin/biosynthesis , Podophyllum/enzymology , Podophyllum/metabolism , Protein Isoforms/metabolism , Acetates/pharmacology , Alcohol Oxidoreductases/genetics , Cyclopentanes/pharmacology , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Oxylipins/pharmacology , Podophyllum/drug effects , Protein Isoforms/genetics , Transcriptome/drug effects , Transcriptome/genetics , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
MAIN CONCLUSION: Xyloglucan endo-transglycosylase/hydrolase ( Ph XET/H) regulates Podophyllum seed germination via GA mediated up-accumulation of Ph XET protein and subsequent endosperm weakening. Xyloglucan endo-transglycosylase/hydrolase (XET/H) belong to glycosyl hydrolase family 16, which play an important role in endosperm weakening and embryonic expansion during seed germination. Podophyllum hexandrum is a high altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. During seed germination in Podophyllum, accumulation of XET/H transcripts was recorded. This data confirmed its possible role in determining the fate of seed for germination. Full length cDNA of a membrane bound XET/H (here onwards PhXET) was cloned from the germinating seeds of Podophyllum. Analysis of nucleotide sequence revealed PhXET with an open reading frame of 720 bp encoding a protein of 239 amino acids with a molecular mass of 28 kDa and pI of 7.58. In silico structure prediction of PhXET showed homology with that of Populus tremula (1UN1). PhXET was predicted to have a potential GPI-anchor domain and was located in plasma membrane. It was found that the exogenously applied phytohormones (GA and ABA) regulate the expression of PhXET. The obtained data showed that the PhXET regulates seed germination in Podophyllum by supplementing its activity along with other endosperm weakening and embryo expansion genes.
