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Transcriptomic studies reveal a key metabolic pathway contributing to a well-maintained photosynthetic system under drought stress in foxtail millet (Setaria italica L.)

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      Abstract

      Drought stress is one of the most important abiotic factors limiting crop productivity. A better understanding of the effects of drought on millet ( Setaria italica L.) production, a model crop for studying drought tolerance, and the underlying molecular mechanisms responsible for drought stress responses is vital to improvement of agricultural production. In this study, we exposed the drought resistant F 1 hybrid, M79, and its parental lines E1 and H1 to drought stress. Subsequent physiological analysis demonstrated that M79 showed higher photosynthetic energy conversion efficiency and drought tolerance than its parents. A transcriptomic study using leaves collected six days after drought treatment, when the soil water content was about ∼20%, identified 3066, 1895, and 2148 differentially expressed genes (DEGs) in M79, E1 and H1 compared to the respective untreated controls, respectively. Further analysis revealed 17 Gene Ontology (GO) enrichments and 14 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in M79, including photosystem II (PSII) oxygen-evolving complex, peroxidase (POD) activity, plant hormone signal transduction, and chlorophyll biosynthesis. Co-regulation analysis suggested that these DEGs in M79 contributed to the formation of a regulatory network involving multiple biological processes and pathways including photosynthesis, signal transduction, transcriptional regulation, redox regulation, hormonal signaling, and osmotic regulation. RNA-seq analysis also showed that some photosynthesis-related DEGs were highly expressed in M79 compared to its parental lines under drought stress. These results indicate that various molecular pathways, including photosynthesis, respond to drought stress in M79, and provide abundant molecular information for further analysis of the underlying mechanism responding to this stress.

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      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

       K Livak,  T Schmittgen (2001)
      The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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        Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms

        High-throughput mRNA sequencing (RNA-Seq) holds the promise of simultaneous transcript discovery and abundance estimation 1-3 . We introduce an algorithm for transcript assembly coupled with a statistical model for RNA-Seq experiments that produces estimates of abundances. Our algorithms are implemented in an open source software program called Cufflinks. To test Cufflinks, we sequenced and analyzed more than 430 million paired 75bp RNA-Seq reads from a mouse myoblast cell line representing a differentiation time series. We detected 13,692 known transcripts and 3,724 previously unannotated ones, 62% of which are supported by independent expression data or by homologous genes in other species. Analysis of transcript expression over the time series revealed complete switches in the dominant transcription start site (TSS) or splice-isoform in 330 genes, along with more subtle shifts in a further 1,304 genes. These dynamics suggest substantial regulatory flexibility and complexity in this well-studied model of muscle development.
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          TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions

          TopHat is a popular spliced aligner for RNA-sequence (RNA-seq) experiments. In this paper, we describe TopHat2, which incorporates many significant enhancements to TopHat. TopHat2 can align reads of various lengths produced by the latest sequencing technologies, while allowing for variable-length indels with respect to the reference genome. In addition to de novo spliced alignment, TopHat2 can align reads across fusion breaks, which can occur after genomic translocations. TopHat2 combines the ability to identify novel splice sites with direct mapping to known transcripts, producing sensitive and accurate alignments, even for highly repetitive genomes or in the presence of pseudogenes. TopHat2 is available at http://ccb.jhu.edu/software/tophat.
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            Author and article information

            Affiliations
            [1 ]College of Agronomy, Shanxi Agricultural University , Taigu, China
            [2 ]Biotechnology Research Institute, Chinese Academy of Agricultural Sciences , Beijing, China
            [3 ]College of Agronomy, Yangzhou University , Yangzhou, China
            [4 ]Industrial Crop Institute, Shanxi Academy of Agricultural Sciences , Fenyang, China
            [5 ]Department of Next Generation Sequencing, Vazyme Biotech Company Ltd. , Nanjing, China
            Contributors
            Journal
            PeerJ
            PeerJ
            peerj
            peerj
            PeerJ
            PeerJ Inc. (San Francisco, USA )
            2167-8359
            8 May 2018
            2018
            : 6
            5947103 4752 10.7717/peerj.4752
            ©2018 Shi et al.

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

            Funding
            Funded by: Project of China Agriculuture Research System
            Award ID: CARS-06-13.5-A28
            Funded by: National Key R&D Program of Shanxi Province
            Award ID: 201703D211007
            Funded by: Key Scientific and Technological Project of Shanxi Province
            Award ID: 2015-TN-09
            Funded by: Science & Technology Innovation Foundation of Shanxi Agricultural University
            Award ID: 2016YJ05
            Funded by: Program for the Top Young Innovative Talents of Shanxi Agricultural University
            Award ID: TYIT201406
            This work was supported by grants from the Project of China Agriculuture Research System (CARS-06-13.5-A28), the National Key R&D Program of Shanxi Province (201703D211007), the Key Scientific and Technological Project of Shanxi Province (2015-TN-09), Science & Technology Innovation Foundation of Shanxi Agricultural University (2016YJ05), the Program for the Top Young Innovative Talents of Shanxi Agricultural University (TYIT201406). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Molecular Biology
            Plant Science

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