Stress-Responsive cis-Regulatory Elements Underline Podophyllotoxin Biosynthesis and Better Performance of Sinopodophyllum hexandrum Under Water Deficit Conditions.

Sinopodophyllum hexandrum is an endangered medicinal herb known for its bioactive lignan podophyllotoxin (PTOX), which is used for the preparation of anticancer drugs. In its natural habitat, S. hexandrum is exposed to a multitude of adversities, such as fluctuating temperatures, water deficit, and high UV radiations. Transcriptional regulation of genes, which is regulated by the condition-specific binding of transcriptional factors to precise motifs in the promoter region, underlines responses to an environmental cue. Therefore, analysis of promoter sequences could ascertain the spatio-temporal expression of genes and overall stress responses. Unavailability of genomic information does not permit such analysis in S. hexandrum, especially on regulation of PTOX pathway. Accordingly, this study describes isolation and in silico analysis of 5'-upstream regions of ShPLR (PINORESINOL-LARICIRESINOL REDUCTASE) and ShSLD (SECOISOLARICIRESINOL DEHYDROGENASE), the two key genes of the PTOX biosynthetic pathway. Data showed a range of motifs related to basal transcription, stress-responsive elements, such as those for drought, low temperature, and light, suggesting that the expression of these genes and resulting PTOX accumulation would be affected by, at least, these environmental cues. While the impact of temperature and light on PTOX accumulation is well studied, the effect of water deficit on the physiology of S. hexandrum and PTOX accumulation remains obscure. Given the presence of drought-responsive elements in the promoters of the key genes, the impact of water deficit on growth and development and PTOX accumulation was studied. The results showed decline in relative water content and net photosynthetic rate, and increase in relative electrolyte leakage with stress progression. Plants under stress exhibited a reduction in transpiration rate and chlorophyll content, with a gradual increase in osmoprotectant content. Besides, stressed plants showed an increase in the expression of genes involved in the phenylpropanoid pathway and PTOX biosynthesis, and an increase in PTOX accumulation. Upon re-watering, non-irrigated plants showed a significant improvement in biochemical and physiological parameters. Summarily, our results demonstrated the importance of osmoprotectants during water deficit and the revival capacity of the species from water deficit, wherein PTOX synthesis was also modulated. Moreover, isolated promoter sequences could be employed in genetic transformation to mediate the expression of stress-induced genes in other plant systems.

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.

Change in protein content during seed germination of a high altitude plant Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle (=Sinopodophyllum hexandrum) is a high-altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. An effective, conventional propagation method is by seed. However, seed germination is erratic, and seedling survival is low. A marginal increase in Podophyllum seed germination was attained with organic solvents. In the present study an attempt was made to decipher the physiological and biochemical barriers in terms of change in proteins during seed germination of Podophyllum. Comparative 2-DE analysis between un-germinated (dormant) and germinating seeds revealed nearly 113 differentially expressed proteins, whereas Peptide Mass Fingerprint (PMF) analysis of 97 protein spots revealed appearance of 27 proteins, up-accumulation of 11 proteins, down-accumulation of 19 proteins and disappearance of 40 proteins with germination. Identified 59 proteins in the homology search were involved in metabolism (carbohydrate and amino acid metabolism; 20 proteins), ABA/GA signaling (17 proteins) and stress (15 proteins) related proteins. Seven proteins were with unknown function. Two-DE, and MS/MS analysis in conjunction with semi-quantitative RT-PCR data of cell wall hydrolyzing genes, revealed that in Podophyllum the radicle protrusion occurs might be because of the up-accumulation of cell wall hydrolases i.e. ß-1, 3-glucanase and XET which weakens the thick walled micropylar endosperm.