SummaryEnhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16±UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T 0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T 1 families corresponded well with the T 0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T 1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of¯anking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T 1 families that were ®eld-tested, and segregation in more than one-third of the families ®t the 3 : 1 ratio. It was concluded that GAL4/VP16±UAS elements provided a useful system for enhancer trap in rice.

Some plants acquire competence to flower in spring after experiencing a seasonal temperature drop-winter cold, in a process termed vernalization. In Arabidopsis thaliana, prolonged exposure to cold induces epigenetic silencing of the potent floral repressor locus FLOWERING LOCUS C (FLC) by Polycomb group (PcG) proteins, and this silencing is stably maintained in subsequent growth and development upon return to warm temperatures. Here we show that a cis-regulatory DNA element in the nucleation region for PcG silencing at FLC and two homologous trans-acting epigenome readers, VAL1 and VAL2, control vernalization-mediated FLC silencing. The sequence-specific readers recognize both the cis element (termed the cold memory element) and a repressive mark, trimethylation of histone H3 at lysine 27 (H3K27me3), and directly associate with LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), leading to establishment of the H3K27me3 peak in the nucleation region at FLC during vernalization. Thus, our work describes a mechanism for PcG-mediated silencing by a DNA sequence-specific epigenome reader.

Polycomb group (PcG) complexes such as PRC1 mediate transcriptional repression. Here, we show that the plant-specific EMBRYONIC FLOWER1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1, and a histone H3 lysine-4 demethylase form a distinct PcG complex, termed EMF1c, that plays PRC1-like roles and is crucial for regulation of the florigen gene FLOWERING LOCUS T (FT) in Arabidopsis. Long-day photoperiods promote FT expression activation in leaf veins specifically at dusk through the photoperiod pathway to induce Arabidopsis flowering. We found that before dusk and at night, a vascular EMF1c directly represses FT expression to prevent photoperiod-independent flowering, whereas at dusk EMF1 binding to FT chromatin is disrupted by the photoperiod pathway, leading to proper FT activation. Furthermore, a MADS-domain transcription factor and potent floral repressor binds EMF1 to repress FT expression. Our study reveals that the vascular EMF1c integrates inputs from several flowering-regulatory pathways to synchronize flowering time to environmental cues.

Summary Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF ‐Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF ‐Y heterotrimer complex NF ‐ YB 1‐ YC 12‐ bHLH 144, which is formed by the binding of NF ‐ YB 1 to NF ‐ YC 12 and then bHLH 144 in a sequential order. Knock‐out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf‐yb1 and crnf‐yc12 . RNA ‐seq analysis identified 1496 genes that were commonly regulated by NF ‐ YB 1 and NF ‐ YC 12 , including the key granule‐bound starch synthase gene Wx . NF ‐ YC 12 and bHLH 144 maintain NF ‐ YB 1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF ‐ YB 1 directly binds to the ‘G‐box’ domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF ‐ YB 1‐ YC 12‐ bHLH 144 complex, which has great potential for rice genetic improvement.

No abstract

No abstract

SUMMARYMeiosis is essential for eukaryotic sexual reproduction and important for genetic diversity among individuals. Although a number of genes regulating homologous chromosome pairing and synapsis have been identified in the plant kingdom, their molecular basis remains poorly understood. In this study, we identified a novel gene, PAIR3 (HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS 3), required for homologous chromosome pairing and synapsis in rice. Two independent alleles, designated pair3-1 and pair3-2, were identified in our T-DNA insertional mutant library which could not form bivalents due to failure of homologous chromosome pairing and synapsis at diakinesis, resulting in sterility in both male and female gametes. Suppression of PAIR3 by RNAi produced similar results to the T-DNA insertion lines. PAIR3 encodes a protein that contains putative coiled-coil motifs, but does not have any close homologs in other organisms. PAIR3 is preferentially expressed in reproductive organs, especially in pollen mother cells and the ovule tissues during meiosis. Our results suggest that PAIR3 plays a crucial role in homologous chromosome pairing and synapsis in meiosis.

Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.