The florigen interactor BdES43 represses flowering in the model temperate grass Brachypodium distachyon.

FLOWERING LOCUS T (FT) protein, physiologically florigen, has been identified as a system integrator of numerous flowering time pathways in many studies, and its homologs are found throughout the plant lineage. It is important to uncover how precisely florigenic homologs contribute to flowering initiation and how these factors interact genetically. Here we dissected the function of Brachypodium FT orthologs BdFTL1 and BdFTL2 using overexpression and gene-editing experiments. Transgenic assays showed that both BdFTL1 and BdFTL2 could promote flowering, whereas BdFTL2 was essential for flowering initiation. Notably, BdFTL1 is subject to alternative splicing (AS), and its transcriptional level and AS are significantly affected by BdFTL2. Additionally, BdFTL2 could bind with the PHD-containing protein BdES43, an H3K4me3 reader. Furthermore, BdES43 was antagonistic to BdFTL2 in flowering initiation in a transcription-dependent manner and significantly affected BdFTL1 expression. BdFTL2, BdES43 and H3K4me3 also had highly similar distribution patterns within the BdFTL1 locus, indicating their interplay in regulating target genes. Taken together, florigen BdFTL2 functions as a potential epigenetic effector of BdFTL1 by interacting with a BdES43-H3K4me3 complex. This finding provides an additional insight for the regulatory mechanism underlying the multifaceted roles of florigen.

CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer.

Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity. NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins. We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter. We term this complex NF-CO. Using saturation mutagenesis, electrophoretic mobility shift assays, and RNA-sequencing profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site. CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo. The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA. In summary, a unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence specificity to HFD/DNA interactions. It is likely that members of the large CCT family participate in similar complexes with At-NF-YB and At-NF-YC, broadening HFD combinatorial possibilities in terms of trimerization, DNA binding specificities, and transcriptional regulation.

NUCLEAR FACTOR Y, Subunit A (NF-YA) Proteins Positively Regulate Flowering and Act Through FLOWERING LOCUS T.

Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal "CORE" elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.

A distal CCAAT/NUCLEAR FACTOR Y complex promotes chromatin looping at the FLOWERING LOCUS T promoter and regulates the timing of flowering in Arabidopsis.

For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.

A supermatrix phylogeny of corvoid passerine birds (Aves: Corvides).

The Corvides (previously referred to as the core Corvoidea) are a morphologically diverse clade of passerine birds comprising nearly 800 species. The group originated some 30 million years ago in the proto-Papuan archipelago, to the north of Australia, from where lineages have dispersed and colonized all of the world's major continental and insular landmasses (except Antarctica). During the last decade multiple species-level phylogenies have been generated for individual corvoid families and more recently the inter-familial relationships have been resolved, based on phylogenetic analyses using multiple nuclear loci. In the current study we analyse eight nuclear and four mitochondrial loci to generate a dated phylogeny for the majority of corvoid species. This phylogeny includes 667 out of 780 species (85.5%), 141 out of 143 genera (98.6%) and all 31 currently recognized families, thus providing a baseline for comprehensive macroecological, macroevolutionary and biogeographical analyses. Using this phylogeny we assess the temporal consistency of the current taxonomic classification of families and genera. By adopting an approach that enforces temporal consistency by causing the fewest possible taxonomic changes to currently recognized families and genera, we find the current familial classification to be largely temporally consistent, whereas that of genera is not.

The promiscuous life of plant NUCLEAR FACTOR Y transcription factors.

The CCAAT box is one of the most common cis-elements present in eukaryotic promoters and is bound by the transcription factor NUCLEAR FACTOR Y (NF-Y). NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC. Unlike animals and fungi, plants have significantly expanded the number of genes encoding NF-Y subunits. We provide a comprehensive classification of NF-Y genes, with a separation of closely related, but distinct, histone fold domain proteins. We additionally review recent experiments that have placed NF-Y at the center of many developmental stress-responsive processes in the plant lineage.

Evidence of taxon cycles in an Indo-Pacific passerine bird radiation (Aves: Pachycephala).

Many insular taxa possess extraordinary abilities to disperse but may differ in their abilities to diversify and compete. While some taxa are widespread across archipelagos, others have disjunct (relictual) populations. These types of taxa, exemplified in the literature by selections of unrelated taxa, have been interpreted as representing a continuum of expansions and contractions (i.e. taxon cycles). Here, we use molecular data of 35 out of 40 species of the avian genus Pachycephala (including 54 out of 66 taxa in Pachycephala pectoralis (sensu lato), to assess the spatio-temporal evolution of the group. We also include data on species distributions, morphology, habitat and elevational ranges to test a number of predictions associated with the taxon-cycle hypothesis. We demonstrate that relictual species persist on the largest and highest islands across the Indo-Pacific, whereas recent archipelago expansions resulted in colonization of all islands in a region. For co-occurring island taxa, the earliest colonists generally inhabit the interior and highest parts of an island, with little spatial overlap with later colonists. Collectively, our data support the idea that taxa continuously pass through phases of expansions and contractions (i.e. taxon cycles).

Gene Family Analysis of the Arabidopsis NF-YA Transcription Factors Reveals Opposing Abscisic Acid Responses During Seed Germination.

In the plant kingdom, each of the NUCLEAR FACTOR-Y (NF-Y) transcription factor families, NF-YA, NF-YB, and NF-YC, has undergone a great expansion compared to the animal kingdom. For example, Arabidopsis thaliana has 10 members of each gene family compared to only one in humans. Progress towards understanding the significance of this expansion is limited due to a lack of studies looking at the complete gene family during plant development. In the current study, transgenic overexpression lines were created for all 10 Arabidopsis NF-YA genes and examined for general development and alterations in abscisic acid (ABA)-mediated seed germination. NF-YA overexpression typically led to severe growth retardation and developmental defects, which extended from embryogenesis through to adult plants. Although overexpression of all NF-YA family members consistently led to growth retardation, some transgenic lines were hypersensitive to ABA during germination while others were hyposensitive. The opposing germination phenotypes were associated with the phylogenetic relationships between the NF-YA members. In addition, ABA marker genes were misregulated and ABA induction of gene expression was reduced in the overexpressors. Collectively, this study demonstrates that although NF-Ys have retained high degrees of similarity, they have evolved unique and sometimes opposing roles during plant development.

Analysis of gene duplication within the Arabidopsis NUCLEAR FACTOR Y, subunit B (NF-YB) protein family reveals domains under both purifying and diversifying selection.

Gene duplication is an evolutionary mechanism that provides new genetic material. Since gene duplication is a major driver for molecular evolution, examining the fate of duplicated genes is an area of active research. The fate of duplicated genes can include loss, subfunctionalization, and neofunctionalization. In this manuscript, we chose to experimentally study the fate of duplicated genes using the Arabidopsis NUCLEAR FACTOR Y (NF-Y) transcription factor family. NF-Y transcription factors are heterotrimeric complexes, composed of NF-YA, NF-YB, and NF-YC. NF-YA subunits are responsible for nucleotide-specific binding to a CCAAT cis-regulatory element. NF-YB and NF-YC subunits make less specific, but essential complex-stabilizing contacts with the DNA flanking the core CCAAT pentamer. While ubiquitous in eukaryotes, each NF-Y family has expanded by duplication in the plant lineage. For example, the model plant Arabidopsis contains 10 each of the NF-Y subunits. Here we examine the fate of duplicated NF-YB proteins in Arabidopsis, which are composed of central histone fold domains (HFD) and less conserved flanking regions (N- and C-termini). Specifically, the principal question we wished to address in this manuscript was to what extent can the 10 Arabidopsis NF-YB paralogs functionally substitute the genes NF-YB2 and NF-YB3 in the promotion of photoperiodic flowering? Our results demonstrate that the conserved histone fold domains (HFD) may be under pressure for purifying (negative) selection, while the non-conserved N- and C-termini may be under pressure for diversifying (positive) selection, which explained each paralog's ability to substitute. In conclusion, our data demonstrate that the N- and C-termini may have allowed the duplicated genes to undergo functional diversification, allowing the retention of the duplicated genes.

NF-YC3, NF-YC4 and NF-YC9 are required for CONSTANS-mediated, photoperiod-dependent flowering in Arabidopsis thaliana.

NF-Y transcription factors represent a complex of three proteins called NF-YA, NF-YB and NF-YC. Each protein is highly conserved in eukaryotes, and in the plant lineage has undergone numerous rounds of duplication. Individual NF-Y are emerging as important regulators of several essential plant processes, including embryogenesis, drought resistance, maintenance of meristems in nitrogen-fixing nodules and photoperiod-dependent flowering time. Building on the recent finding that NF-YB2 and NF-YB3 have overlapping functionality in Arabidopsis photoperiod-dependent flowering (Kumimoto et al., 2008), we have identified three NF-YC (NF-YC3, NF-YC4, and NF-YC9) that are also required for flowering, and physically interact in vivo with both NF-YB2 and NF-YB3. Furthermore, NF-YC3, NF-YC4 and NF-YC9 can physically interact with full-length CONSTANS (CO), and are genetically required for CO-mediated floral promotion. Collectively, the present data greatly strengthens and extends the argument that CO utilizes NF-Y transcription factor complexes for the activation of FLOWERING LOCUS T (FT) during photoperiod-dependent floral initiation.

Construction of high quality Gateway™ entry libraries and their application to yeast two-hybrid for the monocot model plant Brachypodium distachyon.

BACKGROUND: Monocots, especially the temperate grasses, represent some of the most agriculturally important crops for both current food needs and future biofuel development. Because most of the agriculturally important grass species are difficult to study (e.g., they often have large, repetitive genomes and can be difficult to grow in laboratory settings), developing genetically tractable model systems is essential. Brachypodium distachyon (hereafter Brachypodium) is an emerging model system for the temperate grasses. To fully realize the potential of this model system, publicly accessible discovery tools are essential. High quality cDNA libraries that can be readily adapted for multiple downstream purposes are a needed resource. Additionally, yeast two-hybrid (Y2H) libraries are an important discovery tool for protein-protein interactions and are not currently available for Brachypodium. RESULTS: We describe the creation of two high quality, publicly available Gateway™ cDNA entry libraries and their derived Y2H libraries for Brachypodium. The first entry library represents cloned cDNA populations from both short day (SD, 8/16-h light/dark) and long day (LD, 20/4-h light/dark) grown plants, while the second library was generated from hormone treated tissues. Both libraries have extensive genome coverage (~5 × 107 primary clones each) and average clone lengths of ~1.5 Kb. These entry libraries were then used to create two recombination-derived Y2H libraries. Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors. CONCLUSIONS: Accessible community resources are a hallmark of successful biological model systems. Brachypodium has the potential to be a broadly useful model system for the grasses, but still requires many of these resources. The Gateway™ compatible entry libraries created here will facilitate studies for multiple user-defined purposes and the derived Y2H libraries can be immediately applied to large scale screening and discovery of novel protein-protein interactions. All libraries are freely available for distribution to the research community.

Controlled silanization of silica nanoparticles to stabilize foams, climbing films, and liquid marbles.

We describe a method for the synthesis of multigram amounts of silica nanoparticles which are controllably hydrophobized to different extents using a room temperature vapor phase silanization process. The extent of hydrophobization of the particles can be adjusted by changing the amount of dichlorodimethylsilane reagent used in the reaction. The method produces particles with good uniformity of surface coating; the silane coating varies from monolayer coverage at low extents of hydrophobization to approximately trilayer at high extents of hydrophobization. Acid-base titration using conductivity detection was used to characterize the extent of hydrophobization which is expressed as the percent of surface silanol groups remaining after silanization. Particles with %SiOH ranging from 100% (most hydrophilic) to 20% (most hydrophobic) were hand shaken with water/methanol mixtures and produced either a particle dispersion, foam, climbing films, or liquid marbles. The type of colloidal structure produced is discussed in terms of the liquid-air-particle contact angle and the energy of adsorption of the particles to the liquid-air surface.

An evolutionarily conserved mediator of plant disease resistance gene function is required for normal Arabidopsis development.

Plants recognize many pathogens through the action of a diverse family of proteins called disease resistance (R) genes. The Arabidopsis R gene RPM1 encodes resistance to specific Pseudomonas syringae strains. We describe an RPM1-interacting protein that is an ortholog of TIP49a, previously shown to interact with the TATA binding protein (TBP) complex and to modulate c-myc- and beta-catenin-mediated signaling in animals. Reduction of Arabidopsis TIP49a (AtTIP49a) mRNA levels results in measurable increases of two R-dependent responses without constitutively activating defense responses, suggesting that AtTIP49a can act as a negative regulator of at least some R functions. Further, AtTIP49a is essential for both sporophyte and female gametophyte viability. Thus, regulators of R function overlap with essential modulators of plant development.

Humboldt's enigma: What causes global patterns of mountain biodiversity?

Mountains contribute disproportionately to the terrestrial biodiversity of Earth, especially in the tropics, where they host hotspots of extraordinary and puzzling richness. With about 25% of all land area, mountain regions are home to more than 85% of the world's species of amphibians, birds, and mammals, many entirely restricted to mountains. Biodiversity varies markedly among these regions. Together with the extreme species richness of some tropical mountains, this variation has proven challenging to explain under traditional climatic hypotheses. However, the complex climatic characteristics of rugged mountain regions differ fundamentally from those of lowland regions, likely playing a key role in generating and maintaining diversity. With ongoing global changes in climate and land use, the role of mountains as refugia for biodiversity may well come under threat.

Building mountain biodiversity: Geological and evolutionary processes.

Mountain regions are unusually biodiverse, with rich aggregations of small-ranged species that form centers of endemism. Mountains play an array of roles for Earth's biodiversity and affect neighboring lowlands through biotic interchange, changes in regional climate, and nutrient runoff. The high biodiversity of certain mountains reflects the interplay of multiple evolutionary mechanisms: enhanced speciation rates with distinct opportunities for coexistence and persistence of lineages, shaped by long-term climatic changes interacting with topographically dynamic landscapes. High diversity in most tropical mountains is tightly linked to bedrock geology-notably, areas comprising mafic and ultramafic lithologies, rock types rich in magnesium and poor in phosphate that present special requirements for plant physiology. Mountain biodiversity bears the signature of deep-time evolutionary and ecological processes, a history well worth preserving.

NUCLEAR FACTOR-Y: still complex after all these years?

The NUCLEAR FACTOR-Y (NF-Y) families of transcription factors are important regulators of plant development and physiology. Though NF-Y regulatory roles have recently been suggested for numerous aspects of plant biology, their roles in flowering time, early seedling development, stress responses, hormone signaling, and nodulation are the best characterized. The past few years have also seen significant advances in our understanding of the mechanistic function of the NF-Y, and as such, increasingly complex and interesting questions are now more approachable. This review will primarily focus on these developmental, physiological, and mechanistic roles of the NF-Y in recent research.

A global mismatch in the protection of multiple marine biodiversity components and ecosystem services.

The global loss of biodiversity threatens unique biota and the functioning and services of ecosystems essential for human wellbeing. To safeguard biodiversity and ecosystem services, designating protected areas is crucial; yet the extent to which the existing placement of protection is aligned to meet these conservation priorities is questionable, especially in the oceans. Here we investigate and compare global patterns of multiple biodiversity components (taxonomic, phylogenetic and functional), ecosystem services and human impacts, with the coverage of marine protected areas across a nested spatial scale. We demonstrate a pronounced spatial mismatch between the existing degree of protection and all the conservation priorities above, highlighting that neither the world's most diverse, nor the most productive ecosystems are currently the most protected ecosystems. Furthermore, we show that global patterns of biodiversity, ecosystem services and human impacts are poorly correlated, hence complicating the identification of generally applicable spatial prioritization schemes. However, a hypothetical "consensus approach" would have been able to address all these conservation priorities far more effectively than the existing degree of protection, which at best is only marginally better than a random expectation. Therefore, a holistic perspective is needed when designating an appropriate degree of protection of marine conservation priorities worldwide.

The multifaceted roles of NUCLEAR FACTOR-Y in Arabidopsis thaliana development and stress responses.

NUCLEAR FACTOR-Y (NF-Y) is a heterotrimeric transcription factor (TF) consisting of evolutionarily distinct NF-YA, NF-YB and NF-YC subunits. The functional NF-Y heterotrimer binds to CCAAT elements in eukaryotic gene promoters and influences their expression. The genome of the model organism Arabidopsis thaliana encodes 10 distinct NF-YA, NF-YB, and NF-YC proteins, allowing for enormous combinatorial and functional diversity. Two decades of research have elucidated the importance of NF-Ys in plant growth, development and stress responses; however, the molecular mechanisms of action remain largely unexplored. Intriguingly, recent evidence suggests that NF-Ys are frequently associated with other groups of TFs, expanding the potential NF-Y combinatorial complexity. Further, information regarding the regulation of individual NF-Y subunits at the transcriptional and post-transcriptional level is beginning to emerge. In this review, we will identify developing trends within the NF-Y field and discuss recent progress towards a better understanding of NF-Y function, molecular action, and regulation in the context of Arabidopsis. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.