Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

Summary: The LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the α‐enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c‐myc binding protein (MBP‐1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while α‐enolase is found in the cytoplasm. We report here that an Arabidopsis MBP‐1‐like protein (AtMBP‐1) is alternatively translated from full‐length LOS2 transcripts using a second start codon. Like mammalian MBP‐1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N‐terminal region of LOS2 is critical for catalysis. AtMBP‐1 has a short half‐life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin‐dependent proteasome pathway. Arabidopsis plants that over‐express AtMBP‐1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP‐1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co‐expression of these proteins resulted in destabilization of AtMBP‐1 in vivo and abolished the developmental defects associated with AtMBP‐1 over‐expression. Thus, AtMBP‐1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin‐dependent destabilization, apparently mediated by AtSAP5.

Summary: DELAY OF GERMINATION1 (DOG1) is a primary regulator of seed dormancy. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination in Arabidopsis. B3 domain‐containing transcriptional repressors HSI2/VAL1 and HSL1/VAL2 silence seed dormancy and enable the subsequent germination and seedling growth. However, the roles of HSI2 and HSL1 in regulation of DOG1 expression and seed dormancy remain elusive. Seed dormancy was analysed by measurement of maximum germination percentage of freshly harvested Arabidopsis seeds. In vivo protein–protein interaction analysis, ChIP‐qPCR and EMSA were performed and suggested that HSI2 and HSL1 can form dimers to directly regulate DOG1. HSI2 and HSL1 dimers interact with RY elements at DOG1 promoter. Both B3 and PHD‐like domains are required for enrichment of HSI2 and HSL1 at the DOG1 promoter. HSI2 and HSL1 recruit components of polycomb‐group proteins, including CURLY LEAF (CLF) and LIKE HETERCHROMATIN PROTEIN 1 (LHP1), for consequent deposition of H3K27me3 marks, leading to repression of DOG1 expression. Our findings suggest that HSI2‐ and HSL1‐dependent histone methylation plays critical roles in regulation of seed dormancy during seed germination and early seedling growth.