How peonidin functions as a HIF, e. g. with regard to its active form and receptor http://www.selleckchem.com/products/MDV3100.html on the parasite, also awaits further studies. Both TvQR1 and TvPirin genes have orthologs in non parasitic plants. Thus, further phylogenic investigations of these genes in different plant taxa might elucidate the mechanism by which parasitic plants have, during the evolutionary course from complete autotrophy Inhibitors,Modulators,Libraries to heterotrophy, co opted a quinone oxidoreductase and a transcription co factor from their existing genetic Inhibitors,Modulators,Libraries reservoir and adapted them to fulfill the new function of developing the organ that embodies plant parasitism the haustorium. Methods Plant and chemical materials T. versicolor seeds were collected and pooled from wild populations growing in the grasslands of Northern California, USA. For T.
versicolor cDNA clones and Northern blot analysis, seeds were collected in Napa. For in vitro haustorium and root Inhibitors,Modulators,Libraries necrosis assays, qRT PCR experiments, and popula tion genetics study, seeds were collected in Marysville. DMBQ and juglone were obtained from Pflatz Bauer and Alfa Aesar. Peonidin was obtained from Indofine Chemicals and Santa Cruz Biotechnology, Inc. Haustorium induction and toxicity assays T. versicolor seeds were surface sterilized, germinated, grown on agar plates, and seedlings treated with various haustorial inducers and non inducers, treatment combinations, or mock treated with water or 0. 1% EtOH as described previously. Each plate contained 10 20 seedlings. For each treatment, haustorium formation and root necrosis were scored 24 h after treatments in 60 66 seedlings.
Nucleic acid extraction For Northern blot analysis, total RNA was isolated from root tips, remaining roots, and shoots of 100 200 T. versicolor plants treated with water, 0. 1% ethanol, 20 uM DMBQ, 10 uM Inhibitors,Modulators,Libraries juglone, or 30 uM peonidin at 1, 3, and 5 h after treatment. For RT qPCR, 20 uM DMBQ or 30 uM peonidin was applied twice to the same seedling plates at 2 day intervals and haustorium formation was scored Inhibitors,Modulators,Libraries 24 h after each time to identify responsive and non responsive plants. Then the third treatment was applied, and 2 h later, root tips were collected from three batches of 20 each inducer responsive plants representing three biological replicates. One application was performed for mock or 10 uM juglone treatments to collect root tips for three biological replicates.
Total RNA was extracted from root tips by the Trizol method. From the haustorium formation assays performed for the RT qPCR experiments, 20 individual plants responsive or non responsive to DMBQ or peonidin were randomly selected for genomic DNA extraction using CTAB and chloroform, followed by isopropanol precipitation and kinase inhibitor Crizotinib ethanol washes. Transcription assays Northern blot hybridization was performed with 10 ug of total RNA from each sample as described elsewhere.