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Preparing students to explore, understand, and resolve societal challenges such as global climate change is an important task for evolutionary and ecological biologists that will require novel and innovative pedagogical approaches. Recent calls to reform undergraduate science education emphasize the importance of engaging students in inquiry-driven, active, and authentic learning experiences. We believe that the vast digital resources (i.e., “big data”) associated with natural history collections provide invaluable but underutilized opportunities to create such experiences for undergraduates in biology. Here, we describe an online, open-access educational module that we have developed that harnesses the power of collections-based information to introduce students to multiple conceptual and analytical elements of climate change, evolutionary, and ecological biology research. The module builds upon natural history specimens and data collected over the span of nearly a century in Yosemite National Park, California, to guide students through a series of exercises aimed at testing hypotheses regarding observed differences in response to climate change by two closely related and partially co-occurring species of chipmunks. The content of the module can readily be modified to meet the pedagogical goals and instructional levels of different courses while the analytical strategies outlined can be adapted to address a wide array of questions in evolutionary and ecological biology. In sum, we believe that specimen-based natural history data represent a powerful platform for reforming undergraduate instruction in biology. Because these efforts will result in citizens who are better prepared to understand complex biological relationships, the benefits of this approach to undergraduate education will have widespread benefits to society.

Research has shown the highest level of understanding in the geosciences to be the ability to think about the Earth as a dynamic system. The Earth system contains four spheres: the geosphere, the hydrosphere, the biosphere, and the atmosphere. These spheres are linked through the biogeochemical cycles that move matter and energy through the various parts of the Earth system. This study aims to fill a gap in the literature by examining how undergraduate students perceive fluxes and reservoirs of important elements within the Earth system: namely carbon, nitrogen, and phosphorus. Through interviews, concept drawings, and surveys, undergraduate students' conceptions and alternate conceptions about the Earth System and biogeochemical cycles were collected. This study revealed that undergraduate students in both STEM and non-STEM fields tend to hold a "bio-centric" view of the carbon cycle and have more limited conceptions in terms of detail and breadth of the nitrogen and phosphorous cycles. Students who took more STEM courses and were in more interdisciplinary fields tended to have more nuanced (though not necessarily complete) conceptions of these cycles. Implications for this study involve the improvement of teaching biogeochemical concepts across disciplines, but also inform our knowledge about using these cycles in the context of systems thinking. This work also provides a baseline for future work on developing learning progressions for biogeochemical cycles and complex Earth systems and in assessing systems thinking abilities through student knowledge of biogeochemical cycles.

This study measured student engagement in real time through the use of skin biosensors, specifically galvanic skin response (GSR), in a large undergraduate lecture classroom. The study was conducted during an intervention in an introductory-level biology course (N = 420) in which one section of the course was taught with active-learning approaches and the other with traditional didactic teaching. GSR results were aligned and correlated with the Classroom Observation Protocol for Undergraduate STEM [science, technology, engineering, and mathematics], or COPUS, and student self-reflections on their own engagement. Results showed that the active-learning section spent more time working in groups, resulting in GSR measures that trended higher and self-reported engagement, while showing indications of higher content learning gains compared with the traditional lecture section. Comparisons between COPUS scores and GSR readings indicate that engagement increased during group work and decreased during listening activities. Throughout a class period, GSR activity of the active-learning group showed increased trends compared with baseline measures, while the traditional lecture group showed decreased trends. Results indicate that GSR is a promising measure of real-time student engagement in the undergraduate classroom, bringing a new technique to discipline-based education researchers who aim to better measure student engagement; however, some limitations exist for broad-scale implementation.

Youth-led participatory action research (YPAR) has been documented to increase youth's social, emotional, and cognitive outcomes, foster critical thinking and academic success, and encourage civic participation. YPAR allows for young to develop their voices, direct their own learning and act as change agents as they take steps to influence their community's civic health. This article offers an account of one urban, public high school's effort to integrate YPAR as a year-long project for all enrolled 9th graders. Drawing on the work of 100 students who participated in this year-long project, this inquiry examines the topic identification phase of the school's first-ever enactment of YPAR as part of the everyday curriculum. Specific attention is paid to understanding the issues and questions that surfaced during the topic identification stage of the action research process within a formalized school setting. This work has implications for teachers and school leaders interested in adopting YPAR as part of the school-sanctioned curriculum for all students.

Our world is in the midst of unprecedented change-climate shifts and sustained, widespread habitat degradation have led to dramatic declines in biodiversity rivaling historical extinction events. At the same time, new approaches to publishing and integrating previously disconnected data resources promise to help provide the evidence needed for more efficient and effective conservation and management. Stakeholders have invested considerable resources to contribute to online databases of species occurrences. However, estimates suggest that only 10% of biocollections are available in digital form. The biocollections community must therefore continue to promote digitization efforts, which in part requires demonstrating compelling applications of the data. Our overarching goal is therefore to determine trends in use of mobilized species occurrence data since 2010, as online systems have grown and now provide over one billion records. To do this, we characterized 501 papers that use openly accessible biodiversity databases. Our standardized tagging protocol was based on key topics of interest, including: database(s) used, taxa addressed, general uses of data, other data types linked to species occurrence data, and data quality issues addressed. We found that the most common uses of online biodiversity databases have been to estimate species distribution and richness, to outline data compilation and publication, and to assist in developing species checklists or describing new species. Only 69% of papers in our dataset addressed one or more aspects of data quality, which is low considering common errors and biases known to exist in opportunistic datasets. Globally, we find that biodiversity databases are still in the initial stages of data compilation. Novel and integrative applications are restricted to certain taxonomic groups and regions with higher numbers of quality records. Continued data digitization, publication, enhancement, and quality control efforts are necessary to make biodiversity science more efficient and relevant in our fast-changing environment.

Behavioral economic theory suggests that increased engagement in constructive, substance-free activities that are in the service of long-term goals (e.g., college graduation, career development, health) can decrease alcohol use and related problems. However, engaging in activities such as these in the high-risk college environment requires the ability to self-regulate by avoiding rewarding but risky behaviors (e.g., drinking) while also effectively organizing behavior in the pursuit of delayed academic and career-related rewards. The current secondary data analyses evaluated self-regulation as a potential mechanism of behavior change in an alcohol intervention trial that compared a standard alcohol-focused brief motivational intervention (BMI) plus a behavioral economic substance-free activity session (SFAS) with an alcohol BMI plus relaxation training (reaction time [RT]) session (Murphy et al., 2012). Participants were 82 first-year undergraduate students (50% women; Mage = 18.5, SD = .71) who reported 2 or more past-month heavy drinking episodes. After completing a baseline assessment and an individual alcohol-focused BMI, participants were randomized to either the SFAS or the RT session. The BMI + SFAS condition reported greater mean self-regulation at 1 month compared with BMI + RT. Furthermore, self-regulation at 1 month significantly mediated the relation between condition and alcohol-related outcomes at 6-month follow-up. Although preliminary, these results suggest that brief behavioral economic intervention elements that an attempt to increase future goal pursuit and substance-free activities can enhance the short-term efficacy of standard alcohol BMIs and that this effect may be due in part to increases in self-regulation. (PsycINFO Database Record

Increasing our understanding of Earth's biodiversity and responsibly stewarding its resources are among the most crucial scientific and social challenges of the new millennium. These challenges require fundamental new knowledge of the organization, evolution, functions, and interactions among millions of the planet's organisms. Herein, we present a perspective on the Earth BioGenome Project (EBP), a moonshot for biology that aims to sequence, catalog, and characterize the genomes of all of Earth's eukaryotic biodiversity over a period of 10 years. The outcomes of the EBP will inform a broad range of major issues facing humanity, such as the impact of climate change on biodiversity, the conservation of endangered species and ecosystems, and the preservation and enhancement of ecosystem services. We describe hurdles that the project faces, including data-sharing policies that ensure a permanent, freely available resource for future scientific discovery while respecting access and benefit sharing guidelines of the Nagoya Protocol. We also describe scientific and organizational challenges in executing such an ambitious project, and the structure proposed to achieve the project's goals. The far-reaching potential benefits of creating an open digital repository of genomic information for life on Earth can be realized only by a coordinated international effort.

Symptoms of posttraumatic stress (PTS) show significant comorbidity with alcohol use, but little is known about the mechanisms that might account for this comorbidity. Deficits in reward functioning have long been implicated in alcohol misuse and more recently in PTS reactions, but no study has examined whether reward deprivation may serve as a transdiagnostic risk factor for comorbid PTS-alcohol misuse. The current cross-sectional study sought to test the behavioral economic hypothesis that reward deprivation would be related to both PTS symptoms and alcohol problems, and would mediate the relation between PTS symptoms and alcohol problems in college students. We recruited a diverse sample of urban college students (N = 203, Mage = 21.5 years, SD = 5.5; 79.5% female; 56.8% White, 28.1% Black, .9% Asian, 9.8% Multiracial) who endorsed both alcohol use and PTS symptoms. Reward deprivation (lack of access to, and ability to, experience reward) was related to alcohol problems, and a lack of access to reward was related to PTS symptoms. Furthermore, reward access mediated the relation between PTS symptoms and alcohol problems and craving, after controlling for alcohol use, age, gender, and race. These data provide preliminary support for behavioral economic models of alcohol comorbidity and suggest that treatments for combined PTS and alcohol misuse should attempt to reduce barriers to accessing natural rewards. (PsycINFO Database Record

Premise of the Study:Herbarium specimens provide a robust record of historical plant phenology (the timing of seasonal events such as flowering or fruiting). However, the difficulty of aggregating phenological data from specimens arises from a lack of standardized scoring methods and definitions for phenological states across the collections community. Methods and Results:To address this problem, we report on a consensus reached by an iDigBio working group of curators, researchers, and data standards experts regarding an efficient scoring protocol and a data-sharing protocol for reproductive traits available from herbarium specimens of seed plants. The phenological data sets generated can be shared via Darwin Core Archives using the Extended MeasurementOrFact extension. Conclusions:Our hope is that curators and others interested in collecting phenological trait data from specimens will use the recommendations presented here in current and future scoring efforts. New tools for scoring specimens are reviewed.

College student drinking is a major public health concern and can result in a range of negative consequences, from acute health risks to decreased academic performance and drop out. Harm reduction interventions have been developed to reduce problems associated with drinking but there is a need to identify specific risk/protective factors related to academic performance among college drinkers. Behavioral economics suggests that chronic alcohol misuse reflects a dysregulated behavioral process or reinforcer pathology-alcohol is overvalued and the value of prosocial rewards are sharply discounted due, in part, to their delay. This study examined delay discounting, consideration of future consequences (CFC) and protective behavioral strategies (PBS) as predictors of academic success (grade point average; GPA) and engagement (time devoted to academic activities) among 393 college drinkers (61% female). In multivariate models, PBS were associated with greater academic engagement, but were not with academic success. Lower discounting of delayed rewards and greater CFC were associated with both academic success and engagement among drinkers. Previous research suggests that future time orientation is malleable, and the current results provide support for efforts to enhance future time orientation as part of alcohol harm-reduction approaches. (PsycINFO Database Record

The timing of phenological events, such as leaf-out and flowering, strongly influence plant success and their study is vital to understanding how plants will respond to climate change. Phenological research, however, is often limited by the temporal, geographic, or phylogenetic scope of available data. Hundreds of millions of plant specimens in herbaria worldwide offer a potential solution to this problem, especially as digitization efforts drastically improve access to collections. Herbarium specimens represent snapshots of phenological events and have been reliably used to characterize phenological responses to climate. We review the current state of herbarium-based phenological research, identify potential biases and limitations in the collection, digitization, and interpretation of specimen data, and discuss future opportunities for phenological investigations using herbarium specimens.

Iron is the most abundant transition metal in the Earth's crust, and naturally occurring iron oxide minerals play a commanding role in environmental redox reactions. Although iron oxide redox reactions are well-studied, their precise mechanisms are not fully understood. Recent work has shown that these involve electron transfer pathways within the solid, suggesting that overall reaction rates could be dependent upon electron mobility. Initial ultrafast spectroscopy studies of iron oxide nanoparticles sensitized by fluorescein derivatives supported a model for electron mobility based on polaronic hopping of electron charge carriers between iron sites, but the constitutive relationships between hopping mobilities and interfacial charge transfer processes has remained obscured. We developed a coarse-grained lattice Monte Carlo model to simulate the collective mobilities and lifetimes of these photoinjected electrons with respect to recombination with adsorbed dye molecules for essential nanophase ferrihydrite and tested predictions made by the simulations using pump-probe spectroscopy. We acquired optical transient absorption spectra as a function of the particle size and under a variety of solution conditions and used cryogenic transmission electron microscopy to determine the aggregation state of the nanoparticles. We observed biphasic electron recombination kinetics over time scales that spanned from picoseconds to microseconds, the slower regime of which was fit with a stretched exponential decay function. The recombination rates were weakly affected by the nanoparticle size and aggregation state, suspension pH, and injection of multiple electrons per nanoparticle. We conclude that electron mobility indeed limits the rate of interfacial electron transfer in these systems, with the slowest processes relating to escape from deep traps, the presence of which outweighs the influence of environmental factors, such as pH-dependent surface charge.

Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants.

We developed and tested different pedagogical treatments using an Augmented Reality (AR) Sandbox to teach introductory geoscience students about reading topographic maps at five institutions in both pilot and full implementation studies. The AR Sandbox treatments were characterized as 1) unstructured play, 2) a semi-structured lesson, and 3) a structured lesson. The success of each was contrasted with the control condition of a traditional topographic map lab without the AR Sandbox. Students completed a subset of questions from the Topographic Maps Assessment (TMA) and a series of mental rotation questions post-implementation. No significant differences were found on TMA post-test scores between groups who used the unstructured Sandbox play treatment compared to the control condition. Semi-structured and structured lesson formats similarly failed to produce a statistically significant difference on the TMA post-test. This indicates that no single treatment worked universally better than another. However, regression analysis showed two factors significantly predicted performance on the TMA, including spatial performance and self-assessed knowledge (or confidence) of topographic maps. Of the groups that used the Sandbox, students with low and high scores on the mental rotation test performed best on the TMA following the structured treatment.

Augmented reality (AR) sandboxes are useful tools in helping students develop a three-dimensional understanding of topographic maps and landforms. Research on the AR sandbox indicates that students enjoy it, but evidence about student learning gains is inconclusive. This study aims to understand how students engage with these tools as a precedent to understanding learning gains. Technology in the form of skin biosensors allows researchers to measure skin conductance (or electrodermal activity -- EDA) as a proxy for student engagement. This study used wrist biosensors to monitor the engagement of students exposed to different instructional treatments of an AR sandbox in an introductory lab course. The data were supplemented by dynamic video of the students interacting with the sandbox. Statistical analysis of the EDA data suggests that the use of an AR sandbox, particularly during structured lab activities, increases student engagement. Regression modeling supports a relationship between spatial reasoning skills. Based on video data, students' collaborative and social interactions also showed a significant relationship with EDA. These results suggest that instructors focus on developing and using structured and collaborative activities when incorporating AR sandboxes in their courses in order to best engage students.

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.

BackgroundReward deprivation has been implicated in major depressive disorder and severe substance abuse, but its potential relation to alcohol use disorder (AUD) symptoms in non-treatment-seeking young adult drinkers is less clear. Depression is often comorbid with alcohol misuse, so relations of AUD with reward deprivation might be due in part to the presence of depressive symptoms in young adults. Behavioral economic theory views addiction as a state that is related in part to deficits in drug-free rewards, and therefore requires an investigation into whether reward deprivation has a direct relation to alcohol misuse that is, at least partially, independent of mood.MethodsThis study evaluates the contribution of 2 facets of reward deprivation (reward availability and experience) to alcohol use, AUD symptoms, and depression in a sample of young adult heavy episodic drinkers. Data were collected from 392 undergraduates (60.4% female, 85.1% Caucasian) who reported recent heavy drinking (83.7% with at least 1 AUD symptom).ResultsLow reward availability (environmental suppression) was significantly associated with both DSM-5 AUD symptoms and alcohol-related problems after controlling for age, gender, depressive symptomatology, and drinking level.ConclusionsThis study provides support for behavioral economic models that emphasize reward deprivation as a unique risk factor for AUD that is independent of mood and drinking level. Limited access to natural rewards may be a risk and/or maintaining factor for AUD symptoms in college student drinkers.

BackgroundThe ATP-binding cassette (ABC) transporter gene superfamily is ubiquitous among extant organisms and prominently represented in plants. ABC transporters act to transport compounds across cellular membranes and are involved in a diverse range of biological processes. Thus, the applicability to biotechnology is vast, including cancer resistance in humans, drug resistance among vertebrates, and herbicide and other xenobiotic resistance in plants. In addition, plants appear to harbor the highest diversity of ABC transporter genes compared with any other group of organisms. This study applied transcriptome analysis to survey the kingdom-wide ABC transporter diversity in plants and suggest biotechnology applications of this diversity.ResultsWe utilized sequence similarity-based informatics techniques to infer the identity of ABC transporter gene candidates from 1295 phylogenetically-diverse plant transcriptomes. A total of 97,149 putative (approximately 25 % were full-length) ABC transporter gene members were identified; each RNA-Seq library (plant sample) had 88 ± 30 gene members. As expected, simpler organisms, such as algae, had fewer unique members than vascular land plants. Differences were also noted in the richness of certain ABC transporter subfamilies. Land plants had more unique ABCB, ABCC, and ABCG transporter gene members on average (p

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.

Iron oxide nanoparticles are present throughout the Earth and undergo mineral phase transformations that affect their stability and reactivity. The formation of the iron oxide hematite from a 2-line ferrihydrite (2lnFh) precursor requires both phase transformation and growth. Whether phase transformation occurs before or after substantial particle growth, and by which mechanisms particles grow, remain unclear. We conducted time-resolved studies employing X-ray diffraction, room temperature and cryogenic transmission electron microscopy, preferential dissolution by oxalate buffer, and low temperature SQuID magnetometry to investigate the kinetics and mechanism of hematite formation from 2lnFh. A novel form of magnetic measurement was found to be exquisitely sensitive to the presence of hematite, detecting its formation at far lower concentrations than possible with X-ray diffraction. These results indicate that small hematite domains were present even in as-prepared 2lnFh suspensions as a side-product of 2lnFh synthesis and that the hematite domains increased in size and crystallinity with aging time at elevated temperatures. Second-order kinetics reveals that the hematite growth is consistent with a particle-mediated growth mechanism, possibly oriented attachment.

The reaction of peroxides with peroxidases oxidizes the heme iron from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical. X-ray-generated hydrated electrons rapidly reduce Fe(IV), thereby requiring very short exposures using many crystals, and, even then, some reduction cannot be avoided. The new generation of X-ray free electron lasers capable of generating intense X-rays on the tenths of femtosecond time scale enables structure determination with no reduction or X-ray damage. Here, we report the 1.5-Å crystal structure of cytochrome c peroxidase (CCP) compound I (CmpI) using data obtained with the Stanford Linear Coherent Light Source (LCLS). This structure is consistent with previous structures. Of particular importance is the active site water structure that can mediate the proton transfer reactions required for both CmpI formation and reduction of Fe(IV)=O to Fe(III)-OH. The structures indicate that a water molecule is ideally positioned to shuttle protons between an iron-linked oxygen and the active site catalytic His. We therefore have carried out both computational and kinetic studies to probe the reduction of Fe(IV)=O. Kinetic solvent isotope experiments show that the transfer of a single proton is critical in the peroxidase rate-limiting step, which is very likely the proton-coupled reduction of Fe(IV)=O to Fe(III)-OH. We also find that the pKa of the catalytic His substantially increases in CmpI, indicating that this active site His is the source of the proton required in the reduction of Fe(IV)=O to Fe(IV)-OH.

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (

Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

Amborella trichopoda is strongly supported as the single living species of the sister lineage to all other extant flowering plants, providing a unique reference for inferring the genome content and structure of the most recent common ancestor (MRCA) of living angiosperms. Sequencing the Amborella genome, we identified an ancient genome duplication predating angiosperm diversification, without evidence of subsequent, lineage-specific genome duplications. Comparisons between Amborella and other angiosperms facilitated reconstruction of the ancestral angiosperm gene content and gene order in the MRCA of core eudicots. We identify new gene families, gene duplications, and floral protein-protein interactions that first appeared in the ancestral angiosperm. Transposable elements in Amborella are ancient and highly divergent, with no recent transposon radiations. Population genomic analysis across Amborella's native range in New Caledonia reveals a recent genetic bottleneck and geographic structure with conservation implications.

Combining quantitative genetics studies with metabolomics/metabolic profiling platforms, genomics, and transcriptomics is creating significant progress in identifying the causal genes controlling natural variation in metabolite accumulations and profiles. In this review, we discuss key mechanistic and evolutionary insights that are arising from these studies. This includes the potential role of transport and other processes in leading to a separation of the site of mechanistic causation and metabolic consequence. A reilluminated observation is the potential for genomic variation in the organelle to alter phenotypic variation alone and in epistatic interaction with the nuclear genetic variation. These studies are also highlighting new aspects of metabolic pleiotropy both in terms of the breadth of loci altering metabolic variation as well as the potential for broader effects on plant defense regulation of the metabolic variation than has previously been predicted. We also illustrate caveats that can be overlooked when translating quantitative genetics descriptors such as heritability and per-locus r(2) to mechanistic or evolutionary interpretations.

Scientific modeling and systems thinking (SMST) is central to the geosciences, yet few studies have documented how and to what extent undergraduate geoscience courses emphasize SMST, as well as factors that might help explain or predict these trends. Here, we present research based on data (n = 2056) from the most recent (2016) administration of the National Geoscience Faculty Survey, administered to a national sample of postsecondary geoscience instructors in the United States. We investigated instructor- and course-related variables as they relate to a set of 9 survey items that serve as a composite measure for SMST. Significant variation was observed in reported frequencies of individual SMST practices in undergraduate geoscience courses. The highest levels of reported SMST were associated with faculty from atmospheric and environmental sciences, those who emphasized research-based, student-centered pedagogical practices, those who recently made changes to both course content and teaching methods, and those who reported high levels of engagement in instructional improvement activities (workshops, presentations, seminars). Reported SMST practices were similar for faculty identifying as geoscientists and geoscience education researchers, and both were significantly higher than for teaching-focused faculty who do not conduct research. A linear regression model including variables found to be significant in the analyses was able to predict 17% of the overall variance in reported SMST practices. These findings illustrate the importance of instructors' disciplinary orientation and active engagement in instructional innovation as related to SMST, and provide important points of impact for enhancing SMST in undergraduate geoscience courses through course design and faculty development. However, the relatively modest predictive power of the regression model indicates there are many other factors influencing SMST that warrant future research.

Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) has been previously linked to its catalytic function, but the extent to which the different conformations of these residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature synchrotron crystallography and fixed-target X-ray free-electron laser (XFEL) crystallography. The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and above, yet others remain populated or become populated at 180 K and below. These results point to a complex evolution of conformational heterogeneity between 180-–240 K that involves both thermal deactivation and solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in which exchange between rotamer states of a large aromatic ring in the middle of the network shifts the conformational ensemble for the other residues in the network. Together, our multitemperature analyses and XFEL data motivate a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (

How standing genetic variation within a pathogen contributes to diversity in host/pathogen interactions is poorly understood, partly because most studied pathogens are host-specific, clonally reproducing organisms which complicates genetic analysis. In contrast, Botrytis cinerea is a sexually reproducing, true haploid ascomycete that can infect a wide range of diverse plant hosts. While previous work had shown significant genomic variation between two isolates, we proceeded to assess the level and frequency of standing variation in a population of B. cinerea. To begin measuring standing genetic variation in B. cinerea, we re-sequenced the genomes of 13 different isolates and aligned them to the previously sequenced T4 reference genome. In addition one of these isolates was resequenced from four independently repeated cultures. A high level of genetic diversity was found within the 13 isolates. Within this variation, we could identify clusters of genes with major effect polymorphisms, i.e., polymorphisms that lead to a predicted functional knockout, that surrounded genes involved in controlling vegetative incompatibility. The genotype at these loci was able to partially predict the interaction of these isolates in vegetative fusion assays showing that these loci control vegetative incompatibility. This suggests that the vegetative incompatibility loci within B. cinerea are associated with regions of increased genetic diversity. The genome re-sequencing of four clones from the one isolate (Grape) that had been independently propagated over 10 years showed no detectable spontaneous mutation. This suggests that B. cinerea does not display an elevated spontaneous mutation rate. Future work will allow us to test if, and how, this diversity may be contributing to the pathogen's broad host range.

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.