Characterization of a knock-down RNA interference line of OsNRPD1 in rice (Oryza sativa ssp japonica cv Nipponbare)
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ABSTRACT:
ORGANISM(S): Oryza sativa Japonica Group
PROVIDER: GSE135738 | GEO | 2019/08/12
REPOSITORIES: GEO
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Project description:In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
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Project description:BACKGROUND AND AIMS:Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice. METHODOLOGY:We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H(2)O(2) treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts. PRINCIPAL RESULTS:Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H(2)O(2). Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor. CONCLUSIONS:OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
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Project description:BACKGROUND: FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses. RESULTS: FKBP and CYP proteins in rice (Oryza sativa cv. Japonica) were identified and classified, and given the appropriate name for each IMM, considering the ortholog-relation with Arabidopsis and Chlamydomonas or molecular weight of the proteins. 29 FKBP and 27 CYP genes can putatively be identified in rice; among them, a number of genes can be putatively classified as orthologs of Arabidopsis IMMs. However, some genes were novel, did not match with those of Arabidopsis and Chlamydomonas, and several genes were paralogs by genetic duplication. Among 56 IMMs in rice, a significant number are regulated by salt and/or desiccation stress. In addition, their expression levels responding to the water-stress have been analyzed in different tissues, and some subcellular IMMs located by means of tagging with GFP protein. CONCLUSION: Like other green photosynthetic organisms such as Arabidopsis (23 FKBPs and 29 CYPs) and Chlamydomonas (23 FKBs and 26 CYNs), rice has the highest number of IMM genes among organisms reported so far, suggesting that the numbers relate closely to photosynthesis. Classification of the putative FKBPs and CYPs in rice provides the information about their evolutional/functional significance when comparisons are drawn with the relatively well studied genera, Arabidopsis and Chlamydomonas. In addition, many of the genes upregulated by water stress offer the possibility of manipulating the stress responses in rice.
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Project description:Oryza sativa Japonica (rice) is the staple food for all over world population. At the time of germination, the exposure to light may play an important role in the early development of the rice seedling. In order to survey the genes, their functions and role in biological processes, an RNA-Seq based study of 6 libraries prepared from poly-A rich mRNA fraction was carried out to explore transcriptional programs operating in dark and light conditions. For each treatment type, three individual plants were used as biological replicates.
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Project description:Coronatine (COR) is a structural and functional analog of methyl jasmonic acid (MeJA), which can alleviate stress on plant. We studied the effects of COR on the drought stress of rice (Oryza sativa L.). Pre-treatment with COR significantly increased the biomass, relative water and proline content, and DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging activity, decreased the electrolyte leakage and MDA (Malondialdehyde) content in order to maintain the stability of cell membrane. Meanwhile, we determined how COR alleviates water stress by Nipponbare gene expression profiles and cDNA microarray analyses. Seedlings were treated with 0.1 μmol L-1 COR at the three leafed stage for 12 h, followed with 17.5% polyethylene glycol (PEG). Whole genome transcript analysis was determined by employing the Rice Gene Chip (Affymetrix), a total of 870 probe sets were identified to be up or downregulated due to COR treatment under drought stress. Meanwhile, the real-time quantitative PCR (RT-qPCR) method was used to verify some genes; it indicated that there was a good agreement between the microarray data and RT-qPCR results. Our data showed that the differentially expressed genes were involved in stress response, signal transduction, metabolism and tissue structure development. Some important genes response to stress were induced by COR, which may enhance the expression of functional genes implicated in many kinds of metabolism, and play a role in defense response of rice seedling to drought stress. This study will aid in the analysis of the expressed gene induced by COR.
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Project description:We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is approximately 32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.
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Project description:Genes encoding wheat low-molecular-weight glutenin subunits (LMW-GSs) that confer dough strength and extensibility were previously identified from Korean wheat cultivars. To improve low viscoelasticity of rice (Oryza sativa L.) dough caused by the lack of seed storage proteins comparable to wheat gluten, two genes, LMW03 and LMW28, encoding LMW-GSs are cloned from Korean wheat cultivar Jokyoung. The LMW genes are inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter. Transgenic rice plants expressing LMW03 or LMW28 in their seeds are generated using Agrobacterium-mediated transformation. The expression of recombinant wheat LMW-GS in the transgenic rice seeds was confirmed by SDS-PAGE and immunoblot analysis. Their accumulation in the endosperm and aleurone layers of rice seeds was observed through in situ immuno-hybridization.
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