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A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

ABSTRACT: Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5' flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5'UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8-21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5'-UTR significantly enhances the level of gene expression. We termed this phenomenon as "microsatellite mediated enhancement" (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids.

SUBMITTER: Kumar S

PROVIDER: S-EPMC5020687 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

Kumar Santosh S Bhatia Sabhyata S

Scientific reports 20160913

Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5' flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5'UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both ...[more]

PMID: 27623355

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Project description: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.

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.

Dataset Information

A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

Publications

A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

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