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      Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice

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          Abstract

          MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice.

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          Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

          Erika Varkonyi-Gasic, Rongmei Wu, Marion Wood … (2007)
          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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            Understanding mechanisms of novel gene expression in polyploids.

            Thomas Osborn, J. Chris Pires, James Birchler … (2003)
            Polyploidy has long been recognized as a prominent force shaping the evolution of eukaryotes, especially flowering plants. New phenotypes often arise with polyploid formation and can contribute to the success of polyploids in nature or their selection for use in agriculture. Although the causes of novel variation in polyploids are not well understood, they could involve changes in gene expression through increased variation in dosage-regulated gene expression, altered regulatory interactions, and rapid genetic and epigenetic changes. New research approaches are being used to study these mechanisms and the results should provide a more complete understanding of polyploidy.
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              The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.

              Anthony A Millar, Frank Gubler (2005)
              The functions of the vast majority of genes encoding R2R3 MYB domain proteins remain unknown. The closely related MYB33 and MYB65 genes of Arabidopsis thaliana have high sequence similarity to the barley (Hordeum vulgare) GAMYB gene. T-DNA insertional mutants were isolated for both genes, and a myb33 myb65 double mutant was defective in anther development. In myb33 myb65 anthers, the tapetum undergoes hypertrophy at the pollen mother cell stage, resulting in premeiotic abortion of pollen development. However, myb33 myb65 sterility was conditional, where fertility increased both under higher light or lower temperature conditions. Thus, MYB33/MYB65 facilitate, but are not essential for, anther development. Neither single mutant displayed a phenotype, implying that MYB33 and MYB65 are functionally redundant. Consistent with functional redundancy, promoter-beta-glucuronidase (GUS) fusions of MYB33 and MYB65 gave identical expression patterns in flowers (sepals, style, receptacle, anther filaments, and connective but not in anthers themselves), shoot apices, and root tips. By contrast, expression of a MYB33:GUS translational fusion in flowers was solely in young anthers (consistent with the male sterile phenotype), and no staining was seen in shoot meristems or root tips. A microRNA target sequence is present in the MYB genes, and mutating this sequence in the MYB33:GUS fusion results in an expanded expression pattern, in tissues similar to that observed in the promoter-GUS lines, implying that the microRNA target sequence is restricting MYB33 expression. Arabidopsis transformed with MYB33 containing the mutated microRNA target had dramatic pleiotrophic developmental defects, suggesting that restricting MYB33 expression, especially in the shoot apices, is essential for proper plant development.
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                Author and article information

                Contributors
                Jianhua Zhu: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Int J Mol Sci
                Journal ID (iso-abbrev): Int J Mol Sci
                Journal ID (publisher-id): ijms
                Title: International Journal of Molecular Sciences
                Publisher: MDPI
                ISSN (Electronic): 1422-0067
                Publication date (Electronic): 12 April 2016
                Publication date Collection: April 2016
                Volume: 17
                Issue: 4
                Electronic Location Identifier: 499
                Affiliations
                State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China; andyslee@ 123456126.com (X.L.); qasim@ 123456scau.edu.cn (M.Q.S.); wjw0106@ 123456126.com (J.W.); wanglan@ 123456scau.edu.cn (L.W.)
                Author notes
                [* ]Correspondence: xdliu@ 123456scau.edu.cn (X.L.); yglu@ 123456scau.edu.cn (Y.L.); Tel./Fax: +86-20-3829-4893 (X.L. & Y.L.)
                [†]

                These authors contributed equally to this work.

                Article
                Publisher ID: ijms-17-00499
                DOI: 10.3390/ijms17040499
                PMC ID: 4848955
                PubMed ID: 27077850
                SO-VID: b6cbef9f-9909-4b44-99af-9c040d6653cd
                Copyright © © 2016 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 31 December 2015
                Date accepted : 28 March 2016
                Categories
                Subject: Article

                ScienceOpen disciplines: Molecular biology
                Keywords: meiosis,micrornas,polyploidy,pre-meiotic interphase (pma),single microspore stage (scp),sirnas
                Data availability:
                ScienceOpen disciplines: Molecular biology
                Keywords: meiosis, micrornas, polyploidy, pre-meiotic interphase (pma), single microspore stage (scp), sirnas

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