The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling.

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.

Gene Prioritization by Compressive Data Fusion and Chaining.

Data integration procedures combine heterogeneous data sets into predictive models, but they are limited to data explicitly related to the target object type, such as genes. Collage is a new data fusion approach to gene prioritization. It considers data sets of various association levels with the prediction task, utilizes collective matrix factorization to compress the data, and chaining to relate different object types contained in a data compendium. Collage prioritizes genes based on their similarity to several seed genes. We tested Collage by prioritizing bacterial response genes in Dictyostelium as a novel model system for prokaryote-eukaryote interactions. Using 4 seed genes and 14 data sets, only one of which was directly related to the bacterial response, Collage proposed 8 candidate genes that were readily validated as necessary for the response of Dictyostelium to Gram-negative bacteria. These findings establish Collage as a method for inferring biological knowledge from the integration of heterogeneous and coarsely related data sets.

The ABC transporter, AbcB3, mediates cAMP export in D. discoideum development.

Extracellular cAMP functions as a primary ligand for cell surface cAMP receptors throughout Dictyostelium discoideum development, controlling chemotaxis and morphogenesis. The developmental consequences of cAMP signaling and the metabolism of cAMP have been studied in great detail, but it has been unclear how cells export cAMP across the plasma membrane. Here we show pharmacologically and genetically that ABC transporters mediate cAMP export. Using an evolutionary-developmental biology approach, we identified several candidate abc genes and characterized one of them, abcB3, in more detail. Genetic and biochemical evidence suggest that AbcB3 is a component of the cAMP export mechanism in D. discoideum development.

Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.

Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR) causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.

Analysis of mutations that dissociate G(2) and essential S phase functions of human ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase.

ATR (ataxia telangiectasia-mutated and Rad3-related) contains 16 conserved candidate autophosphorylation sites that match its preferred S/TQ consensus. To determine whether any is functionally important, we mutated the 16 candidate residues to alanine in a single cDNA to create a 16A-ATR mutant. The 16A-ATR mutant maintains kinase and G(2) checkpoint activities. However, it fails to rescue the essential function of ATR in maintaining cell viability and fails to promote replication recovery from a transient exposure to replication stress. Further analysis identified T1566A/T1578A/T1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate that these residues occur in a region between ATR HEAT repeats 31R and 32R that aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Although this region is important for ATR function, the 3A-ATR residues do not appear to be sites of autophosphorylation. Nevertheless, our analysis identifies an important regulatory region of ATR that is shared among the PI3K-related protein kinase family. Furthermore, our data indicate that the essential function of ATR for cell viability is linked to its function in promoting proper replication in the context of replication stress and is independent of G(2) checkpoint activity.

Thr-1989 phosphorylation is a marker of active ataxia telangiectasia-mutated and Rad3-related (ATR) kinase.

The DNA damage response kinases ataxia telangiectasia-mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ataxia telangiectasia-mutated and Rad3-related (ATR) signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR Thr-1989 as a DNA damage-regulated phosphorylation site. Selective inhibition of ATR prevents Thr-1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to Thr-1989 provide a proximal marker of ATR activation.

ATR signalling: more than meeting at the fork.

Preservation of genome integrity via the DNA-damage response is critical to prevent disease. ATR (ataxia telangiectasia mutated- and Rad3-related) is essential for life and functions as a master regulator of the DNA-damage response, especially during DNA replication. ATR controls and co-ordinates DNA replication origin firing, replication fork stability, cell cycle checkpoints and DNA repair. Since its identification 15 years ago, a model of ATR activation and signalling has emerged that involves localization to sites of DNA damage and activation through protein-protein interactions. Recent research has added an increasingly detailed understanding of the canonical ATR pathway, and an appreciation that the canonical model does not fully capture the complexity of ATR regulation. In the present article, we review the ATR signalling process, focusing on mechanistic findings garnered from the identification of new ATR-interacting proteins and substrates. We discuss how to incorporate these new insights into a model of ATR regulation and point out the significant gaps in our understanding of this essential genome-maintenance pathway.

Temperature effects on particulate matter emissions from light-duty, gasoline-powered motor vehicles.

The Kansas City Light-Duty Vehicle Emissions Study (KCVES) measured exhaust emissions of regulated and unregulated pollutants from 496 vehicles recruited in the Kansas City metropolitan area in 2004 and 2005. Vehicle emissions testing occurred during the summer and winter, with the vehicles operated at ambient temperatures. One key component of this study was the investigation of the influence of ambient temperature on particulate matter (PM) emissions from gasoline-powered vehicles. A subset of the recruited vehicles were tested in both the summer and winter to further elucidate the effects of temperature on vehicle tailpipe emissions. The study results indicated that PM emissions increased exponentially as temperature decreased. In general, PM emissions doubled for every 20 degrees F drop in ambient temperature, with these increases independent of vehicle model year. The effects of temperature on vehicle emissions was most pronounced during the initial start-up of the vehicle (cold start phase) when the vehicle was still cold, leading to inefficient combustion, inefficient catalyst operation, and the potential for the vehicle to be operating under fuel-rich conditions. The large data set available from this study also allowed for the development of a model to describe temperature effects on PM emission rates due to changing ambient conditions. This study has been used as the foundation to develop PM emissions rates, and to model the impact of ambient temperature on these rates, for gasoline-powered vehicles in the EPA's new regulatory motor vehicle emissions model, MOVES.

SOSS1/2: Sensors of single-stranded DNA at a break.

In this issue of Molecular Cell, Huang et al. (2009) describe two heterotrimeric single-stranded DNA binding complexes, SOSS1 and SOSS2, that function downstream of the MRN complex to promote DNA repair and the G2/M checkpoint.

Dpb11 activates the Mec1-Ddc2 complex.

The Saccharomyces cerevisiae Mec1-Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1-Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1-Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1-Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans.

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis.

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.

Real-world vehicle emissions: A summary of the 15th coordinating research council on-road vehicle emissions workshop.

The Coordinating Research Council held its 15th workshop in April 2005, with nearly 90 presentations describing the most recent mobile source-related emissions research. In this paper, the authors summarize the presentations from researchers who are engaged in improving our understanding of the contribution of mobile sources to air quality. Participants in the workshop discussed emission models and emission inventories, results from gas- and particle-phase emissions studies from spark-ignition and diesel-powered vehicles (with an emphasis in this workshop on particle emissions), effects of fuels on emissions, evaluation of in-use emissions control programs, and efforts to improve our capabilities in performing on-board emissions measurements, as well as topics for future research.