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.

Community development, implementation, and assessment of a NIBLSE bioinformatics sequence similarity learning resource.

As powerful computational tools and 'big data' transform the biological sciences, bioinformatics training is becoming necessary to prepare the next generation of life scientists. Furthermore, because the tools and resources employed in bioinformatics are constantly evolving, bioinformatics learning materials must be continuously improved. In addition, these learning materials need to move beyond today's typical step-by-step guides to promote deeper conceptual understanding by students. One of the goals of the Network for Integrating Bioinformatics into Life Sciences Education (NIBSLE) is to create, curate, disseminate, and assess appropriate open-access bioinformatics learning resources. Here we describe the evolution, integration, and assessment of a learning resource that explores essential concepts of biological sequence similarity. Pre/post student assessment data from diverse life science courses show significant learning gains. These results indicate that the learning resource is a beneficial educational product for the integration of bioinformatics across curricula.

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.

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.

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.

NUCLEAR FACTOR Y, Subunit C (NF-YC) Transcription Factors Are Positive Regulators of Photomorphogenesis in Arabidopsis thaliana.

Recent reports suggest that NF-Y transcription factors are positive regulators of skotomorphogenesis in Arabidopsis thaliana. Three NF-YC genes (NF-YC3, NF-YC4, and NF-YC9) are known to have overlapping functions in photoperiod dependent flowering and previous studies demonstrated that they interact with basic leucine zipper (bZIP) transcription factors. This included ELONGATED HYPOCOTYL 5 (HY5), which has well-demonstrated roles in photomorphogenesis. Similar to hy5 mutants, we report that nf-yc3 nf-yc4 nf-yc9 triple mutants failed to inhibit hypocotyl elongation in all tested light wavelengths. Surprisingly, nf-yc3 nf-yc4 nf-yc9 hy5 mutants had synergistic defects in light perception, suggesting that NF-Ys represent a parallel light signaling pathway. As with other photomorphogenic transcription factors, nf-yc3 nf-yc4 nf-yc9 triple mutants also partially suppressed the short hypocotyl and dwarf rosette phenotypes of CONSTITUTIVE PHOTOMORPHOGENIC 1 (cop1) mutants. Thus, our data strongly suggest that NF-Y transcription factors have important roles as positive regulators of photomorphogenesis, and in conjunction with other recent reports, implies that the NF-Y are multifaceted regulators of early seedling development.

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.

Alternative splicing of Arabidopsis IBR5 pre-mRNA generates two IBR5 isoforms with distinct and overlapping functions.

The INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5) gene encodes a dual specificity phosphatase that regulates plant auxin responses. IBR5 has been predicted to generate two transcripts through alternative splicing, but alternative splicing of IBR5 has not been confirmed experimentally. The previously characterized ibr5-1 null mutant exhibits many auxin related defects such as auxin insensitive primary root growth, defective vascular development, short stature and reduced lateral root development. However, whether all these defects are caused by the lack of phosphatase activity is not clear. Here we describe two new auxin insensitive IBR5 alleles, ibr5-4, a catalytic site mutant, and ibr5-5, a splice site mutant. Characterization of these new mutants indicates that IBR5 is post-transcriptionally regulated to generate two transcripts, AT2G04550.1 and AT2G04550.3, and consequently two IBR5 isoforms, IBR5.1 and IBR5.3. The IBR5.1 isoform exhibits phosphatase catalytic activity that is required for both proper degradation of Aux/IAA proteins and auxin-induced gene expression. These two processes are independently regulated by IBR5.1. Comparison of new mutant alleles with ibr5-1 indicates that all three mutant alleles share many phenotypes. However, each allele also confers distinct defects implicating IBR5 isoform specific functions. Some of these functions are independent of IBR5.1 catalytic activity. Additionally, analysis of these new mutant alleles suggests that IBR5 may link ABP1 and SCF(TIR1/AFBs) auxin signaling pathways.

NUCLEAR FACTOR Y transcription factors have both opposing and additive roles in ABA-mediated seed germination.

In the model organism Arabidopsis thaliana the heterotrimeric transcription factor NUCLEAR FACTOR Y (NF-Y) has been shown to play multiple roles in facilitating plant growth and development. Although NF-Y itself represents a multi-protein transcriptional complex, recent studies have shown important interactions with other transcription factors, especially those in the bZIP family. Here we add to the growing evidence that NF-Y and bZIP form common complexes to affect many processes. We carried out transcriptional profiling on nf-yc mutants and through subsequent analyses found an enrichment of bZIP binding sites in the promoter elements of misregulated genes. Using NF-Y as bait, yeast two hybrid assays yielded interactions with bZIP proteins that are known to control ABA signaling. Accordingly, we find that plants mutant for several NF-Y subunits show characteristic phenotypes associated with the disruption of ABA signaling. While previous reports have shown additive roles for NF-YC family members in photoperiodic flowering, we found that they can have opposing roles in ABA signaling. Collectively, these results demonstrated the importance and complexity of NF-Y in the integration of environmental and hormone signals.

Identification and characterization of NF-Y transcription factor families in the monocot model plant Brachypodium distachyon.

BACKGROUND: Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins. Using the dicot plant model system Arabidopsis thaliana (Arabidopsis), NF-Y were previously shown to control a variety of agronomically important traits, including drought tolerance, flowering time, and seed development. The aim of the current research was to identify and characterize NF-Y families in the emerging monocot model plant Brachypodium distachyon (Brachypodium) with the long term goal of assisting in the translation of known dicot NF-Y functions to the grasses. METHODOLOGY/PRINCIPAL FINDINGS: We identified, annotated, and further characterized 7 NF-YA, 17 NF-YB, and 12 NF-YC proteins in Brachypodium (BdNF-Y). By examining phylogenetic relationships, orthology predictions, and tissue-specific expression patterns for all 36 BdNF-Y, we proposed numerous examples of likely functional conservation between dicots and monocots. To test one of these orthology predictions, we demonstrated that a BdNF-YB with predicted orthology to Arabidopsis floral-promoting NF-Y proteins can rescue a late flowering Arabidopsis mutant. CONCLUSIONS/SIGNIFICANCE: The Brachypodium genome encodes a similar complement of NF-Y to other sequenced angiosperms. Information regarding NF-Y phylogenetic relationships, predicted orthologies, and expression patterns can facilitate their study in the grasses. The current data serves as an entry point for translating many NF-Y functions from dicots to the genetically tractable monocot model system Brachypodium. In turn, studies of NF-Y function in Brachypodium promise to be more readily translatable to the agriculturally important grasses.

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.