Abstract Prevention of autonomous division of the egg apparatus and central cell in a female gametophyte before fertilization ensures successful reproduction in flowering plants. Here we show that rice ovules with PRC2 Osfie1 and Osfie2 double mutations exhibit asexual embryo and autonomous endosperm formation at a high frequency, while ovules with a single Osfie2 mutation display asexual pre-embryo-like structures at a lower frequency without fertilization. Confocal microscopy images indicate that the asexual embryos were mainly derived from eggs in the double mutants, while the asexual pre-embryos likely originated from eggs or synergids. Early onsetting, higher penetrance and better development of asexual embryos in the double mutants compared with those in Osfie2 suggest that autonomous endosperm facilitated the asexual embryo development. Transcriptomic analysis showed pluripotency factors such as male genome expressed OsBBM1 and OsWOX8/9 were activated in the asexual embryos. Similarly, the maternal alleles of the paternally expressed imprinted genes were activated in the autonomous endosperm. Our results suggest that the egg apparatus and central cell convergently adopt PRC2 to suppresses asexual embryo and autonomous endosperm formation possibly through silencing male genome-expressed genes.

Prevention of autonomous division of the egg apparatus and central cell in a female gametophyte before fertilization ensures successful reproduction in flowering plants. Here we show that rice ovules of Polycomb repressive complex 2 (PRC2) Osfie1 and Osfie2 double mutants exhibit asexual embryo and autonomous endosperm formation at a high frequency, while ovules of single Osfie2 mutants display asexual pre-embryo-like structures at a lower frequency without fertilization. Earlier onset, higher penetrance and better development of asexual embryos in the double mutants compared with those in Osfie2 suggest that the autonomous endosperm facilitated asexual embryo development. Transcriptomic analysis showed that male genome-expressed OsBBM1 and OsWOX8/9 were activated in the asexual embryos. Similarly, the maternal alleles of the paternally expressed imprinted genes were activated in the autonomous endosperm, suggesting that the egg apparatus and central cell convergently adopt PRC2 to maintain the non-dividing state before fertilization, possibly through silencing of the maternal alleles of male genome-expressed genes.

The reproductive success of many plants depends on their capacity to respond appropriately to their environment. One environmental cue that triggers flowering is the extended cold of winter, which promotes the transition from vegetative to reproductive growth in a response known as vernalization. In annual plants of the Brassicaceae , the floral repressor, FLOWERING LOCUS C ( FLC ), is downregulated by exposure to low temperatures. Repression is initiated during winter cold and then maintained as the temperature rises, allowing plants to complete their life cycle during spring and summer. The two stages of FLC repression, initiation and maintenance, are distinguished by different chromatin states at the FLC locus. Initiation involves the removal of active chromatin marks and the deposition of the repressive mark H3K27me3 over a few nucleosomes in the initiation zone, also known as the nucleation region. H3K27me3 then spreads to cover the entire locus, in a replication dependent manner, to maintain FLC repression. FLC is released from repression in the next generation, allowing progeny of a vernalized plant to respond to winter. Activation of FLC in this generation has been termed resetting to denote the restoration of the pre-vernalized state in the progeny of a vernalized plant. It has been assumed that resetting must differ from the activation of FLC expression in progeny of plants that have not experienced winter cold. Considering that there is now strong evidence indicating that chromatin undergoes major modifications during both male and female gametogenesis, it is time to challenge this assumption.

Brassica rapa L. is an important vegetable and oilseed crop. We investigated the distribution of the histone mark tri-methylation of H3K27 (H3K27me3) in B. rapa and its role in the control of gene expression at two stages of development (2-day cotyledons and 14-day leaves) and among paralogs in the triplicated genome. H3K27me3 has a similar distribution in two inbred lines, while there was variation of H3K27me3 sites between tissues. Sites that are specific to 2-day cotyledons have increased transcriptional activity, and low levels of H3K27me3 in the gene body region. In 14-day leaves, levels of H3K27me3 were associated with decreased gene expression. In the triplicated genome, H3K27me3 is associated with paralogs that have tissue-specific expression. Even though B. rapa and Arabidopsis thaliana are not closely related within the Brassicaceae, there is conservation of H3K27me3-marked sites in the two species. Both B. rapa and A. thaliana require vernalization for floral initiation with FLC being the major controlling locus. In all four BrFLC paralogs, low-temperature treatment increases H3K27me3 at the proximal nucleation site reducing BrFLC expression. Following return to normal temperature growth conditions, H3K27me3 spreads along all four BrFLC paralogs providing stable repression of the gene.

Disruption of gene silencing by Polycomb protein complexes leads to homeotic transformations and altered developmental-phase identity in plants. Here we define short genomic fragments, known as Polycomb response elements (PREs), that direct Polycomb repressive complex 2 (PRC2) placement at developmental genes regulated by silencing in Arabidopsis thaliana. We identify transcription factor families that bind to these PREs, colocalize with PRC2 on chromatin, physically interact with and recruit PRC2, and are required for PRC2-mediated gene silencing in vivo. Two of the cis sequence motifs enriched in the PREs are cognate binding sites for the identified transcription factors and are necessary and sufficient for PRE activity. Thus PRC2 recruitment in Arabidopsis relies in large part on binding of trans-acting factors to cis-localized DNA sequence motifs.

Endosperm cellularization is essential for embryo development and viable seed formation. Loss of function of the FERTILIZATION INDEPENDENT SEED (FIS) class Polycomb genes, which mediate trimethylation of histone H3 lysine27 (H3K27me3), as well as imbalanced contributions of parental genomes interrupt this process. The causes of the failure of cellularization are poorly understood. In this study we identified PICKLE RELATED 2 (PKR2) mutations which suppress seed abortion in fis1/mea by restoring endosperm cellularization. PKR2, a paternally expressed imprinted gene (PEG), encodes a CHD3 chromatin remodeler. PKR2 is specifically expressed in syncytial endosperm and its maternal copy is repressed by FIS1. Seed abortion in a paternal genome excess interploidy cross was also partly suppressed by pkr2. Simultaneous mutations in PKR2 and another PEG, ADMETOS (ADM), additively rescue the seed abortion in fis1 and in the interploidy cross, suggesting that PKR2 and ADM modulate endosperm cellularization independently and reproductive isolation between plants of different ploidy is established by imprinted genes. Genes upregulated in fis1 and downregulated in the presence of pkr2 are enriched in glycosyl-hydrolyzing activity, while genes downregulated in fis1 and upregulated in the presence of pkr2 are enriched with microtubule motor activity, consistent with the cellularization patterns in fis1 and the suppressor line. The antagonistic functions of FIS1 and PKR2 in modulating endosperm development are similar to those of PICKLE (PKL) and CURLY LEAF (CLF), which antagonistically regulate root meristem activity. Our results provide further insights into the function of imprinted genes in endosperm development and reproductive isolation.

Abstract. Timing of life history events (phenology) is a key driver for the adaptation of grain crops to their environments. Anthesis (flowering) date is the critical phenological stage that has been most extensively studied. Maximum crop yield is achieved by maximising the duration of the pre-anthesis biomass accumulation phase and hence yield potential, while minimising the risk of water stress and temperature stress (heat and cold) during flowering and grain-filling stages. In this article, we review our understanding of phenology of the valuable oilseed crop canola (oilseed rape, Brassica napus L.) from the perspectives of biophysical modelling and genetics. In conjunction, we review the genomic resources for canola and how they could be used to develop models that can accurately predict flowering date in any given set of environmental conditions. Finally, we discuss how molecular marker tools can help canola breeders to continue to improve canola productivity in the light of climate changes and to broaden its adaptation into new agricultural areas.

Timing of life history events (phenology) is a key driver for the adaptation of grain crops to their environments. Anthesis (flowering) date is the critical phenological stage that has been most extensively studied. Maximum crop yield is achieved by maximising the duration of the pre-anthesis biomass accumulation phase and hence yield potential, while minimising the risk of water stress and temperature stress (heat and cold) during flowering and grain-filling stages. In this article, we review our understanding of phenology of the valuable oilseed crop canola (oilseed rape, Brassica napus L.) from the perspectives of biophysical modelling and genetics. In conjunction, we review the genomic resources for canola and how they could be used to develop models that can accurately predict flowering date in any given set of environmental conditions. Finally, we discuss how molecular marker tools can help canola breeders to continue to improve canola productivity in the light of climate changes and to broaden its adaptation into new agricultural areas.

Vernalization is the promotion of flowering in response to prolonged exposure to low temperatures. In Arabidopsis, FLOWERING LOCUS C (FLC), a suppressor of flowering, is repressed by low temperatures but the mechanism leading to the initial decrease in FLC transcription remains a mystery. No mutants that block the repression of FLC at low temperatures have been identified to date. If the failure to identify such a mutant is assumed to imply that no such mutant exists, then it follows that the first response to the drop in temperature is physical, not genetic. In this Opinion article we propose that the drop in temperature first causes a simple change in the topology of the chromatin polymer, which in turn initiates the repression of FLC transcription.

For a plant to flower, its development must switch from the vegetative to the flowering state in order to make the seeds for the next generation. For cereals and the model plant Arabidopsis, the switching occurs in response to a cold event, signalling to the plant that it is winter with spring and summer soon to follow. Much is understood about the nature of this switching process genetically in terms of how the expression of the FLOWERING LOCUS C (F L C) gene, orchestrating the vegetative development, is repressed. Only recently has a mechanism been proposed that explains how the cold initiates the repression process that switches OFF the expression of Arabidopsis F L C. This paper will discuss how the mathematics of rubber elasticity has been the conceptualization step in the formulation of a model for this proposed mechanism for the initiation, in response to a cold event, of the genetics steps that initiate the repression. In part, the goal is to use the modelling of the switching of a plant from its vegetative to its flowering phase as an illustration of the pivotal role played by the conceptualization step in model formulation.The successful implementation of the conceptualization process in modelling and simulating real-world processes hinges on two separate matters:The need is required not only for(i) direct involvement, experience and familiarity with and understanding of different successful real-world (industrial) modelling endeavours,but also for(ii) a complete and comprehensive understanding of the workings of the particular real-world system being studied along with the motivation for and the essence of the questions to be answered.The importance of (i) is the need for modelers to gain experience by being directly involved with the solving of real-world problems, such as industrial mathematics study group meetings and related endeavours. That of (ii) relates to the need to have a close and productive collaboration with the originators of the real-world problem that results in a win-win relationship with them.For example, the modeler must be certain that in the conceptualization step, the outcome must not only capture the essence of the actions responsible for the observed phenomenon, it must not be inconsistent with other features, particularly those related to actions resulting from the observed phenomenon. It is also important, for a model to be truly useful, that it not only captures the observed phenomenon or event but also makes predictions of events not yet observed, which could subsequently be used to test the validity of the model. It is only with such validation that the accuracy/success of the conceptualization step be truthfully measured.

RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4–siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein–RNA interactions in plants.

BACKGROUND:Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes.RESULTS:We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs).CONCLUSIONS:Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3.

Short noncoding RNAs have been demonstrated to play important roles in regulation of gene expression and stress responses, but the repertoire and functions of long noncoding RNAs (lncRNAs) remain largely unexplored, particularly in plants. To explore the role of lncRNAs in disease resistance, we used a strand-specific RNA-sequencing approach to identify lncRNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana. Antisense transcription was found in c. 20% of the annotated A. thaliana genes. Several noncoding natural antisense transcripts responsive to F. oxysporum infection were found in genes implicated in disease defense. While the majority of the novel transcriptionally active regions (TARs) were adjacent to annotated genes and could be an extension of the annotated transcripts, 159 novel intergenic TARs, including 20 F. oxysporum-responsive lncTARs, were identified. Ten F. oxysporum-induced lncTARs were functionally characterized using T-DNA insertion or RNA-interference knockdown lines, and five were demonstrated to be related to disease development. Promoter analysis suggests that some of the F. oxysporum-induced lncTARs are direct targets of transcription factor(s) responsive to pathogen attack. Our results demonstrated that strand-specific RNA sequencing is a powerful tool for uncovering hidden levels of transcriptome and that IncRNAs are important components of the antifungal networks in A. thaliana.

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

BACKGROUND:The transitions from juvenile to adult and adult to reproductive phases of growth are important stages in the life cycle of plants. The regulators of these transitions include miRNAs, in particular miR156 and miR172 which are part of a regulatory module conserved across the angiosperms. In Arabidopsis miR171 represses differentiation of axillary meristems by repressing expression of SCARECROW-LIKE(SCL) transcription factors, however the role of miR171 has not been examined in other plants.RESULTS:To investigate the roles of mir171 and its target genes in a monocot, the Hvu pri-miR171a was over-expressed in barley (Hordeum vulgare L. cv. Golden promise) leading to reduced expression of at least one HvSCL gene. The resulting transgenic plants displayed a pleiotropic phenotype which included branching defects, an increased number of short vegetative phytomers and late flowering. These phenotypes appear to be the consequence of changes in the organisation of the shoot meristem. In addition, the data show that miR171 over-expression alters the vegetative to reproductive phase transition by activating the miR156 pathway and repressing the expression of the TRD (THIRD OUTER GLUME) and HvPLA1 (Plastochron1) genes.CONCLUSIONS:Our data suggest that some of the roles of miR171 and its target genes that have been determined in Arabidopsis are conserved in barley and that they have additional functions in barley including activation of the miR156 pathway.

Background During the early stages of seed development many genes are under dynamic regulation to ensure the proper differentiation and establishment of the tissue that will constitute the mature grain. To investigate how miRNA regulation contributes to this process in barley, a combination of small RNA and mRNA degradome analyses were used to identify miRNAs and their targets. Results Our analysis identified 84 known miRNAs and 7 new miRNAs together with 96 putative miRNA target genes regulated through a slicing mechanism in grain tissues during the first 15 days post anthesis. We also identified many potential miRNAs including several belonging to known miRNA families. Our data gave us evidence for an increase in miRNA-mediated regulation during the transition between pre-storage and storage phases. Potential miRNA targets were found in various signalling pathways including components of four phytohormone pathways (ABA, GA, auxin, ethylene) and the defence response to powdery mildew infection. Among the putative miRNA targets we identified were two essential genes controlling the GA response, a GA3oxidase1 and a homolog of the receptor GID1 , and a homolog of the ACC oxidase which catalyses the last step of ethylene biosynthesis. We found that two MLA genes are potentially miRNA regulated, establishing a direct link between miRNAs and the R gene response. Conclusion Our dataset provides a useful source of information on miRNA regulation during the early development of cereal grains and our analysis suggests that miRNAs contribute to the control of development of the cereal grain, notably through the regulation of phytohormone response pathways.