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      Durum wheat miRNAs in response to nitrogen starvation at the grain filling stage

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          Abstract

          Durum wheat highly depends on nitrogen for seed development and yield, and the obtainment of varieties with a better nitrogen use efficiency is crucial to reduce production costs and environmental pollution. In this study, sequencing of two small RNA libraries obtained from tissues of Ciccio and Svevo cultivars grown under nitrogen starvation conditions produced 84 novel, and 161 conserved miRNAs. Of these, 7 novel and 13 known miRNAs were newly identified in this work. Quantitative PCR analysis of selected miRNAs highlighted that the expression levels of some of them depends on the tissue and on the cultivar, Svevo being the most responsive to nitrogen starvation. A number of target genes were predicted to be involved in nitrogen metabolism. An inverse correlation for the qPCR expression data of miRNA/target pairs miR399b/ PHO2, miR393c/ AFB2, ttu-novel-61/ CCAAT-TF was observed in specific tissues or cultivar. Especially, ttu-novel-61 was down-regulated and its target CCAAT-TF up-regulated in almost all tissues both in Svevo and in Ciccio. Moreover, CCAAT-TF was confirmed to be cleaved by ttu-novel-61 at the expected site. The discovery of miRNAs involved in the response to nitrogen stress represents an important step towards functional analyses, with the final aim to design strategies for improving nitrogen use efficiency in durum wheat.

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          Most cited references46

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          Origin, biogenesis, and activity of plant microRNAs.

          MicroRNAs (miRNAs) are key posttranscriptional regulators of eukaryotic gene expression. Plants use highly conserved as well as more recently evolved, species-specific miRNAs to control a vast array of biological processes. This Review discusses current advances in our understanding of the origin, biogenesis, and mode of action of plant miRNAs and draws comparisons with their metazoan counterparts.
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            Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture.

            Productive agriculture needs a large amount of expensive nitrogenous fertilizers. Improving nitrogen use efficiency (NUE) of crop plants is thus of key importance. NUE definitions differ depending on whether plants are cultivated to produce biomass or grain yields. However, for most plant species, NUE mainly depends on how plants extract inorganic nitrogen from the soil, assimilate nitrate and ammonium, and recycle organic nitrogen. Efforts have been made to study the genetic basis as well as the biochemical and enzymatic mechanisms involved in nitrogen uptake, assimilation, and remobilization in crops and model plants. The detection of the limiting factors that could be manipulated to increase NUE is the major goal of such research. An overall examination of the physiological, metabolic, and genetic aspects of nitrogen uptake, assimilation and remobilization is presented in this review. The enzymes and regulatory processes manipulated to improve NUE components are presented. Results obtained from natural variation and quantitative trait loci studies are also discussed. This review presents the complexity of NUE and supports the idea that the integration of the numerous data coming from transcriptome studies, functional genomics, quantitative genetics, ecophysiology and soil science into explanatory models of whole-plant behaviour will be promising.
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              Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

              MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.
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                Author and article information

                Contributors
                Role: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysis
                Role: Investigation
                Role: Formal analysisRole: Investigation
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                16 August 2017
                2017
                : 12
                : 8
                : e0183253
                Affiliations
                [1 ] Institute of Biosciences and Bioresources, National Research Council, Bari, Italy
                [2 ] Institute for Electronics and Magnetism, National Research Council (CNR), Parma, Italy
                Dokuz Eylul Universitesi, TURKEY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0003-0993-6828
                Article
                PONE-D-17-08998
                10.1371/journal.pone.0183253
                5558935
                28813501
                31b8292d-0092-4bb2-b820-b8cf3588a9d3
                © 2017 Zuluaga et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 March 2017
                : 1 August 2017
                Page count
                Figures: 5, Tables: 0, Pages: 18
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003407, Ministero dell’Istruzione, dell’Università e della Ricerca;
                Award ID: PON01_01145 ISCOCEM “Sviluppo tecnologico e innovazione per la sostenibilità e competitività della cerealicoltura meridionale”
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003407, Ministero dell’Istruzione, dell’Università e della Ricerca;
                Award ID: PRIN 2010-2011 DD 23/10/2012 n. 719 “Identificazione e caratterizzazione di geni utili ad incrementare la produttività e sostenibilità del frumento duro”
                Award Recipient :
                This research was supported by the MIUR projects PON01_01145 ISCOCEM “Sviluppo tecnologico e innovazione per la sostenibilità e competitività della cerealicoltura meridionale”, and PRIN 2010-2011 DD 23/10/2012 n. 719 “Identificazione e caratterizzazione di geni utili ad incrementare la produttività e sostenibilità del frumento duro” to GS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                MicroRNAs
                Biology and Life Sciences
                Organisms
                Plants
                Grasses
                Wheat
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Leaves
                Biology and Life Sciences
                Plant Science
                Plant Physiology
                Plant Defenses
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Plant Science
                Plant Pathology
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Ecology
                Plant Ecology
                Plant-Environment Interactions
                Plant Resistance to Abiotic Stress
                Ecology and Environmental Sciences
                Ecology
                Plant Ecology
                Plant-Environment Interactions
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Plant Science
                Plant Ecology
                Plant-Environment Interactions
                Plant Resistance to Abiotic Stress
                Biology and Life Sciences
                Agriculture
                Crop Science
                Crops
                Cereal Crops
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Transcription Factors
                Biology and Life Sciences
                Genetics
                Gene Expression
                Gene Regulation
                Transcription Factors
                Biology and Life Sciences
                Biochemistry
                Proteins
                Regulatory Proteins
                Transcription Factors
                Research and Analysis Methods
                Experimental Organism Systems
                Model Organisms
                Arabidopsis Thaliana
                Research and Analysis Methods
                Model Organisms
                Arabidopsis Thaliana
                Biology and Life Sciences
                Organisms
                Plants
                Brassica
                Arabidopsis Thaliana
                Research and Analysis Methods
                Experimental Organism Systems
                Plant and Algal Models
                Arabidopsis Thaliana
                Biology and Life Sciences
                Genetics
                Gene Expression
                Custom metadata
                All miRNA sequences are available st SRA-NCBI database ( https://www.ncbi.nlm.nih.gov/sra), accession numbers SRP069277 and SRP069277.

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