We made a series of improved Gateway binary vectors (pGWBs) for plant transformation. Fifteen different reporters and tags, sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, 6ÂHis, FLAG, 3ÂHA, 4ÂMyc, 10ÂMyc, GST, T7, and TAP, were employed. Some vectors carry the 2Â35S-promoter for higher-level expression. The kanamycin-and hygromycin-resistant markers are independently available for each of the 43 types of vectors, thus an additional transformation of once-transformed plants can be carried out easily. Their small size and high-copy number in Escherichia coli make possible easier handling at plasmid preparation and sequencing. Improved pGWBs should be a powerful tool for transgenic research in plants.

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RE-SPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIPquantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.ChIP-seq | plant T he circadian clock in plants regulates a broad range of biological processes. For example, hypocotyl elongation is observed before dawn and cold-stress responses reach maximal levels in the afternoon in Arabidopsis thaliana (1, 2), all largely because of circadian coordination of these biological processes (clock-output) with daily cycles. The circadian clock mechanism controls the temporal regulation of numerous genes involved in output processes (3-5).A number of recent studies have described the genetic components of the clock in Arabidopsis. CIRCADIAN CLOCK-ASSOCI-ATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) encode morning-expressed MYB transcription factors (TFs) that directly repress TIMING OF CAB EXPRESSION 1 [TOC1, also called PSEUDO-RESPONSE REGULATOR 1 (PRR1)], EARLY FLOWERING 3 (ELF3), ELF4, and LUXARRHYTHMO (LUX) (6-10). ELF3 and LUX associate with upstream region of PRR9, and repress PRR9 expression (11, 12). Expression of PRR9 and PRR7 are activated by CCA1 and LHY (13). CCA1 and LHY are in turn repressed by four PRR proteins, PRR9, PRR7, PRR5, and TOC1 from early daytime through to around midnight (14-16). These TFs form a negative feedback loop for clock function (12,17,18). Evidence is accumulating that these TFs directly regulate the expression of genes involved in clock-output pathways. LUX, ELF3, and ELF4 together form the "evening complex" that directly represses expression of PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and P...

The circadian clock is a biological timekeeping system that provides organisms with the ability to adapt to day-night cycles. Timing of the expression of four members of the Arabidopsis thaliana PSEUDO-RESPONSE REGULATOR (PRR) family is crucial for proper clock function, and transcriptional control of PRRs remains incompletely defined. Here, we demonstrate that direct regulation of PRR5 by CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) determines the repression state of PRR5 in the morning. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses indicated that CCA1 associates with three separate regions upstream of PRR5. CCA1 and its homolog LATE ELONGATED HYPOCOTYL (LHY) suppressed PRR5 promoter activity in a transient assay. The regions bound by CCA1 in the PRR5 promoter gave rhythmic patterns with troughs in the morning, when CCA1 and LHY are at high levels. Furthermore, ChIP-seq revealed that CCA1 associates with at least 449 loci with 863 adjacent genes. Importantly, this gene set contains genes that are repressed but upregulated in cca1 lhy double mutants in the morning. This study shows that direct binding by CCA1 in the morning provides strong repression of PRR5, and repression by CCA1 also temporally regulates an evening-expressed gene set that includes PRR5.

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the postmitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M-specific genes repressed in post-mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor-type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome-wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M-specific genes and to E2F target genes. MYB3R3 associates with the repressor-type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post-mitotic quiescent state determining organ size.

Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.grain size | weight | yield | plant biomass | rice