Unknown,Transcriptomics,Genomics,Proteomics

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Arabidopsis thaliana strain:Col 0 Transcriptome or Gene expression

ABSTRACT: We obtained an Arabidopsis mutant from the Arabidopsis Biological Resource Center stock collection and verified that it was homozygous for a T-DNA insertion in the first exon of ORRM1 (SALK_072648, designated here as orrm1). The homozygous mutant did not show any phenotypic defect when grown under growth room conditions. We examined the organelle transcriptome of the mutant for editing defects because other proteins carrying RIP domains have been shown to be editing factors. We analyzed the plastid RNA editing extent with a new methodology based on RNA-seq. Briefly, total RNA is isolated from leaves and RT-PCR products corresponding to known organelle genes are obtained by using gene-specific primers. The products are mixed in equimolar ratio, sheared, and used as templates to produce an Illumina TruSeq library. This RNA-seq analysis demonstrated that ORRM1 is a plastid editing factor; 12 among 34 plastid sites exhibit a severe reduction of editing extent in the mutant relative to the wild-type

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: StÃ©phane Bentolila

PROVIDER: E-MTAB-4378 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize.

Sun Tao T Germain Arnaud A Giloteaux Ludovic L Hammani Kamel K Barkan Alice A Hanson Maureen R MR Bentolila Stéphane S

Proceedings of the National Academy of Sciences of the United States of America 20130304 12

Plant RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of both mitochondria and plastids. Specific targeting of particular Cs is achieved by pentatricopeptide proteins that recognize cis elements upstream of the C that is edited. Members of the RNA-editing factor interacting protein (RIP) family in Arabidopsis have recently been shown to be essential components of the plant editosome. We have identified a gene that contains a pair of truncated RIP domains ( ...[more]

PMID: 23487777

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Project description:Introduction: The Plant Organelle RNA Recognition (PORR) domain proteins are nucleus-encoded RNA-binding proteins that have acquired specific roles in organelle RNA metabolism as splicing factors of chloroplast group II introns. LEFKOTHEA (At5g62990) is a nuclear gene encoding a PORR domain protein that carries a transit peptide (TP) and monopartite or bipartite nuclear localization signals (NLS). These motifs result in dual-targeting of LEFKOTHEA to the nucleus and chloroplasts implying a role in the splicing of chloroplast group II introns and nuclear pre-mRNA introns. Therefore, we examined the splicing efficiency of plastid and nuclear genes in lefko2 mutant. Methods: The lefko2 mutant is represented by the Salk T-DNA collection unicode SALK_044699/emb1692 and was obtained from the Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). Embryo development in homozygous lefko2 plants is ceased abruptly arresting embryogenesis upon globular-to-heart stage transition. Total RNA was extracted using plant RNA kit spin columns with an on-column DNase treatment from heart-stage lefko2 mutant and wild-type Arabidopsis embryos. The quantity and integrity of the RNA was assessed using a NanoDrop 1000 spectrophotometer and agarose gel electrophoresis. RNA-seq libraries were generated using the TruSeq Low Input kit according to the manufacturer’s instructions (Illumina). Sequencing was performed on an Illumina HiSeq 2000 instrument at BGI (Beijing Genomics Institute). Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (TAIR10). RNA-seq data were analyzed using the SOAPnuke (version v.1.5.2) with parameters “-l 15 -q 0.2 -n 0.05” and the HISAT2 pipeline (version 2.0.4) with parameters “--phred64 --sensitive --no-discordant --no-mixed -I 1 -X 1000”. Differential alternative splicing events were detected for lefko1 and wild-type using rMATS and visualized using the Integrative Genomics Viewer (IGV) tool. Results: RNA-seq uncovered an outstanding number of coding or non-coding novel transcripts in lefko2 mutant embryos. Splicing defects were observed in numerous nuclear and plastid genes of lefko2 embryos compared to wild type. Among them, intron retention (IR) events were the most prominent. Further, the fidelity of 5’ splice site (5’SS) donor and 3’SS acceptor splicing was highly disturbed in lefko2 embryos. To less extend, exon skipping (ES) defects were also detected. Conclusions: Detailed nuclear and chloroplast splicing events were widely observed in lefko2 aborted mutant embryos demonstrating a prevalent role of LEFKOTHEA in the splicing of nuclear pre-mRNA introns and chloroplast group II introns.

Project description:Shortly after the release of singlet oxygen (1O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment. Experiment Overall Design: Two individual biologica replicates, each containing material of five mature plants of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, respectively, were used for the microarray analysis. Plants were germinated on soil and kept under continuous light until the beginning of bolting and then transferred to the dark for 8 h. Dark-incubated mature plants were reilluminated for 30 min and subsequently harvested for RNA extraction. Experiment Overall Design: The EX1 (At4g33630) T-DNA insertion line SALK 002088 and EX2 (At1g27510) T-DNA insertion line SALK 012127 were obtained from the European Arabidopsis Stock Centre (NASC). Homozygous mutant lines were identified by PCR analysis by using T-DNA-, EX1- and EX2-specific primers. Both T-DNA-lines were crossed with a flu Col-0 line that had been obtained by 5 backcrosses of flu1-1 in Landsberg erecta with wild-type Columbia. The ex1/flu and ex2/flu mutant lines were crossed, and within the segregating F2 population triple mutants were identified by PCR-based genotyping. For the cultivation of mature plants, seeds of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, all in Col-0 ecotype, were sown on soil and plants were grown under continuous light (100 mol m 2 s 1).

Dataset Information

Arabidopsis thaliana strain:Col 0 Transcriptome or Gene expression

Publications

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize.

Similar Datasets

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