During Arabidopsis seed development, the growing embryo invades and consumes the surrounding endosperm tissue. The signalling pathways that coordinate the separation of the embryo from the endosperm and the concomitant breakdown of the endosperm are poorly understood. We have identified a novel bHLH transcription factor, ZHOUPI (ZOU), which mediates these processes. ZOU is expressed exclusively in the endosperm of developing seeds. It is activated in the central cell immediately after fertilization and is initially expressed uniformly in endosperm, subsequently resolving to the embryo surrounding region (ESR). However, zou mutant embryos have defects in cuticle formation and in epidermal cell adhesion, suggesting that ZOU functions non-autonomously to regulate embryonic development. In addition, the endosperm of zou mutant seeds fails to separate from the embryo, restricting embryo expansion and resulting in the production of shrivelled collapsed seeds. zou seeds retain more endosperm than do wild-type seeds at maturity, suggesting that ZOU also controls endosperm breakdown. We identify several target genes whose expression in the ESR is regulated by ZOU. These include ABNORMAL LEAF SHAPE1, which encodes a subtilisinlike protease previously shown to have a similar role to ZOU in regulating endosperm adhesion and embryonic epidermal development. However, expression of several other ESR-specific genes is independent of ZOU. Therefore, ZOU is not a general regulator of endosperm patterning, but rather controls specific signalling pathways that coordinate embryo invasion and breakdown of surrounding endosperm tissues.

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

In Arabidopsis, mutations in the Pc-G gene CURLY LEAF (CLF) give early flowering plants with curled leaves. This phenotype is caused by mis-expression of the floral homeotic gene AGAMOUS (AG) in leaves, so that ag mutations largely suppress the clf phenotype. Here, we identify three mutations that suppress clf despite maintaining high AG expression. We show that the suppressors correspond to mutations in FPA and FT, two genes promoting flowering, and in SEPALLATA3 (SEP3) which encodes a co-factor for AG protein. The suppression of the clf phenotype is correlated with low SEP3 expression in all case and reveals that SEP3 has a role in promoting flowering in addition to its role in controlling floral organ identity. Genetic analysis of clf ft mutants indicates that CLF promotes flowering by reducing expression of FLC, a repressor of flowering. We conclude that SEP3 is the key target mediating the clf phenotype, and that the antagonistic effects of CLF target genes masks a role for CLF in promoting flowering.

Soybean (Glycine max) is one of the most important crops in the world, and its yield is largely determined by grain weight and grain size. However, the genes that regulate soybean seed size have not been identified. CYP78A, which is highly conserved within terrestrial plants, regulates organ development. In Arabidopsis, AtCYP78A5/KLU has been shown to determine seed size. In the present study, soybean CYP78A72 (GmCYP78A72), one of the orthologs of KLU, was over-expressed in both Arabidopsis and soybean to examine its function in plant development. GmCYP78A72 heterologous expression in Arabidopsis resulted in enlarged sepals, petals, seeds and carpel. Over-expression of GmCYP78A72 in soybean resulted in increased pea size, which is an extremely desirable trait for enhancing productivity. Moreover, knock-down of GmCYP78A72 does not reduce grain size. However, silencing of GmCYP78A57, GmCYP78A70 and GmCYP78A72 genes in triplet reduces the seed size significantly indicating functional redundancy of these three GmCYP78A genes. In conclusion, we investigated the role of CYP78A in soybean seed regulation, and our strategy can be effectively used to engineer large seed traits in soybean varieties as well as other crops.