MOLECULAR CHARACTERIZATION OF THE ARABIDOPSIS NUCLEAR FACTOR-Y TRANSCRIPTION FACTOR FAMILY

NUCLEAR FACTOR-Y (NF-Y) transcription factors, composed of three independent families: NF-YA, NF-YB, and NF-YC, have greatly expanded in plants in comparison to animals. For example, while humans have only one member of each subunit, the modal plant Arabidopsis thaliana (Arabidopsis) has 10 members. However, due to lack of studies, the significance of the expansion in the plant lineage is poorly understood. Plant NF-Ys have primarily been studied on their role regulating flowering, abscisic acid (ABA) responses, embryogenesis, and nodulation in legumes. However, key questions remain on how the NF-Y regulates these processes. Here I answer two fundamental questions about the NF-YA subunit during regulation of ABA responses and flowering. ABA mediated seed germination is one of the plant developmental responses regulated by the NF-Y. Three NF-YC subunits, NF-YC3, NF-YC4, and NF-YC9 have opposing responses during abscisic acid (ABA) mediated seed germination, demonstrating that members of this closely related gene family have evolved unique regulatory roles. Since the mature NF-Y complex binds DNA as a trimer I hypothesized that the NF-YA and NF-YB family members should also have opposing regulatory functions. However, opposing functions have not been identified for the NF-YA or NF-YB. In Chapter 2, I studied the germination responses of all 10 Arabidopsis NF-YA genes by creating overexpression constructs. Germination responses on ABA containing media showed that the closely related paralogs NF-YA1 and NF-YA9 are insensitive and NF-YA2, NF-YA4, NF-YA7, NF-YA8, and NF-YA10 were hypersensitive to ABA. The result supported my hypothesis and show that the closely related NF-YA family members have evolved opposing roles regulating ABA mediated seed germination. The NF-Ys have been extensively studied during its regulation of photoperiod dependent flowering. NF-YB and NF-YC subunits are known positive regulators of this pathway, however the role of NF-YA subunits remained a fundamental question that had not been answered. Prior research had proposed a model where NF-YA acts as negative regulators of flowering. However, several recent publications demonstrated that NF-YA should be acting as positive regulators. In Chapter 3, I tried to understand the role played by NF-YA during floral regulation. My hypothesis was that NF-YAs are positive regulators of flowering. Since loss of function NF-YA are lethal or do not have flowering phenotypes, I used two approaches to test if NF-YA are positive regulators. First, to indirectly test if NF-YAs are need for the NF-YB/NF-YC dimer to regulate flowering, I created a mutant version of NF-YB that loses interaction with NF-YA. The mutant was overexpressed in the late flowering nf-yb2 nf-yb3 double mutant and was found not to complement the late flowering phenotype. However, the mutant protein was strongly expressed and was able to localize to the nucleus. This result strongly indicated that NF-YA should be required as positive regulators of flowering. My second approach was to study overexpressors of NF-YA. Here I was able to identify that NF-YA2 overexpressors drove early flowering and led to the upregulation of a key floral gene FT. These results strongly suggested that NF-YA2 might be a positive regulator of flowering. Further, a recent publication had demonstrated that CO provides an activation domain for the NF-Y complex. The NF-Ys were able to induce flowering in the absence of CO (in a co mutant background) when an activation domain called EDLL was attached to NF-YB2. I hypothesized that the NF-YB2 mutant that loses interaction with NF-YA will not be able to induce flowering, however NF-YA2 will be able to induce flowering in the absence of CO when attached to the EDLL domain. The results supported the hypothesis. As a conclusion, my data strongly suggests that NF-YAs are required as positive regulators of flowering. In addition to studying the roles played during seed germination and flowering, I also studied protein interactions of the NF-Y and how the NF-Ys are regulated. In Chapter 4, I studied the protein-protein interactions of NF-YC9 and its targets. A combination of deletions and point mutations were used to understand how NF-YC9 interacts with its targets. The results demonstrated that while the conserved domain is required for protein function, the non-conserved regions are also necessary for the interaction between NF-YC9 and most of its targets. In Chapter 6, I have presented a collection of yeast 2-hybrid (Y2H) experiments done to better understand the nature of NF-Y protein interactions. This chapter demonstrated the NF-Ys are able to interact with a variety of other plant proteins including transcription factors. It also demonstrated inter-species protein interactions in plants. In Chapter 5, I studied the regulation of NF-YA transcript and protein. Here I was able to show that NF-YA family transcripts and proteins are regulated by light. In conclusion this dissertation added to the literature on NF-Y in several aspects; 1) opposing role for the NF-Y were identified during seed germination, 2) NF-YA were strongly suggested to be positive regulators of flowering, 3) protein interactions of the NF-Y were dissected through Y2H analysis, 4) NF-YA transcripts and proteins were shown to be regulated by light

NUCLEAR FACTOR Y Transcription Factors Have Both Opposing and Additive Roles in ABA-Mediated Seed Germination

The authors thank Bing Zhang and Taylor Fore for use of the Zeiss AxioImager microscope and technical assistance. We additionally thank Krishna Suthar and Ashley Robbins for technical support in the lab.Conceived and designed the experiments: BFH RWK. Performed the experiments: RWK CLS KKG JRR NS. Analyzed the data: RWK BFH. Wrote the paper: RWK BFH.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.Yeshttp://www.plosone.org/static/editorial#pee

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.This work was funded by the National Science Foundation (US, http://www.nsf.gov/) award 1149822 to BFH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ye

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

Regulation of High-Temperature Stress Response by Small RNAs

Temperature extremes constitute one of the most common environmental stresses that adversely affect the growth and development of plants. Transcriptional regulation of temperature stress responses, particularly involving protein-coding gene networks, has been intensively studied in recent years. High-throughput sequencing technologies enabled the detection of a great number of small RNAs that have been found to change during and following temperature stress. The precise molecular action of some of these has been elucidated in detail. In the present chapter, we summarize the current understanding of small RNA-mediated modulation of high- temperature stress-regulatory pathways including basal stress responses, acclimation, and thermo-memory. We gather evidence that suggests that small RNA network changes, involving multiple upregulated and downregulated small RNAs, balance the trade-off between growth/development and stress responses, in order to ensure successful adaptation. We highlight specific characteristics of small RNA-based tem- perature stress regulation in crop plants. Finally, we explore the perspectives of the use of small RNAs in breeding to improve stress tolerance, which may be relevant for agriculture in the near future

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

We thank Dr. Ben Smith (University of Oklahoma) for assistance with FLIM-FRET measurements and Dr. Min Ni (University of Minnesota) for critical reading of the manuscript. The cop1-4 mutant allele and cop1-4 co-9 cross were kindly provided by George Coupland (Max Planck Institute).Author Summary Light perception is critically important for the fitness of plants in both natural and agricultural settings. Plants not only use light for photosynthesis, but also as a cue for proper development. As a seedling emerges from soil it must determine the light environment and adopt an appropriate growth habit. When blue and red wavelengths are the dominant sources of light, plants will undergo photomorphogenesis. Photomorphogenesis describes a number of developmental responses initiated by light in a seedling, and includes shortened stems and establishing the ability to photosynthesize. The genes regulating photomorphogenesis have been studied extensively, but a complete picture remains elusive. Here we describe the finding that NUCLEAR FACTOR-Y (NF-Y) genes are positive regulators of photomorphogenesis—i.e., in plants where NF-Y genes are mutated, they display some characteristics of dark grown plants, even though they are in the light. Our data suggests that the roles of NF-Y genes in light perception do not fit in easily with those of other described pathways. Thus, studying these genes promises to help develop a more complete picture of how light drives plant development.Yeshttp://www.plosgenetics.org/static/editorial#pee

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

Abstract 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.</p

NF-YC3, NF-YC4 and NF-YC9 protein-protein interaction network.

<p>Both demonstrated and GeneMANIA predicted protein-protein interaction data for NF-YC3, NF-YC4, and NF-YC9 were visualized using Cytoscape <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Cline1" target="_blank">[46]</a>. Predicted physical interactions are depicted as dashed lines, while demonstrated interactions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Wenkel1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Kumimoto2" target="_blank">[16]</a> and this work) are depicted as solid lines. Input nodes NF-YC3, NF-YC4 and NF-YC9 are shown as squares. Circle nodes are those predicted data from GeneMANIA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-WardeFarley1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Mostafavi1" target="_blank">[45]</a>. Octagonal nodes represent demonstrated physical interactions (e.g., Y2H, some shown in this report - see below). Related protein nodes are color coded as follows: red-bZIP; blue – CCT; green - H2A; orange/tan - NF-YB; yellow – NF-YA; gray – unclassified interacting proteins. A Microsoft Excel file is available to recreate/manipulate this data (File S1). Common names were used where available - File S1 contains all AGI numbers and references for sources of data.</p

Misregulated genes in the <i>nf-yc triple</i> mutant have ABRE-like promoter elements.

<p>A) ABRE-like motif discovered through MEME analysis. B) Positional distribution of MEME motif within the promoter set. TSS - transcriptional start site. To help assess the relationships between Arabidopsis NF-Y proteins discussed here and below, note that phylogenetic trees were previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Siefers1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Gusmaroli1" target="_blank">[76]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059481#pone.0059481-Gusmaroli2" target="_blank">[77]</a>.</p