Homologous NF-YC2 Subunit from Arabidopsis and Tobacco Is Activated by Photooxidative Stress and Induces Flowering

The transcription factor NF-Y consists of the three subunits A, B and C, which are encoded in Arabidopsis in large gene families. The multiplicity of the genes implies that NF-Y may act in diverse combinations of each subunit for the transcriptional control. We aimed to assign a function in stress response and plant development to NF-YC subunits by analyzing the expression of NF-Y genes and exploitation of nf-y mutants. Among the subunit family, NF-YC2 showed the strongest inducibility towards oxidative stress, e.g. photodynamic, light, oxidative, heat and drought stress. A tobacco NF-YC homologous gene was found to be inducible by photooxidative stress generated by an accumulation of the tetrapyrrole metabolite, coproporphyrin. Despite the stress induction, an Arabidopsis nf-yc2 mutant and NF-YC2 overexpressors did not show phenotypical differences compared to wild-type seedlings in response to photooxidative stress. This can be explained by the compensatory potential of other members of the NF-YC family. However, NF-YC2 overexpression leads to an early flowering phenotype that is correlated with increased FLOWERING LOCUS T-transcript levels. It is proposed that NF-YC2 functions in floral induction and is a candidate gene among the NF-Y family for the transcriptional activation upon oxidative stress

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

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

Effect of Secondary Echo Signals in Spin-Systems with a Large Inhomogeneous Broadening of NMR Line

The possibility of comparatively simple and fast determination of characteristic relaxation parameters T1, T2 and T3 for nuclear spin-systems with strong Larmor and Rabi inhomogeneous broadenings of NMR lines using the secondary echo signal effect was experimentally shown. Resides, this method gives opportunity to obtain a valuable infomation on the inhomogeneous NMR broadening which reflects the character of magnetic field microscopic destribution in such systems, as example, multidomain magnetics and superconductors.Comment: 12 pages, 5 figure

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

PwHAP5, a CCAAT-binding transcription factor, interacts with PwFKBP12 and plays a role in pollen tube growth orientation in Picea wilsonii

The HAP complex occurs in many eukaryotic organisms and is involved in multiple physiological processes. Here it was found that in Picea wilsonii, HAP5 (PwHAP5), a putative CCAAT-binding transcription factor gene, is involved in pollen tube development and control of tube orientation. Quantitative real-time reverse transcription-PCR showed that PwHAP5 transcripts were expressed strongly in germinating pollen and could be induced by Ca2+. Overexpression of PwHAP5 in pollen altered pollen tube orientation, whereas the tube with PwHAP5RNAi showed normal growth without diminishing pollen tube growth. Furthermore, PwFKBP12, which encodes an FK506-binding protein (FKBP) was screened and a bimolecular fluorescence complementation assay performed to confirm the interaction of PwHAP5 and PwFKBP12 in vivo. Transient expression of PwFKBP12 in pollen showed normal pollen tube growth, whereas the tube with PwFKBP12RNAi bent. The phenotype of overexpression of HAP5 on pollen tube was restored by FKBP12. Altogether, our study supported the role of HAP5 in pollen tube development and orientation regulation and identified FKBP12 as a novel partner to interact with HAP5 involved in the process

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

Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

BACKGROUND: North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. METHODS: We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop) from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. RESULTS: Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55%) tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. CONCLUSIONS: Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies

Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions