RICE FLOWERING LOCUS T 1 (RFT1/FT-L3) is the closest homologue of Heading date 3a (Hd3a), which is thought to encode a mobile flowering signal and promote floral transition under short-day (SD) conditions. RFT1 is located only 11.5 kb from Hd3a on chromosome 6. Although RFT1 RNAi plants flowered normally, double RFT1-Hd3a RNAi plants did not flower up to 300 days after sowing (DAS), indicating that Hd3a and RFT1 are essential for flowering in rice. RFT1 expression was very low in wild-type plants, but there was a marked increase in RFT1 expression by 70 DAS in Hd3a RNAi plants, which flowered 90 DAS. H3K9 acetylation around the transcription initiation site of the RFT1 locus had increased by 70 DAS but not at 35 DAS. In the absence of Hd3a and RFT1 expression, transcription of OsMADS14 and OsMADS15, two rice orthologues of Arabidopsis APETALA1, was strongly reduced, suggesting that they act downstream of Hd3a and RFT1. These results indicate that Hd3a and RFT1 act as floral activators under SD conditions, and that RFT1 expression is partly regulated by chromatin modification.

Successful sexual reproduction in flowering plants depends on the accurate timing of flowering, which transits from vegetative stages to reproductive stages. Floral transition is regulated by both endogenous and environmental signals. Photoperiodic flowering is one of the most important factors in controlling floral transition among these various signals and is regulated both by day length and by the endogenous circadian rhythm (Thomas and Vince, 1977). Plants fall into one of three photoperiod-sensing classes: long-day plants (LDP), which promote flowering by sensing long-day (LD) photoperiods, short-day plants (SDP), which promote flowering by sensing short-day (SD) photoperiods, and day-natural plants, which are not regulated by photoperiod. The signaling cascades of photoperiodic flowering have been extensively studied in Arabidopsis thaliana (LDP) (Baurle and Dean, 2006;Imaizumi and Kay, 2006) and rice (SDP) (Izawa, 2007; Tuji et al., 2008). A number of signaling cascade genes have been identified and characterized. In Arabidopsis, GIGANTEA (GI) integrates cellular signals from light sensory transduction and the circadian clock, and activates CONSTANS (CO), which encodes a zinc-finger transcriptional activator (Park et al., 1999;Samach et al., 2000). CO induces FLOWERING LOCUS T (FT), which encodes a mobile flowering signal under LD conditions (Corbesier et al., 2007;Jaeger and Wigge, 2007;Lin et al., 2007;Mathieu et al., 2007). The GI-CO-FT pathway is conserved in rice (OsGI-Hd1-Hd3a) (Yano et al., 2000;Kojima et al., 2002;Hayama et al., 2002). Expression of Hd3a, the rice ortholog of FT, is also induced by Ehd1, a B-type response regulator that functions independently of Hd1 under SD conditions (Doi et al., 2004). OsMADS51, which is regulated by OsGI, functions upstream of Ehd1 (Kim et al., 2007). It was recently reported that RID1/Ehd2/OsId1 is a positive regulator of both SD and LD flowering in rice (Wu et al., 2008;Matsubara et al., 2008;Park et al., 2008). By contrast, under LD conditions, Hd1 suppresses the expression of Hd3a and causes delayed flowering (Hayama et al., 2003). Ghd7 encodes a transcription factor with a CCT motif, which acts as an LD-specific repressor of flowering (Xue et al., 2008). Thus, these studies revealed that rice flowering is regulated both by a 'SD activation pathway' and a 'LD suppression pathway' as an SDP. However, cultivated rice is grown extensively throughout Asia, and at the northern extremes of rice cultivation, including Japan and northern provinces of China and Korea, natural day length during rice cultivation is nearly LD (13-14.5 hours light) (Izawa, 2007), making LD flowering agronomically important in these regions. However, the genetic pathways governing LD flowering in rice are not well understood.FT/Hd3a, which is a common floral inducer in Arabidopsis thaliana (LDP) and rice (SDP), encodes florigen, the mobile flowering signal (Tamaki et al., 2007;Corbesier et al., 2007;Jaeger and Wigge, 2007;Lin et al., 2007;Mathieu et al., 2007), although the regulation ...

SUMMARYSmall RNAs that interact with Argonaute (AGO) proteins play central roles in RNA-mediated silencing. MEI-OSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice AGO, has specific functions in the development of pre-meiotic germ cells and the progression of meiosis. Here, we show that MEL1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21-nucleotide phased small interfering RNAs (phasiRNAs) that bear a 5 0 -terminal cytosine. Most phasiRNAs are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non-coding RNAs (lincRNAs) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNAs are processed via DICER-LIKE4 (DCL4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118-dependent and the DCL4-dependent pathways are both required for biogenesis of 21-nt phasiRNAs associated with germlinespecific MEL1 AGO in rice, and over 700 lincRNAs are key factors for induction of this biogenesis during reproductive-specific stages.

It has been almost 30 years since RNA interference (RNAi) was shown to silence genes via double-stranded RNAs (dsRNAs) in Caenorhabditis elegans (Fire et al. 1998). 20–30-nucleotide (nt) small non-coding RNAs are a key element of the RNAi machinery. Recently, phased small interfering RNAs (phasiRNAs), small RNAs that are generated from a long RNA precursor at intervals of 21 to 26-nt, have been identified in plants and animals. In Drosophila, phasiRNAs are generated by the endonuclease, Zucchini (Zuc), in germlines. These phasiRNAs, known as one of PIWI-interacting RNAs (piRNAs), mainly repress transposable elements. Similarly, reproduction-specific phasiRNAs have been identified in the family Poaceae, although DICER LIKE (DCL) protein-dependent phasiRNA biogenesis in rice is distinct from piRNA biogenesis in animals. In plants, phasiRNA biogenesis is initiated when 22-nt microRNAs (miRNAs) cleave single-stranded target RNAs. Subsequently, RNA-dependent RNA polymerase (RDR) forms dsRNAs from the cleaved RNAs, and dsRNAs are further processed by DCLs into 21 to 24-nt phasiRNAs. Finally, the phasiRNAs are loaded to ARGONAUTE (AGO) proteins to induce RNA-silencing. There are diverse types of phasiRNA precursors and the miRNAs that trigger the biogenesis. Their expression patterns also differ among plant species, suggesting that species-specific combinations of these triggers dictate the spatio-temporal pattern of phasiRNA biogenesis during development, or in response to environmental stimuli.

Reproduction-specific small RNAs are vital regulators of germline development in animals and plants. MicroRNA2118 (miR2118) is conserved in plants and induces the production of phased small interfering RNAs (phasiRNAs). To reveal the biological functions of miR2118, we describe here rice mutants with large deletions of the miR2118 cluster. Our results demonstrate that the loss of miR2118 causes severe male and female sterility in rice, associated with marked morphological and developmental abnormalities in somatic anther wall cells. Small RNA profiling reveals that miR2118-dependent 21-nucleotide (nt) phasiRNAs in the anther wall are U-rich, distinct from the phasiRNAs in germ cells. Furthermore, the miR2118-dependent biogenesis of 21-nt phasiRNAs may involve the Argonaute proteins OsAGO1b/OsAGO1d, which are abundant in anther wall cell layers. Our study highlights the site-specific differences of phasiRNAs between somatic anther wall and germ cells, and demonstrates the significance of miR2118/U-phasiRNA functions in anther wall development and rice reproduction.