Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

Journal of Experimental Botany, Vol. 66, No. 13 pp. 4035–4046, 2015 doi:10.1093/jxb/erv209 Advance Access publication 4 May 2015 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice Naoto Sano1, Hanako Ono1, Kazumasa Murata2, Tetsuya Yamada1, Tadashi Hirasawa1 and Motoki Kanekatsu1,* 1 Department of Plant Production, United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan 2 Agricultural Research Institute, Toyama Agricultural, Forestry & Fisheries Research Center, Toyama, Toyama 939-8153, Japan * To whom correspondence should be addressed. E-mail: Abstract Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the longlived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. Key words: Actinomycin D, long-lived mRNA, Oryza sativa, RNA-Seq, seed development, seed germination. Introduction During seed development, various macromolecules, such as carbohydrates, lipids, proteins, and mRNA, accumulate in embryos. Some of these stored molecules play important roles in seed maturation, desiccation and dormancy, but others function in the seed germination process, in which quiescent embryos rapidly derepress genetic activity following imbibition. Early studies on gene expression in germinating cotton seeds revealed that transcription is not required for de novo protein synthesis in imbibed seeds, suggesting that protein synthesis during the initial phase of germination is initiated from mRNA templates stored in mature dry seeds (Dure and Waters, 1965; Waters and Dure, 1966). Consistent with these reports, treatment of seeds from the model plant Arabidopsis thaliana and rice with a translational inhibitor has been shown to impair germination, whereas treatment of seeds with a transcriptional inhibitor had no marked effects (Rajjou et al., 2004; Sano et al., 2012). Taken together, these findings indicate that the translation of stored mRNA Abbreviations: Act D, actinomycin D; DAF, days after flowering; DAI, days after imbibition. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received 26 December 2014; Revised 27 February 2015; Accepted 30 March 2015 4036 | Sano et al. 2012; Gao et al., 2013; Zhai et al., 2013). However, RNASeq has not been applied to a series of developing embryos between 10 DAF and 40 DAF (mature dry stage) in rice. Here, to analyse the temporal accumulation of long-lived mRNAs required for germination in rice, we performed germination tests for developing embryos of rice treated with a transcriptional inhibitor. Two Japonica rice cultivars, Nipponbare and Koshihikari, were used as materials and compared in this study in order to detect the fundamental long-lived mRNAs for germination. Nipponbare is a standard Japonica cultivar, which has been used for the International Rice Genome Sequencing Project (Sasaki and Burr, 2000), while Koshihikari is the most commonly grown Japonica cultivar in Japan and has often been used as a parental line to develop new cultivars for improving eating quality. We also identified the candidates of long-lived mRNAs that are essential for germination by comparing the transcriptomes of developing embryos before and after the completion of transcription for germination using an RNA-Seq technique. Furthermore, the change of candidates for long-lived mRNAs was analysed by real-time RT-PCR using immature embryos germinating after imbibition to confirm that their accumulation is indispensable for the induction of germination. Materials and methods Plant materials and sampling Rice plants (Oryza sativa L. cvs. Nipponbare and Koshihikari) were cultivated during rice growing season (May to September in 2011) under natural conditions in Toyama, Japan (36°37′N, 137°14′E). Spikelets were tagged on the day of female anthesis, and were harvested at 10, 15, 20, 25, 30 and 40 DAF. The seeds harvested at 40 DAF were air-dried and stored for 7 months at room temperature, and were then used as mature dry seeds for germination assays. The other harvested developing seeds were immediately used for germination assays without air-drying treatment. Germination assays Developing embryos were separated from the dehulled seeds collected at 10, 15, 20, 25 and 30 DAF and from the mature dry seeds (40 DAF) using a surgical blade. The dissected embryos were incubated in distilled water with or without 200 µM Act D (Wako) at 28°C under dark conditions. Germination assays were carried out in triplicate using 50 embryos each at 3, 7, 10 and 14 days after imbibition (DAI). The imbibition solutions were replaced with fresh solution at the start of each germination assay. The significance of statistical differences in the germination rates for each treatment was examined using the Tukey–Kramer test. Extraction of total RNA from developing embryos Total RNA was extracted from 20 embryos separated from the dehulled seeds using Fruit-mate for RNA Purification and RNAiso (...truncated)