Abstract
BackgroundAn accurate assessment of transcription ‘rate’ is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription ‘rate’. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a ‘molecular hub’ to understand gene expression profiles.ResultsThe protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli.ConclusionsOur study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear ‘run-on’ transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA-Seq for global nuclear run-on assay (GNRO-Seq) for genome wide in-situ measurement of transcription rate of plant genes.
Highlights
IntroductionIn general, inferred by measuring transcript abundance
Gene expression is, in general, inferred by measuring transcript abundance
We fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of Tryptophan decarboxylase (TDC) gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA)
Summary
In general, inferred by measuring transcript abundance Methods such as RNA gel blotting (Northern blotting), semi-quantitative RT–PCR and real-time quantitative PCR [1,2,3] provide information about the steady-state accumulation of a given RNA transcript at a given time point. Methods to assay transcription rate contribute important information regarding the first kinetics in the central dogma of molecular biology. One such method which helps to accurately assess the rate of transcription is the nuclear run-on assay which provides an in- situ measure of transcription rate of specific genes in transcriptionally active nuclei. Isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and limit an accurate measurement of gene transcription ‘rate’. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and serves as a ‘molecular hub’ to understand gene expression profiles
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