FERTILIZATION INDEPENDENT ENDOSPERM Research Articles

Glucose-driven TOR-FIE-PRC2 signalling controls plant development.

Nutrients and energy have emerged as central modulators of developmental programmes in plants and animals1-3. The evolutionarily conserved target of rapamycin (TOR) kinase is a master integrator of nutrient and energy signalling that controls growth. Despite its key regulatory roles in translation, proliferation, metabolism and autophagy2-5, little is known about how TOR shapes developmental transitions and differentiation. Here we show that glucose-activated TOR kinase controls genome-wide histone H3 trimethylation at K27 (H3K27me3) in Arabidopsis thaliana, which regulates cell fate and development6-10. We identify FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), an indispensable component of Polycomb repressive complex 2 (PRC2), which catalyses H3K27me3(refs.6-8,10-12), as a TOR target. Direct phosphorylation by TOR promotes the dynamic translocation of FIE from the cytoplasm to the nucleus. Mutation of the phosphorylation site on FIE abrogates the global H3K27me3 landscape, reprogrammes the transcriptome and disrupts organogenesis in plants. Moreover, glucose-TOR-FIE-PRC2 signalling modulates vernalization-induced floral transition. We propose that this signalling axis serves as a nutritional checkpoint leading to epigenetic silencing of key transcription factor genes that specify stem cell destiny in shoot and root meristems and control leaf, flower and silique patterning, branching and vegetative-to-reproduction transition. Our findings reveal a fundamental mechanism of nutrient signalling in direct epigenome reprogramming, with broad relevance for the developmental control of multicellular organisms.

BZIP71 delays flowering by suppressing Ehd1 expression in rice.

Flowering time is a fundamental factor determining the global distribution and final yield of rice (Oryza sativa). Although diverse flowering time genes have been reported in this crop, the transcriptional regulation of its key flowering genes are poorly understood. Here, we report that a basic leucine zipper transcription factor, bZIP71, functions as a flowering repressor. The overexpression of bZIP71 delays flowering, while the bzip71 mutant flowers early in both long-day and short-day conditions. A genetic analysis showed that the regulation of flowering by bZIP71 might be independent of Heading date 2 (Hd2), Hd4, and Hd5. Importantly, bZIP71 directly associates with the Early heading date 1 (Ehd1) promoter and represses its transcription, and genetically the function of bZIP71 is impaired in the ehd1 mutant. Moreover, bZIP71 interacts with major components of polycomb repressive complex 2 (PRC2), SET domain group protein 711 (SDG711), and Fertilization independent endosperm 2 (FIE2), through which bZIP71 regulates the H3K27me3 level of Ehd1. Taken together, we present a transcriptional regulatory mechanism in which bZIP71 enhances the H3K27me3 level of Ehd1 and transcriptionally represses its expression, which not only offers a novel insight into a flowering pathway, but also provides a valuable putative target for the genetic engineering and breeding of elite rice cultivars.

Gene Expression in Parthenogenic Maize Proembryos.

Angiosperm plants reproduce both sexually and asexually (by apomixis). In apomictic plants, the embryo and endosperm develop without fertilization. Modern maize seems to have a broken apomixis-triggering mechanism, which still works in Tripsacum and in Tripsacum–maize hybrids. For the first time, maize lines characterized by pronounced and inheritable high-frequency maternal parthenogenesis were generated 40 years ago, but there are no data on gene expression in parthenogenic maize proembryos. Here we examined for the first time gene expression in parthenogenic proembryos isolated from unpollinated embryo sacs (ESs) of a parthenogenic maize line (AT-4). The DNA-methylation genes (dmt103, dmt105) and the genes coding for the chromatin-modifying enzymes (chr106, hdt104, hon101) were expressed much higher in parthenogenic proembryos than in unpollinated ESs. The expression of the fertilization-independent endosperm (fie1) genes was found for the first time in parthenogenic proembryos and unpollinated ESs. In parthenogenic proembryos, the Zm_fie2 gene was expressed up to two times higher than it was expressed in unpollinated ESs.

Expression of some Epigenetic-Related Genes in Regenerated Shoots of Prunus persica

Establishment of an efficient adventitious shoot regeneration system for mature embryo of Prunus persica via shoot organogenesis has been achieved. Three different combinations of plant growth regulators (PGRs) have been applied in the current study; 6-Benzyl adenine (BA) or thidiazuron (TDZ) with Indole acetic acid (IAA) or Indole butyric acid (IBA). The high regeneration frequency was illustrated by 1.5 mg/l BA with 0.5 mg/l IAA combinations. Quantitative expression of eight epigenetic regulatory genes; Chromomethylase 3 (CMT3), Domains Rearranged Methyltransferase2 (DRM2), Fertilization Independent Endosperm (FIE2) and Chromodomain Protein Like Heterochromatin Protein1 (LHP1), Pickle (PKL), Proliferating Cell Nuclear Antigen (PCNA), Shoot Meristemless (STM) and DICER-LIKE1 (DCL1) have been estimated in the peach regenerated shoots. We found that the auxins and cytokinins in different combinations especially BA effect on the expression of some epigenetic genes in relation to the mature embryo.

Allelic variation in rice Fertilization Independent Endosperm 1 contributes to grain width under high night temperature stress.

Summary A higher minimum (night‐time) temperature is considered a greater limiting factor for reduced rice yield than a similar increase in maximum (daytime) temperature. While the physiological impact of high night temperature (HNT) has been studied, the genetic and molecular basis of HNT stress response remains unexplored.We examined the phenotypic variation for mature grain size (length and width) in a diverse set of rice accessions under HNT stress. Genome‐wide association analysis identified several HNT‐specific loci regulating grain size as well as loci that are common for optimal and HNT stress conditions.A novel locus contributing to grain width under HNT conditions colocalized with Fie1, a component of the FIS‐PRC2 complex. Our results suggest that the allelic difference controlling grain width under HNT is a result of differential transcript‐level response of Fie1 in grains developing under HNT stress.We present evidence to support the role of Fie1 in grain size regulation by testing overexpression (OE) and knockout mutants under heat stress. The OE mutants were either unaltered or had a positive impact on mature grain size under HNT, while the knockouts exhibited significant grain size reduction under these conditions.

Functional divergence of two duplicated Fertilization Independent Endosperm genes in rice with respect to seed development.

Fertilization Independent Endosperm (FIE) is an essential member of Polycomb Repressive Complex 2 (PRC2) that plays important roles in the developmental regulation of plants. OsFIE1 and OsFIE2 are two FIE homologs in the rice genome. Here, we showed that OsFIE1 probably duplicated from OsFIE2 after the origin of the tribe Oryzeae, but has a specific expression pattern and methylation landscape. During evolution, OsFIE1 underwent a less intensive purifying selection than did OsFIE2. The mutant osfie1 produced smaller seeds and displayed reduced dormancy, indicating that OsFIE1 predominantly functions in late seed development. Ectopic expression of OsFIE1, but not OsFIE2, was deleterious to vegetative growth in a dose-dependent manner. The newly evolved N-terminal tail of OsFIE1 was probably not the cause of the adverse effects on vegetative growth. The CRISPR/Cas9-derived mutant osfie2 exhibited impaired cellularization of the endosperm, which suggested that OsFIE2 is indispensable for early seed development as a positive regulator of cellularization. Autonomous endosperm was observed in both OsFIE2+- and osfie1/OsFIE2+- but at a very low frequency. Although OsFIE1-PRC2 exhibited H3K27me3 methyltransferase ability in plants, OsFIE1-PRC2 is likely to be less important for development in rice than is OsFIE2-PRC2. Our findings revealed the functional divergence of OsFIE1 and OsFIE2 and shed light on their distinct evolution following duplication.

Integration of Transcriptional Repression and Polycomb-Mediated Silencing of WUSCHEL in Floral Meristems.

Arabidopsis (Arabidopsis thaliana) floral meristems terminate after the carpel primordia arise. This is achieved through the temporal repression of WUSCHEL (WUS), which is essential for stem cell maintenance. At floral stage 6, WUS is repressed by KNUCKLES (KNU), a repressor directly activated by AGAMOUS. KNU was suggested to repress WUS through histone deacetylation; however, how the changes in the chromatin state of WUS are initiated and maintained to terminate the floral meristem remains elusive. Here, we show that KNU integrates initial transcriptional repression with polycomb-mediated stable silencing of WUS After KNU is induced, it binds to the WUS promoter and causes eviction of SPLAYED, which is a known activator of WUS and can oppose polycomb repression. KNU also physically interacts with FERTILIZATION-INDEPENDENT ENDOSPERM, a key polycomb repressive complex2 component, and mediates the subsequent deposition of the repressive histone H3 lysine 27 trimethylation for stable silencing of WUS This multi-step silencing of WUS leads to the termination of floral stem cells, ensuring proper carpel development. Thus, our work describes a detailed mechanism for heritable floral stem cell termination in a precise spatiotemporal manner.

Developmental patterning of the sub-epidermal integument cell layer in Arabidopsis seeds.

Angiosperm seed development is a paradigm of tissue cross-talk. Proper seed formation requires spatial and temporal coordination of the fertilization products – embryo and endosperm – and the surrounding seed coat maternal tissue. In early Arabidopsis seed development, all seed integuments were thought to respond homogenously to endosperm growth. Here, we show that the sub-epidermal integument cell layer has a unique developmental program. We characterized the cell patterning of the sub-epidermal integument cell layer, which initiates a previously uncharacterized extra cell layer, and identified TRANSPARENT TESTA 16 and SEEDSTICK MADS box transcription factors as master regulators of its polar development and cell architecture. Our data indicate that the differentiation of the sub-epidermal integument cell layer is insensitive to endosperm growth alone and to the repressive mechanism established by FERTILIZATION INDEPENDENT ENDOSPERM and MULTICOPY SUPPRESSOR OF IRA1 Polycomb group proteins. This work demonstrates the different responses of epidermal and sub-epidermal integument cell layers to fertilization.

Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice.

Polycomb group (PcG) proteins have been shown to affect growth and development in plants. To further elucidate their role in these processes in rice, we isolated and characterized a rice mutant which exhibits dwarfism, reduced seed setting rate, defective floral organ, and small grains. Map-based cloning revealed that abnormal phenotypes were attributed to a mutation of the Fertilization Independent Endosperm 2 (OsFIE2) protein, which belongs to the PcG protein family. So we named the mutant as osfie2-1. Histological analysis revealed that the number of longitudinal cells in the internodes decreased in osfie2-1, and that lateral cell layer of the internodes was markedly thinner than wild-type. In addition, compared to wild-type, the number of large and small vascular bundles decreased in osfie2-1, as well as cell number and cell size in spikelet hulls. OsFIE2 is expressed in most tissues and the coded protein localizes in both nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that OsFIE2 interacts with OsiEZ1 which encodes an enhancer of zeste protein previously identified as a histone methylation enzyme. RNA sequencing-based transcriptome profiling and qRT-PCR analysis revealed that some homeotic genes and genes involved in endosperm starch synthesis, cell division/expansion and hormone synthesis and signaling are differentially expressed between osfie2-1 and wild-type. In addition, the contents of IAA, GA3, ABA, JA and SA in osfie2-1 are significantly different from those in wild-type. Taken together, these results indicate that OsFIE2 plays an important role in the regulation of plant height and grain yield in rice.

Mechanisms of endosperm initiation.

Key messageOverview of developmental events and signalling during central cell maturation and early endosperm development with a focus on mechanisms of sexual and autonomous endosperm initiation.Endosperm is important for seed viability and global food supply. The mechanisms regulating the developmental transition between Female Gametophyte (FG) maturation and early endosperm development in angiosperms are difficult to study as they occur buried deep within the ovule. Knowledge of the molecular events underlying this developmental window of events has significantly increased with the combined use of mutants, cell specific markers, and plant hormone sensing reporters. Here, we review recent discoveries concerning the developmental events and signalling of FG maturation, fertilization, and endosperm development. We focus on the regulation of the initiation of endosperm development with and without fertilization in Arabidopsis and the apomict Hieracium, comparing this to what is known in monocots where distinct differences in developmental patterning may underlie alternative mechanisms of suppression and initiation. The Polycomb Repressive Complex 2 (PRC2), plant hormones, and transcription factors are iteratively involved in early fertilization-induced endosperm formation in Arabidopsis. Auxin increases and PRC2 complex inactivation can also induce fertilization-independent endosperm proliferation in Arabidopsis. Function of the PRC2 complex member FERTILIZATION-INDEPENDENT ENDOSPERM and two loci AutE and LOP are required for autonomous endosperm development in apomictic Hieracium. A comparative understanding of cues required for early endosperm development will facilitate genetic engineering approaches for the development of resilient seed crops, especially if an option for fertilization-independent endosperm formation was possible to combat stress-induced crop failure.

Imprinted gene OsFIE1 modulates rice seed development by influencing nutrient metabolism and modifying genome H3K27me3.

Imprinted Polycomb group (PcG) genes play a critical role in seed development in Arabidopsis. However, the role of the imprinted gene in cereal plants remains obscure. Here, a transgenic approach was conducted to study the function of the imprinted gene Oryza sativa Fertilization-Independent Endosperm 1 (OsFIE1) during seed development in rice (Oryza sativa ssp. japonica 'ZhongHua11'). RNAi of OsFIE1 and homozygous T-DNA insertion mutant osfie1 led to smaller seeds, delayed embryo development, smaller aleurone layer cells, and decreased seed set rate. OsFIE1 was specifically expressed in endosperm, and mRNA of OsFIE1 was also enriched in the inner seed coat together with the corresponding PcG members OsiEZ1 and OsCLF. Meanwhile, the contents of seed storage proteins and Ile, Leu, and Val were decreased, accompanied by the down-regulation of multiple transcription factors, storage protein synthesis and amino acid metabolism-related genes in OsFIE1-RNAi lines and osfie1. Western blot analysis showed that the complex OsFIE1-PcG in endosperm regulated the expression of target genes by genome H3K27me3 modification. We conclude that the OsFIE1-PcG complex, which was enriched in the inner seed coat and endosperm linked the development of embryo and endosperm by influencing transcription factors and nutrient metabolism and induced a highly differential effect when compared with the OsFIE2-PcG complex.

The Polycomb group protein CLF emerges as a specific tri-methylase of H3K27 regulating gene expression and development in Physcomitrella patens

Packaging of eukaryotic DNA largely depends on histone modifications that affect the accessibility of DNA to transcriptional regulators, thus controlling gene expression. The Polycomb group (PcG) chromatin remodeling complex deposits a methyl group on lysine 27 of histone 3 leading to repressed gene expression. Plants encode homologs of the Enhancer of zeste (E(z)), a component of the PcG complex from Drosophila, one of which is a SET domain protein designated CURLY LEAF (CLF). Although this SET domain protein exhibits a strong correlation with the presence of the H3K27me3 mark in plants, the methyl-transferase activity and specificity of its SET domain have not been directly tested in-vivo.Using the evolutionary early-diverged land plant model species Physcomitrella patens we show that abolishment of a single copy gene PpCLF, as well as an additional member of the PcG complex, FERTILIZATION-INDEPENDENT ENDOSPERM (PpFIE), results in a specific loss of tri-methylation of H3K27. Using site-directed mutagenesis of key residues, we revealed that H3K27 tri-methylation is mediated by the SET domain of the CLF protein. Moreover, the abolishment of H3K27me3 led to enhanced expression of transcription factor genes. This in turn led to the development of fertilization-independent sporophyte-like structures, as observed in PpCLF and PpFIE null mutants. Overall, our results demonstrate the role of PpCLF as a SET protein in tri-methylation of H3K27 in-vivo and the importance of this modification in regulating the expression of transcription factor genes involved in developmental programs of P. patens.

Fertilization Independent Endosperm genes repress NbGH3.6 and regulate the auxin level during shoot development in Nicotiana benthamiana.

The Fertilization Independent Endosperm (FIE) gene is required to restrict endosperm development without fertilization, and it represses flowering during embryo and seedling development in Arabidopsis thaliana However, the regulatory mechanism of the FIE gene in postembryonic shoot development is not well understood. Silencing of Nicotiana benthamiana homologues of the FIE gene, NbFIE1 and NbFIE2, resulted in the enhanced outgrowth of axillary buds and the impairment of secondary xylem differentiation. RNA sequencing analysis found that one of the auxin-responsive GRETCHEN HAGEN 3(GH3) family genes, NbGH3.6, was upregulated and maintained a high expression during the time course of silencing NbFIE genes. Chromatin immunoprecipiation (ChIP)-PCR results showed a lack of H3K27me3 marks on NbGH3.6 chromatin in NbFIE-silenced plants compared with negative control plants, indicating that NbGH3.6 was a direct target of NbFIE genes during postembryonic shoot development. Moreover, the free IAA content was reduced significantly in NbFIE-silenced plants, which might cause the enhanced outgrowth of axillary buds as well as impaired secondary xylem differentiation. These results clearly indicated that NbGH3.6 was a primary target of NbFIE genes during postembryonic shoot development, and NbFIE genes regulated axillary bud growth and secondary xylem formation through tuning endogenous auxin homeostasis, possibly by regulating the expression of the NbGH3.6 gene.

Isolation, expression and evolution of FERTILIZATION INDEPENDENT ENDOSPERM homologs in Podostemaceae.

Podostemaceae is an interesting family of angiosperms with unusual development and morphology. Among these, double fertilization, a defining feature of angiosperms is invariably missing in the family. Consequently, embryo development in the seeds takes place without endosperm. In recent years, the role of polycomb genes has garnered much interest because of their crucial role in seed development. Some of these genes have been reported from many unrelated species, underlining their high conservation. Thus, it becomes exciting to know the role of these genes in podostemads, which are devoid of double fertilization and endosperm. Here, we report the isolation, characterization and expression patterns of homologs of Fertilization Independent Endosperm (FIE) in two species of Podostemaceae, Zeylanidium olivaceum and Polypleurum stylosum. FIE like homologs could be identified in Z. olivaceum (ZoFIE) and P. stylosum (PsFIE). The predicted amino acid sequence of FIE homologs showed similarity to other homologs, containing the conserved seven WD40 repeats. Expression studies revealed that ZoFIE and PsFIE transcripts were present in the vegetative tissue (thallus in Podostemaceae) and the seedlings, similar to the model plants. However, the ZoFIE and PsFIE expression disappeared in the flowering stages. This unique pattern of expression suggests that in the absence of double fertilization and endosperm the expression of FIS complex genes perhaps is obliterated in Podostemaceae.

Ectopic expression of EbFIE from apomictic Eulaliopsis binata in rice results in pleiotropic phenotypes likely due to interaction with OsCLF

FERTILIZATION INDEPENDENT ENDOSPERM (FIE) is a core component of PcG complexes and functions in plant phase transition and seed generation. However, understanding in its function of apomictic monocot plants remains blank. Here an FIE homology EbFIE, has been isolated from apomictic Graminae species Eulaliopsis binata. EbFIE shares higher homology to OsFIE2 than OsFIE1, and has been classified into the monocot FIE2 clade. In addition, the broad expression pattern of EbFIE is also similar to OsFIE2. While, ectopic expression of EbFIE in rice resulted in pleiotropic phenotypes similar to that of OsFIE1 over-expressing plants. Meanwhile, EbFIE could bind OsCLF in vitro as OsFIE1 but different with OsFIE2. Molecular models comparison indicated that both EbFIE and OsFIE1 had a smaller E(z) protein binding groove than OsFIE2. Further site-directed mutagenesis analysis revealed that single amino acid substitution of I194F in OsFIE2 could improve its OsCLF binding capacity. Taken together, our results suggested that EbFIE was a conserved FIE homolog belonging to monocot FIE2 clade, but due to the similarity in protein conformation with FIE1, EbFIE might play a broad role in vegetative and reproductive development regulation by interaction with CLF homolog.

Exogenous steroid hormones stimulate full development of autonomous endosperm in Arabidopsis thaliana

<p>Most flowering plants, including important crops, require double fertilization to form an embryo and endosperm, which nourishes it. Independence from fertilization is a feature of apomictic plants that produce seeds, from which the plants that are clones of the mother plant arise. The phenomenon of apomixis occurs in some sexual plants under specific circumstances. Since the launch of a fertilization-independent mechanism is considered a useful tool for plant breeding, there have been efforts to artificially induce apomixis. We have been able to produce fertilization-independent endosperm in vitro in <em>Arabidopsis</em> over the last few years. This paper demonstrates the methods of improving the quality of the endosperm obtained using plant and mammalian steroid hormones. Additionally, it shows the study on the autonomous endosperm (AE) formation mechanism in vitro.</p><p>This paper examines the effect of exogenous steroid hormones on unfertilized egg and central cell divisions in culture of unpollinated pistils of <em>Arabidopsis</em> Col-0 wild-type and <em>fie-1</em> mutant. All media with hormones used (estrone, androsterone, progesterone, and epibrassinolide) stimulated central cell divisions and fertilization-independent endosperm development. The stages of AE development followed the pattern of <em>Arabidopsis thaliana</em> wild type after fertilization. Subsequent stages of AE were observed from 2-nuclear up to cellular with the most advanced occurring on medium with 24-epibrassinolide and progesterone. The significant influence of mammalian sex hormones on speed of AE development and differentiation was noticed. Using restriction analysis, the changes in methylation of <em>FIE</em> gene was established under in vitro condition. The authors of this paper showed that <em>Arabidopsis thaliana</em> has a high potency to fertilization-independent development.</p>

A reference genetic linkage map of apomictic Hieracium species based on expressed markers derived from developing ovule transcripts.

Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations.

OsFIE2 plays an essential role in the regulation of rice vegetative and reproductive development

Polycomb group (PcG) proteins are gene repressors that help to maintain cellular identity during development via chromatin remodeling. Fertilization-independent endosperm (FIE), a member of the PcG complex, operates extensively in plant development, but its role in rice has not been fully investigated to date. We report the isolation and characterization of a PcG member in rice, which was designated OsFIE2 for Oryza sativa Fertilization-Independent Endosperm 2. OsFIE2 is a single-copy gene in the rice genome and shows a universal expression pattern. The OsFIE2 RNAi lines displayed pleiotropic phenotypes in vegetative and reproductive organ generation. In unfertilized lines, endosperm formation could be triggered without embryo formation, which indicates that FIE is indeed involved in the suppression of autonomous endosperm development in rice. Furthermore, lateral root generation was promoted early in the roots of OsFIE2 RNAi lines, whereas the primary root was premature and highly differentiated. As the root tip stem cell differentiated, QHB, the gene required for stem cell maintenance in the quiescent center, was down-regulated. Our data suggest that the OsFIE2-PcG complex is vital for rice reproduction and endosperm formation. Its role in stem cell maintenance suggests that the gene is functionally conserved in plants as well as animals.

Alterations in Seed Development Gene Expression Affect Size and Oil Content of Arabidopsis Seeds

Seed endosperm development in Arabidopsis (Arabidopsis thaliana) is under control of the polycomb group complex, which includes Fertilization Independent Endosperm (FIE). The polycomb group complex regulates downstream factors, e.g. Pheres1 (PHE1), by genomic imprinting. In heterozygous fie mutants, an endosperm develops in ovules carrying a maternal fie allele without fertilization, finally leading to abortion. Another endosperm development pathway depends on MINISEED3 (a WRKY10 transcription factor) and HAIKU2 (a leucine-rich repeat kinase). While the role of seed development genes in the embryo and endosperm establishment has been studied in detail, their impact on metabolism and oil accumulation remained unclear. Analysis of oil, protein, and sucrose accumulation in mutants and overexpression plants of the four seed development genes revealed that (1) seeds carrying a maternal fie allele accumulate low oil with an altered composition of triacylglycerol molecular species; (2) homozygous mutant seeds of phe1, mini3, and iku2, which are smaller, accumulate less oil and slightly less protein, and starch, which accumulates early during seed development, remains elevated in mutant seeds; (3) embryo-specific overexpression of FIE, PHE1, and MINI3 has no influence on seed size and weight, nor on oil, protein, or sucrose content; and (4) overexpression of IKU2 results in seeds with increased size and weight, and oil content of overexpressed IKU2 seeds is increased by 35%. Thus, IKU2 overexpression represents a novel strategy for the genetic manipulation of the oil content in seeds.

N-terminal region of rice polycomb group protein OsEZ1 is required for OsEZ1–OsFIE2 protein interaction

Polycomb group (PcG) proteins form Polycomb Repressive Complex 2 (PRC2), which regulates seed development by the epigenetic control of gene expression. Interaction assay among Arabidopsis Fertilization-independent-seed2 (FIS) class PcG proteins showed that Fertilization-independent endosperm (FIE) interacts with Medea (MEA), a SET-domain polycomb protein, of which N-terminal region is crucial for the interaction. In this study, rice SET-domain PcG protein OsEZ1, also known as OsiEZ1 in indica rice, was analyzed to identify an interacting domain of OsEZ1 required for OsEZ1–OsFIE2 protein interaction. A series of OsEZ1 deletions were generated and used to determine an interacting domain of OsEZ1 with OsFIE2 using the yeast two-hybrid system. Among OsEZ1 deletions, only OsEZ1∆2 and OsEZ1∆3 interacted with OsFIE2, indicating that the 155K–169R or N-proximal region of OsEZ1 is crucial for OsFIE2–OsEZ1 interaction. To examine the physiological roles of OsEZ1, 35S:OsEZ1Arabidopsis lines were generated. OsEZ1 overexpressors exhibited altered seedling growth and seed size, implying that OsEZ1 may play important roles in seedling and seed development.