Roderick W. Kumimoto

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.

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.