Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.

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.

Xyloglucan endo-transglycosylase/hydrolase (XET/H) gene is expressed during the seed germination in Podophyllum hexandrum: a high altitude Himalayan plant.

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.

Cloning and functional characterization of ß-1, 3-glucanase gene from Podophyllum hexandrum - a high altitude Himalayan plant.

Podophyllum hexandrum is a high-altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. ß-1, 3-glucanase cDNA was cloned from the germinating seeds of Podophyllum (Ph-glucanase). Glucanases belong to pathogenesis related glycohydralase family of proteins, which also play an important role in endosperm weakening and testa rupture during seed germination. Analysis of cloned nucleotide sequence revealed Ph-glucanase with an open reading frame of 852bp encoding a protein of 283 amino acids with a molecular mass of 31kDa and pI of 4.39. In-silico structure prediction of Ph-glucanase showed homology with that of Hevea brasiliensis (3em5B). Structural stability and enhanced catalytic efficiency in harsh climatic conditions possibly due to the presence of glycosyl hydrolase motif (LGIVISESGWPSAG) and a connecting loop towards inner side and well exposed carbohydrate metabolism domain-COG5309, can readily hydrolyse cell wall sugar moieties. Seeds from the transgenic Arabidopsis plants over-expressing Ph-glucanase showed better germination performance against a wide range of temperatures and abscisic acid (ABA) stress. This can be attributed to the accumulation of Ph-glucanase at both transcript and protein levels during the seed germination in transgenic Arabidopsis. Results confirm that the cloned novel seed specific glucanase from a cold desert plant Podophyllum could be used for the manipulation of different plant species seeds against various harsh conditions.

A multi-omics strategy resolves the elusive nature of alkaloids in Podophyllum species.

Podophyllum hexandrum and, to a much lesser extent P. peltatum, are sources of podophyllotoxin, extensively used as a chemical scaffold for various anti-cancer drugs. In this study, integrated omics technologies (including advanced mass spectrometry/metabolomics, transcriptome sequencing/gene assemblies, and bioinformatics) gave unequivocal evidence that both plant species possess a hitherto unknown aporphine alkaloid metabolic pathway. Specifically, RNA-seq transcriptome sequencing and bioinformatics guided gene assemblies/analyses in silico suggested presence of transcripts homologous to genes encoding all known steps in aporphine alkaloid biosynthesis. A comprehensive metabolomics analysis, including UPLC-TOF-MS and MALDI-MS imaging in situ, then enabled detection, identification, localization and quantification of the aporphine alkaloids, magnoflorine, corytuberine and muricinine, in the underground and aerial tissues. Interestingly, the purported presence of alkaloids in Podophyllum species has been enigmatic since the 19th century, remaining unresolved until now. The evolutionary and phylogenetic ramifications of this discovery are discussed.

Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

Temperature-dependent growth and emergence of functional leaves: an adaptive mechanism in the seedlings of the western Himalayan plant Podophyllum hexandrum.

As an adaptive mechanism, hypocotyl dormancy delays emergence of functional leaf until favorable season of growth in Podophyllum hexandrum, an endangered medicinal plant of the western Himalayas. However, upon exposure of the freshly germinated seedlings to favorable temperature (25 degrees C), functional leaves emerged within 20 days. Therefore, we examined regulation mechanisms of growth and development of this alpine plant by temperature under laboratory conditions. The seedlings were exposed to (1) 25 degrees C (temperature prevailing at the time of maximum vegetative growth), (2) 4 degrees C (mean temperature at the onset of winter in its natural habitat), and (3) 10 degrees C (an intermediate temperature). Slackened growth at 4 degrees C was followed by senescence of aerial parts and quiescence of roots and predetermined leaf primordia. Rapid development of leaf primordia at 25 degrees C was associated with increased starch hydrolysis. This was evident from higher alpha-amylase activity and reducing sugars. These parameters decreased on sudden exposure to 4 degrees C. In contrast, the roots (perennating organs) showed a slight increase (1.36-fold) in alpha-amylase activity. Growth and development in seedlings growing at 10 degrees C (temperature less adverse than 4 degrees C) were comparatively faster. The content of reducing sugars and alpha-amylase activity were also higher in all the seedling parts at 10 degrees C as compared to 4 degrees C. This indicated larger requirements for sugar by the seedlings at 10 degrees C. Irrespective of temperature, maximum changes in nitrate and nitrate reductase occurred during the initial 10 days, i.e., when the readily available form of sugars (reducing sugar) was highest. This indicated that a temperature-dependent availability of carbon, but not temperature itself, was an important regulator of uptake and reduction of nitrogen.

Secoisolariciresinol dehydrogenase purification, cloning, and functional expression. Implications for human health protection.

Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the approximately 32-kDa NAD-dependent secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of approximately 32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, secoisolariciresinol and matairesinol, has been elucidated. Last, both secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called "mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers.