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:OsEZ1 Arabidopsis lines were generated. OsEZ1 overexpressors exhibited altered seedling growth and seed size, implying that OsEZ1 may play important roles in seedling and seed development.     

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.     

Rice Fertilization-Independent Endosperm1 Regulates Seed Size under Heat Stress by Controlling Early Endosperm Development

Although heat stress reduces seed size in rice (Oryza sativa), little is known about the molecular mechanisms underlying the observed reduction in seed size and yield. To elucidate the mechanistic basis of heat sensitivity and reduced seed size, we imposed a moderate (34°C) and a high (42°C) heat stress treatment on developing rice seeds during the postfertilization stage. Both stress treatments reduced the final seed size. At a cellular level, the moderate heat stress resulted in precocious endosperm cellularization, whereas severe heat-stressed seeds failed to cellularize. Initiation of endosperm cellularization is a critical developmental transition required for normal seed development, and it is controlled by Polycomb Repressive Complex2 (PRC2) in Arabidopsis (Arabidopsis thaliana). We observed that a member of PRC2 called Fertilization-Independent Endosperm1 (OsFIE1) was sensitive to temperature changes, and its expression was negatively correlated with the duration of the syncytial stage during heat stress. Seeds from plants overexpressing OsFIE1 had reduced seed size and exhibited precocious cellularization. The DNA methylation status and a repressive histone modification of OsFIE1 were observed to be temperature sensitive. Our data suggested that the thermal sensitivity of seed enlargement could partly be caused by altered epigenetic regulation of endosperm development during the transition from the syncytial to the cellularized state.World rice (Oryza sativa) production needs to increase significantly to sustain an increasing population. However, higher average temperatures caused by global warming are predicted to decrease rice yields in many parts of the world, especially Asia. In one study, rice yield was estimated to decrease by 10% for every 1°C rise in minimum growing season temperature (Peng et al., 2004). Comparable yield losses with rising temperatures have been reported for two other major cereal crops: wheat (Triticum spp.) and maize (Zea mays; Wardlaw, 1989; Lobell et al., 2011). Rice, wheat, and maize together are the main sources of calories for most countries (Reynolds et al., 2011). Therefore, it is critical that we understand the agronomic, biological, and economic consequences of high temperature on crop yields.Heat stress during seed development decreases the seed size in many cereals (Nagato and Ebata, 1960; Hunter et al., 1977; Savin et al., 1996) and when coupled with seed number per unit area, determines seed yield. Seed size is largely contributed by the endosperm, a triploid tissue derived from fusion of the sperm cell with the diploid central cell during the double fertilization event. Endosperm development progresses in distinct developmental stages. After fertilization, the endosperm enters the syncytial stage, where triploid nuclei undergo rapid mitotic divisions without cytokinesis, followed by cellularization and finally, differentiation and maturation (Olsen, 2001; Sabelli and Larkins, 2009b). Duration of the syncytial stage and rate of mitotic divisions during this stage are important determinants of seed size (Mizutani et al., 2010). Successful transition from the syncytial to the cellularization stage is critical for normal seed development (Brown et al., 1996).In Arabidopsis (Arabidopsis thaliana), the processes controlling early endosperm development and the transition from syncytial to cellularized stage are associated with the Polycomb Repressive Complex2 (PRC2) genes, which includes Fertilization-Independent Endosperm (FIE), Fertilization-Independent Seed2 (FIS2), Medea (MEA), and Multicopy Suppressor of IRA1 (Guitton and Berger, 2005; Baroux et al., 2006; Huh et al., 2007). The PRC2 complex is involved in gene silencing mediated by a repressive histone modification (H3K27me3; Köhler and Villar, 2008). Loss of function of several of these PRC2 genes results in abnormal endosperm development. A notable phenotype observed in Arabidopsis FIS mutants is endosperm overproliferation and seed failure (Kiyosue et al., 1999; Sørensen et al., 2002). Several endosperm-specific MADS-box genes (such as Pheres1, AGAMOUS-LIKE36 (AGL36), and AGL62 among others) are misregulated in seeds that are deficient in PRC2-encoding genes (Kang et al., 2008; Köhler and Villar, 2008; Walia et al., 2009). A loss-of-function mutation in Arabidopsis AGL62 resulted in precocious cellularization and smaller seeds (Kang et al., 2008). Although the function of the PRC2 complex is conserved in cereals such as rice and maize, orthologs of FIS2 and MEA have not been reported (Spillane et al., 2007; Luo et al., 2009). Orthologs of the Arabidopsis FIE gene have been reported in both rice (OsFIE1 and OsFIE2) and maize (ZmFIE1 and ZmFIE2; Springer et al., 2002; Danilevskaya et al., 2003; Luo et al., 2009). OsFIE1 is expressed only in the endosperm, whereas OsFIE2 is expressed in all tissues tested (Luo et al., 2009; Nallamilli et al., 2013). OsFIE1 is an imprinted gene, and its expression is regulated by DNA and H3K9me2 methylation (Luo et al., 2009; Zhang et al., 2012). OsFIE2 has a critical role in normal endosperm development and grain filling (Nallamilli et al., 2013).Our understanding of the epigenetic regulation of rice seed development has improved significantly (Zemach et al., 2010; Luo et al., 2011; Rodrigues et al., 2013). However, how the epigenetic regulation during seed development is altered during environmental perturbations is not well characterized. Most research efforts in the past have focused on the grain-filling stage (when storage proteins and starch accumulate) under stressful conditions (Yamakawa et al., 2007). However, it is not known if and how an environmental stress that specifically occurs during early seed development impacts seed size in rice. Here, we present evidence that early rice seed development is highly sensitive to heat stress and results in seed size reduction. We suggest a molecular mechanism that involves the rice PRC2 gene OsFIE1 as a potential component involved in regulating seed enlargement under heat stress.     

Expression and Processing of an Arabidopsis 2S Albumin in Transgenic Tobacco

2S albumin seed storage proteins undergo a complex series of posttranslational proteolytic cleavages. In order to determine if this process is correctly carried out in transgenic plants, the gene AT2S1 encoding an Arabidopsis thaliana 2S albumin isoform has been expressed in transgenic tobacco. Initial experiments using a reporter gene demonstrated that the AT2S1 promoter directs seed specific expression in both transgenic tobacco and Brassica napus plants. The entire AT2S1 gene was then transferred into tobacco plants, where it showed a tissue specific and developmentally regulated expression. Arabidopsis 2S albumin accumulates up to 0.1% of the total high-salt extractable seed protein. Protein sequencing demonstrated that the amino termini of the two Arabidopsis 2S albumin subunits were correctly processed, suggesting that the protease(s) necessary for posttranslational processing of 2S albumin precursors may display common specificities among different dicot plant species. Immunocytochemical studies showed that the Arabidopsis 2S albumin is localized in the protein body matrix of tobacco endosperm and embryo. Correct processing and targeting of the 2S albumin in transgenic plants suggests that modified versions could be expressed, allowing the study of 2S albumin processing and in particular the possible roles of the processed fragments in protein stability and/or targeting.     

Arabidopsis MSI1 connects LHP1 to PRC2 complexes

Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1–MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication.