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Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay.

ABSTRACT: BACKGROUND:An 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. RESULTS:The 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. CONCLUSIONS:Our 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.

SUBMITTER: Kumar S

PROVIDER: S-EPMC4449189 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Project description:Somatic embryogenesis is an important and wonderful biotechnological tool used to develop whole plant from a single or a group of somatic cells. The differentiated somatic cells become totipotent stem cells by drastic reprogramming of a wide range of cellular activities, leading to the acquisition of embryogenic competence. After acquiring competence, the cells pass through globular, heart, torpedo and cotyledonary stages of embryo; however, all advanced embryos do not convert into full plant, produce adventive embryos or callus instead, thus reverses the programming. This is a big limitation in propagation of many plants. Understanding and unraveling the proteins at this 'embryo to plantlet' transition stage will help to get more numbers of plants. Thus, our study was aimed at an identification of differentially abundant proteins between two important advanced stages, i.e. cotyledonary-(T1) and maturation stage (T2) of somatic embryos in Catharanthus roseus. A total of 2949 and 3030 proteins were identified in cotyledonary and maturation stage, respectively. Of these, 1129 proteins were common to both. Several proteins were found to be differentially accumulated in two different embryo stages in which over 60 proteins were most accumulated during somatic embryo maturation time. More chlorophyll accumulation was noted at this time under the influence of gibberellic acid (GA3). Proteins like Mg-protoporphyrin IX chelatase, chlorophyll a-b-binding protein, photosystem I iron-sulfur center, photosystem II Psb, photosystem II subunit P-1, P-II domain-containing protein, RuBisCO large chain, RuBisCO small chain, RuBisCO activase, RuBisCO large subunit-binding proteins were synthesized. Some of the identified proteins are linked to chlorophyll synthesis, carbohydrate metabolism and stress. The identified proteins are categorized into different groups on the basis of their cellular location, role and other metabolic processes. Biochemical attributes like protein, sugar, proline, antioxidant enzyme (APX, SOD and CAT) activities were high in T2 as compared to T1. The proteins like peroxidases, pathogenesis-related proteins, the late-embryogenesis abundant proteins, argonaute, germin and others have been discussed in C. roseus somatic embryo maturation process.

Project description:BackgroundTo combat infection to biotic stress plants elicit the biosynthesis of numerous natural products, many of which are valuable pharmaceutical compounds. Jasmonate is a central regulator of defense response to pathogens and accumulation of specialized metabolites. Catharanthus roseus produces a large number of terpenoid indole alkaloids (TIAs) and is an excellent model for understanding the regulation of this class of valuable compounds. Recent work illustrates a possible role for the Catharanthus WRKY transcription factors (TFs) in regulating TIA biosynthesis. In Arabidopsis and other plants, the WRKY TF family is also shown to play important role in controlling tolerance to biotic and abiotic stresses, as well as secondary metabolism.ResultsHere, we describe the WRKY TF families in response to jasmonate in Arabidopsis and Catharanthus. Publically available Arabidopsis microarrays revealed at least 30% (22 of 72) of WRKY TFs respond to jasmonate treatments. Microarray analysis identified at least six jasmonate responsive Arabidopsis WRKY genes (AtWRKY7, AtWRKY20, AtWRKY26, AtWRKY45, AtWRKY48, and AtWRKY72) that have not been previously reported. The Catharanthus WRKY TF family is comprised of at least 48 members. Phylogenetic clustering reveals 11 group I, 32 group II, and 5 group III WRKY TFs. Furthermore, we found that at least 25% (12 of 48) were jasmonate responsive, and 75% (9 of 12) of the jasmonate responsive CrWRKYs are orthologs of AtWRKYs known to be regulated by jasmonate.ConclusionOverall, the CrWRKY family, ascertained from transcriptome sequences, contains approximately 75% of the number of WRKYs found in other sequenced asterid species (pepper, tomato, potato, and bladderwort). Microarray and transcriptomic data indicate that expression of WRKY TFs in Arabidopsis and Catharanthus are under tight spatio-temporal and developmental control, and potentially have a significant role in jasmonate signaling. Profiling of CrWRKY expression in response to jasmonate treatment revealed potential associations with secondary metabolism. This study provides a foundation for further characterization of WRKY TFs in jasmonate responses and regulation of natural product biosynthesis.

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Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay.

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