SUMMARYNuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPCassociated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

SummaryIn Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting¯ower meristem identity. Later in¯ower development, the AP1 gene is required for normal development of sepals and petals. Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during¯ower development. To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay. Among the ®ve MADS-box genes identi®ed in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study. Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1. Furthermore, the early¯owering phenotype of plants constitutively expressing AP1 is signi®cantly enhanced by constitutive SEP3 expression. Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal¯ower development.

Summary Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ‐identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F‐box protein that interacts with Arabidopsis SKP1‐like proteins, both in the yeast two‐hybrid system and in vitro. In yeast and other organisms both F‐box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F‐box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

SWI/SNF-type chromatin remodelers, such as BRAHMA (BRM), and H3K27 demethylases both have active roles in regulating gene expression at the chromatin level1–5, but how they are recruited to specific genomic sites remains largely unknown. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), a plant-unique H3K27 demethylase6, targets genomic loci containing a CTCTGYTY motif via its zinc-finger (ZnF) domains and facilitates the recruitment of BRM. Genome-wide analyses showed that REF6 colocalizes with BRM at many genomic sites with the CTCTGYTY motif. Loss of REF6 results in decreased BRM occupancy at BRM–REF6 co-targets. Furthermore, REF6 directly binds to the CTCTGYTY motif in vitro, and deletion of the motif from a target gene renders it inaccessible to REF6 in vivo. Finally, we show that, when its ZnF domains are deleted, REF6 loses its genomic targeting ability. Thus, our work identifies a new genomic targeting mechanism for an H3K27 demethylase and demonstrates its key role in recruiting the BRM chromatin remodeler.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.

Background Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop’s sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. Results To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40–1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40–1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40–1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. Conclusions Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40–1 expression, whereas higher miR156 overexpression results in drought susceptibility.

The Polycomb Group (PcG) proteins form two protein complexes, PcG Repressive Complex 1 ( PRC 1) and PRC 2, which are key epigenetic regulators in eukaryotes. PRC 2 represses gene expression by catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). In Arabidopsis ( Arabidopsis thaliana ), CURLY LEAF ( CLF ) and SWINGER ( SWN ) are two major H3K27 methyltransferases and core components of PRC 2, playing essential roles in plant growth and development. Despite their importance, genome‐wide binding profiles of CLF and SWN have not been determined and compared yet. In this study, we generated transgenic lines expressing GFP ‐tagged CLF / SWN under their respective native promoters and used them for Ch IP ‐seq analyses to profile the genome‐wide distributions of CLF and SWN in Arabidopsis seedlings. We also profiled and compared the global H3K27me3 levels in wild‐type ( WT ) and PcG mutants ( clf , swn , and clf swn ). Our data show that CLF and SWN bind to almost the same set of genes, except that SWN has a few hundred more targets. Two short DNA sequences, the GAGA ‐like and Telo ‐box‐like motifs, were found enriched in the CLF and SWN binding regions. The H3K27me3 levels in clf , but not in swn , were markedly reduced compared with WT ; and the mark was undetectable in the clf swn double mutant. Further, we profiled the transcriptomes in clf , swn , and clf swn, and compared that with WT . Thus this work provides a useful resource for the plant epigenetics community for dissecting the functions of PRC 2 in plant growth and development.