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      EAT1 transcription factor, a non-cell-autonomous regulator of pollen production, activates meiotic small RNA biogenesis in rice anther tapetum

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          Abstract

          The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24- PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24- PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 ( DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24- PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.

          Author summary

          Meiotic crossover formation shuffles homologous genes between parental genomes, and enables transmission of new gene sets to the offspring. Frequency and positions of crossovers are determined by numerous genetic and epigenetic factors, and low nucleosome-density regions are associated with crossover hot spots in yeasts and Arabidopsis. The epigenetic chromosome landscape is shaped by unevenly distributed modifications of nucleosome components, histones and DNAs. Recently, we found that MEL1 (ARGONAUTE5) promotes large-scale remodeling of meiotic chromosomes with dramatic increases of histone H3 lysine 9 dimethylation, and that loss of MEL1 resulted in early meiotic arrest with few crossovers present. In rice anthers, MEL1-associating small interfering RNAs (masiRNAs) were composed of large amounts of premeiotic 21-nt phasiRNAs, plus low levels of both 24-nt repeat-associated siRNA and meiotic 24-nt phasiRNAs. Production of 24-nt phasiRNA during the meiotic stage was largely EAT1-dependent. Collectively, our findings suggest a possibility that unknown small RNA-mediated signaling regulates male meiosis non-cell-autonomously, probably a downstream output involves large-scale chromosome remodeling promoted by Argonaute proteins, while a possibility of EAT1-dependent, but small RNA-independent signaling cannot be excluded. In any cases, the studies on MEL1 and tapetal bHLH proteins will be a clue to reveal small RNA-mediated processes determining meiotic epigenetic landscape.

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          Most cited references53

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          A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.

          To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.
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            A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

            Background Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue. Results Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts. Conclusions We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.
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              ShortRead: a bioconductor package for input, quality assessment and exploration of high-throughput sequence data

              Summary: ShortRead is a package for input, quality assessment, manipulation and output of high-throughput sequencing data. ShortRead is provided in the R and Bioconductor environments, allowing ready access to additional facilities for advanced statistical analysis, data transformation, visualization and integration with diverse genomic resources. Availability and Implementation: This package is implemented in R and available at the Bioconductor web site; the package contains a ‘vignette’ outlining typical work flows. Contact: mtmorgan@fhcrc.org
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Writing – original draft
                Role: Formal analysis
                Role: Data curationRole: Formal analysis
                Role: Formal analysis
                Role: Formal analysis
                Role: Formal analysis
                Role: ConceptualizationRole: Data curationRole: Funding acquisitionRole: Writing – original draft
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                12 February 2018
                February 2018
                : 14
                : 2
                : e1007238
                Affiliations
                [1 ] Experimental Farm, National Institute of Genetics, Yata, Mishima, Shizuoka, Japan
                [2 ] Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Yata, Mishima, Shizuoka, Japan
                [3 ] Graduate School of Science and Technology, Niigata University, Ikarashi, Nishi-ku, Niigata, Japan
                [4 ] NODAI Genome Research Center, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo, Japan
                [5 ] NODAI Research Institute, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo, Japan
                Donald Danforth Plant Science Center, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0001-6325-5201
                http://orcid.org/0000-0002-2057-543X
                Article
                PGENETICS-D-17-00918
                10.1371/journal.pgen.1007238
                5825165
                29432414
                c8dd6de2-05d1-40aa-b04c-643bed4ff333
                © 2018 Ono 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
                : 8 May 2017
                : 1 February 2018
                Page count
                Figures: 7, Tables: 1, Pages: 27
                Funding
                Funded by: Japan Society for the Promotion of Science (JP)
                Award ID: KAKENHI 25252004
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001700, Ministry of Education, Culture, Sports, Science and Technology;
                Award ID: KAKENHI, Grant-in-Aid for Scientific Research on Innovative Areas 17H05849
                Award Recipient :
                Funded by: NODAI Genome Research Center, Tokyo University of Agriculture
                Award ID: Cooperative Research Grant of the Genome Research for BioResource 16B-10
                Award Recipient :
                This work was supported by KAKENHI Grant No. 25252004 and No. 17H05849 from Japan Society for Promotion of Science (JSPS). Deep sequencing analyses were supported by Cooperative Research Grant of the Genome Research for BioResource, NODAI Genome Research Center, Tokyo University of Agriculture. 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
                Plant Science
                Plant Anatomy
                Flower Anatomy
                Anthers
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                Small interfering RNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Small interfering RNAs
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Grasses
                Rice
                Research and Analysis Methods
                Experimental Organism Systems
                Plant and Algal Models
                Rice
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Cell Cycle and Cell Division
                Meiosis
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Meiosis
                Biology and Life Sciences
                Genetics
                Genetic Loci
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics
                Sequence Analysis
                Sequence Motif Analysis
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Double stranded RNA
                Biology and Life Sciences
                Biotechnology
                Genetic Engineering
                Genetically Modified Organisms
                Genetically Modified Plants
                Biology and Life Sciences
                Biotechnology
                Plant Biotechnology
                Genetically Modified Plants
                Biology and Life Sciences
                Plant Science
                Plant Biotechnology
                Genetically Modified Plants
                Biology and Life Sciences
                Organisms
                Eukaryota
                Plants
                Genetically Modified Plants
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-02-23
                The fastq sequence results of mRNA-seq, sRNA-seq and MEL1-RIP-seq have been deposited in the Sequence Read Archive of the DNA Data Bank of Japan (DDBJ) (accession number DRA005258). All other data are within the paper and its Supporting Information files.

                Genetics
                Genetics

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