The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield

We have increased the productivity and yield of potato (Solanum tuberosum) by developing a novel method to enhance photosynthetic carbon fixation based on expression of a polyprotein (DEFp) comprising all three subunits (D, E and F) of Escherichia coli glycolate dehydrogenase (GlcDH). The engineered polyprotein retained the functionality of the native GlcDH complex when expressed in E. coli and was able to complement mutants deficient for the D, E and F subunits. Transgenic plants accumulated DEFp in the plastids, and the recombinant protein was active in planta, reducing photorespiration and improving CO₂ uptake with a significant impact on carbon metabolism. Transgenic lines with the highest DEFp levels and GlcDH activity produced significantly higher levels of glucose (5.8‐fold), fructose (3.8‐fold), sucrose (1.6‐fold) and transitory starch (threefold), resulting in a substantial increase in shoot and leaf biomass. The higher carbohydrate levels produced in potato leaves were utilized by the sink capacity of the tubers, increasing the tuber yield by 2.3‐fold. This novel approach therefore has the potential to increase the biomass and yield of diverse crops.     

On the mechanism of protein fold‐switching by a molecular sensor

Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild‐type fold (N) to a circularly‐permuted fold (N′), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order–disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D_9k. A simple three‐state model for fold switching holds that N and N′ interconvert through the unfolded state. This model predicts that the fundamental properties of the switch—calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate—can be controlled by altering the relative stabilities of N and N′. We find that selectively destabilizing N or N′ changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole‐molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The value of three cereal aphid species as food for a generalist predator

The value of the cereal aphid species Metopolophium dirhodum (Wlk.), Sitobion avenae (F.) and Rhopalosiphum padi (L.) as prey for the linyphiid spider Erigone atra (Bl.) was assessed. Fecundity of females was determined for spiders fed on eight experimental diets: three single‐species aphid diets, a mixed diet of all three aphid species, three mixed diets with each aphid species in combination with fruit flies Drosophila melanogaster (Meig.), and pure D. melanogaster as a high quality comparison diet. The development and survival of first‐instar juveniles fed on three diets of single aphid species, and on a diet of Collembola were compared with those subjected to starvation. Prey value for adult females was assessed by egg production, hatching success and offspring size. In pure diets all three aphid species were of low value to the spiders, causing a rapid decline in egg production and supporting no growth of significance of first‐instar juveniles. No difference in value of aphid species of single‐species aphid diets was found in the fecundity experiment, while a ranking of aphid species of M. dirhodum > R. padi > S. avenae was revealed in the survivorship experiment. A mixed‐aphid diet was not found to be advantageous compared with single‐species aphid diets, and no advantage of including aphids in mixed diets with fruit flies was found. Metopolophium dirhodum and R. padi were neutral in mixed diets, while a diet of S. avenae and fruit flies caused reduced egg production compared with the pure diet of fruit flies, revealing a toxic effect of S. avenae on the spider. The value‐ranking of aphid species in mixed diets was similar to that of single‐species diets. A similar ranking of aphid species was found for different fitness parameters (fecundity of adult females and development of juveniles). A ranking of aphids by offspring size of mothers on aphid‐only diets was S. avenae > M. dirhodum > R. padi. All aphid‐fruit fly diets resulted in larger offspring than a diet of only D. melanogaster, with the overall largest offspring being produced on the diet of M. dirhodum and fruit flies.     

Model selection as a tool for phylogeographic inference: an example from the willow Salix melanopsis

Phylogeographic inference has typically relied on analyses of data from one or a few genes to provide estimates of demography and population histories. While much has been learned from these studies, all phylogeographic analysis is conditioned on the data, and thus, inferences derived from data that represent a small sample of the genome are unavoidably tenuous. Here, we demonstrate one approach for moving beyond classic phylogeographic research. We use sequence capture probes and Illumina sequencing to generate data from >400 loci in order to infer the phylogeographic history of Salix melanopsis, a riparian willow with a disjunct distribution in coastal and the inland Pacific Northwest. We evaluate a priori phylogeographic hypotheses using coalescent models for parameter estimation, and the results support earlier findings that identified post‐Pleistocene dispersal as the cause of the disjunction in S. melanopsis. We also conduct a series of model selection exercises using IMa2, Migrate‐n and ?a?i. The resulting ranking of models indicates that refugial dynamics were complex, with multiple regions in the inland regions serving as the source for postglacial colonization. Our results demonstrate that new sources of data and new approaches to data analysis can rejuvenate phylogeographic research by allowing for the identification of complex models that enable researchers to both identify and estimate the most relevant parameters for a given system.     

Effects of sucrose supplementation and generation on life‐history traits of Bracon cephi and Bracon lissogaster,parasitoids of the wheat stem sawfly

Knowledge of the life‐history traits that influence the reproductive success of parasitoids could provide useful information to enhance their effectiveness as biological control agents. The wheat stem sawfly Cephus cinctus Norton (Hymenoptera: Cephidae) is a major pest of wheat in the Northern Great Plains of North America. The bivoltine, sympatric and specialist parasitoids Bracon cephi (Gahan) and Bracon lissogaster Muesebeck (Hymenoptera: Braconidae) are the only species known to attack C. cinctus in wheat. In the present study, we quantify the body size, longevity, egg load and egg volume of B. cephi and B. lissogaster aiming to test whether these life‐history traits differ among adult females developing from overwintering and summer generation larvae. We also investigate the effect of sucrose supplementation on the same reproductive parameters. The results obtained show no differences between life‐history traits of B. cephi developing from the overwintering and summer generations. By contrast, the egg load of B. lissogaster is significantly different between generations, with overwintered females producing 1.2‐fold more eggs than summer individuals. Sucrose feeding has a strong positive effect on life‐history traits of both parasitoid species and generations. The longevity of sucrose‐fed females of B. cephi is increased three‐fold, whereas the survival of B. lissogaster is increased almost nine‐fold. Females of B. cephi treated with sucrose have four‐fold more mature eggs, whereas the egg load of B. lissogaster is increased 1.15‐ to 1.25‐fold with sugar feeding. Only B. cephi females increase egg volume with sucrose. The present study provides valuable insights on the life‐history and nutritional requirements of these two important parasitoid species.     

Paleogenetic records of Daphnia pulicaria in two North American lakes reveal the impact of cultural eutrophication

Understanding the evolutionary consequences of the green revolution, particularly in wild populations, is an important frontier in contemporary biology. Because human impacts have occurred at varying magnitudes or time periods depending on the study ecosystem, evolutionary histories may vary considerably among populations. Paleogenetics in conjunction with paleolimnology enable us to associate microevolutionary dynamics with detailed information on environmental change. We used this approach to reconstruct changes in the temporal population genetic structure of the keystone zooplankton grazer, Daphnia pulicaria, using dormant eggs extracted from sediments in two Minnesota lakes (South Center, Hill). The extent of agriculture and human population density in the catchment of these lakes has differed markedly since European settlement in the late 19th century and is reflected in their environmental histories reconstructed here. The reconstructed environments of these two lakes differed strongly in terms of environmental stability and their associated patterns of Daphnia population structure. We detected long periods of stability in population structure and environmental conditions in South Center Lake that were followed by a dramatic temporal shift in population genetic structure after the onset of European settlement and industrialized agriculture in its watershed. In particular, we noted a 24.3‐fold increase in phosphorus (P) flux between pre‐European and modern sediment P accumulation rates (AR) in this lake. In contrast, no such shifts were detected in Hill Lake, where the watershed was not as impacted by European settlement and rates of change were less directional with a much smaller increase in sediment P AR (2.3‐fold). We identify direct and indirect effects of eutrophication proxies on genetic structure in these lake populations and demonstrate the power of using this approach in understanding the consequences of anthropogenic environmental change on natural populations throughout historic time periods.     

PHYTOPLANKTON SELENIUM REQUIREMENTS: THE CASE FOR SPECIES ISOLATED FROM TEMPERATE AND POLAR REGIONS OF THE SOUTHERN HEMISPHERE1

A series of laboratory culture experiments was used to investigate the effect of selenium (Se, 0–10 nM) on the growth, cellular volume, photophysiology, and pigments of two temperate and four polar oceanic phytoplankton species [coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. P. Mohler, cyanobacterium Synechococcus sp., prymnesiophyte Phaeocystis sp., and three diatoms—Fragilariopsis cylindrus (Grunow) Kriegar, Chaetoceros sp., and Thalassiosira antarctica G. Karst.]. Only Synechoccocus sp. and Phaeocystis sp. did not show any requirement for Se. Under Se‐deficient conditions, the growth rate of E. huxleyi was decreased by 1.6‐fold, whereas cellular volume was increased by 1.9‐fold. Se limitation also decreased chl a (2.5‐fold), maximum relative electron transport rate (1.9‐fold), and saturating light intensity (2.8‐fold), suggesting that Se plays a role in photosynthesis or high‐light acclimation. Pigment analysis for Antarctic taxa provided an interesting counterpoint to the physiology of E. huxleyi. For all Se‐dependent Antarctic diatoms, Se limitation decreased growth rate and chl a content, whereas cellular volume was not affected. Pigment analysis revealed that other pigments were affected under Se deficiency. Photoprotective pigments increased by 1.4‐fold, while diadinoxanthin:diatoxanthin ratios decreased by 1.5‐ to 4.9‐fold under Se limitation, supporting a role for Se in photoprotection. Our results demonstrate an Se growth requirement for polar diatoms and indicate that Se could play a role in the biogeochemical cycles of other nutrients, such as silicic acid in the Southern Ocean. Se measurements made during the austral summer in the Southern Ocean and Se biological requirement were used to discuss possible Se limitation in phytoplankton from contrasting oceanographic regions.     

Identification of the ryanodine receptor mutation I4743M and its contribution to diamide insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae)

Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field‐collected population of Spodoptera exigua. The field‐collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH‐S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH‐S strain by crossing WF with WH‐S, followed by three rounds of backcrossing with WH‐S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH‐S strain. Compared with WH‐S, the near‐isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.     

Estimating carbon fixation of plant organs for afforestation monitoring using a process‐based ecosystem model and ecophysiological parameter optimization

Afforestation projects for mitigating CO₂ emissions require to monitor the carbon fixation and plant growth as key indicators. We proposed a monitoring method for predicting carbon fixation in afforestation projects, combining a process‐based ecosystem model and field data and addressed the uncertainty of predicted carbon fixation and ecophysiological characteristics with plant growth. Carbon pools were simulated using the Biome‐BGC model tuned by parameter optimization using measured carbon density of biomass pools on an 11‐year‐old Eucommia ulmoides plantation on Loess Plateau, China. The allocation parameters fine root carbon to leaf carbon (FRC:LC) and stem carbon to leaf carbon (SC:LC), along with specific leaf area (SLA) and maximum stomatal conductance (g_smax) strongly affected aboveground woody (AC) and leaf carbon (LC) density in sensitivity analysis and were selected as adjusting parameters. We assessed the uncertainty of carbon fixation and plant growth predictions by modeling three growth phases with corresponding parameters: (i) before afforestation using default parameters, (ii) early monitoring using parameters optimized with data from years 1 to 5, and (iii) updated monitoring at year 11 using parameters optimized with 11‐year data. The predicted carbon fixation and optimized parameters differed in the three phases. Overall, 30‐year average carbon fixation rate in plantation (AC, LC, belowground woody parts and soil pools) was ranged 0.14–0.35 kg‐C m^?2 y^?1 in simulations using parameters of phases (i)–(iii). Updating parameters by periodic field surveys reduced the uncertainty and revealed changes in ecophysiological characteristics with plant growth. This monitoring method should support management of afforestation projects by carbon fixation estimation adapting to observation gap, noncommon species and variable growing conditions such as climate change, land use change.     

Estimating mass change of migrant songbirds during stopover: comparison of three different methods

ABSTRACT Stopover‐site quality has often been assessed using changes in the body mass of migrants estimated from individuals recaptured on subsequent days or using regression methods. We compared estimates of mass change using these two techniques to estimates of mass change determined from birds recaptured on the same day. Using spring and fall banding data collected on Appledore Island, Maine, from 1990–2007, we examined body mass changes of the five most common species. Over this period, 18,954 individuals of these five species were captured and banded, with 11.6% of birds recaptured at least 1 d after initial capture and 3.1% recaptured on the same day. Using both regression and same‐day recapture methods, all five species had positive hourly mass gains during fall migration; results were mixed for the subsequent‐day analysis method. Trends were less consistent during spring migration. Using all three methods of estimating mass change, Red‐eyed Vireos (Vireo olivaceus) lost mass, American Redstarts (Setophaga ruticilla) and Northern Waterthrushes (Parkesia noveboracensis) gained mass, and results for Yellow‐bellied Flycatchers (Empidonax flaviventris), and Black‐and‐white Warblers (Mniotilta varia) varied with method. We found similar trends in mass change using the same‐day recapture and regression methods. However, we found lower mean mass gain for most species using the same‐day recapture method, suggesting that there may be a short‐term capture and handling effect. Our results provide additional support for the use of regression models to compare changes in mass of migrating songbirds at stopover sites.     

Linking temperature sensitivity of soil organic matter decomposition to its molecular structure,accessibility, and microbial physiology

Temperature sensitivity of soil organic matter (SOM) decomposition may have a significant impact on global warming. Enzyme‐kinetic hypothesis suggests that decomposition of low‐quality substrate (recalcitrant molecular structure) requires higher activation energy and thus has greater temperature sensitivity than that of high‐quality, labile substrate. Supporting evidence, however, relies largely on indirect indices of substrate quality. Furthermore, the enzyme‐substrate reactions that drive decomposition may be regulated by microbial physiology and/or constrained by protective effects of soil mineral matrix. We thus tested the kinetic hypothesis by directly assessing the carbon molecular structure of low‐density fraction (LF) which represents readily accessible, mineral‐free SOM pool. Using five mineral soil samples of contrasting SOM concentrations, we conducted 30‐days incubations (15, 25, and 35 °C) to measure microbial respiration and quantified easily soluble C as well as microbial biomass C pools before and after the incubations. Carbon structure of LFs (<1.6 and 1.6–1.8 g cm^?3) and bulk soil was measured by solid‐state ¹³C‐NMR. Decomposition Q₁₀ was significantly correlated with the abundance of aromatic plus alkyl‐C relative to O‐alkyl‐C groups in LFs but not in bulk soil fraction or with the indirect C quality indices based on microbial respiration or biomass. The warming did not significantly change the concentration of biomass C or the three types of soluble C despite two‐ to three‐fold increase in respiration. Thus, enhanced microbial maintenance respiration (reduced C‐use efficiency) especially in the soils rich in recalcitrant LF might lead to the apparent equilibrium between SOM solubilization and microbial C uptake. Our results showed physical fractionation coupled with direct assessment of molecular structure as an effective approach and supported the enzyme‐kinetic interpretation of widely observed C quality‐temperature relationship for short‐term decomposition. Factors controlling long‐term decomposition Q₁₀ are more complex due to protective effect of mineral matrix and thus remain as a central question.     

Sop‐GPU: Accelerating biomolecular simulations in the centisecond timescale using graphics processors

Theoretical exploration of fundamental biological processes involving the forced unraveling of multimeric proteins, the sliding motion in protein fibers and the mechanical deformation of biomolecular assemblies under physiological force loads is challenging even for distributed computing systems. Using a C_α‐based coarse‐grained self organized polymer (SOP) model, we implemented the Langevin simulations of proteins on graphics processing units (SOP‐GPU program). We assessed the computational performance of an end‐to‐end application of the program, where all the steps of the algorithm are running on a GPU, by profiling the simulation time and memory usage for a number of test systems. The ～90‐fold computational speedup on a GPU, compared with an optimized central processing unit program, enabled us to follow the dynamics in the centisecond timescale, and to obtain the force‐extension profiles using experimental pulling speeds (v_f = 1–10 μm/s) employed in atomic force microscopy and in optical tweezers‐based dynamic force spectroscopy. We found that the mechanical molecular response critically depends on the conditions of force application and that the kinetics and pathways for unfolding change drastically even upon a modest 10‐fold increase in v_f. This implies that, to resolve accurately the free energy landscape and to relate the results of single‐molecule experiments in vitro and in silico, molecular simulations should be carried out under the experimentally relevant force loads. This can be accomplished in reasonable wall‐clock time for biomolecules of size as large as 10⁵ residues using the SOP‐GPU package. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

The crystal structure of the AF2331 protein from Archaeoglobus fulgidus DSM 4304 forms an unusual interdigitated dimer with a new type of α + β fold

The structure of AF2331, a 11‐kDa orphan protein of unknown function from Archaeoglobus fulgidus, was solved by Se‐Met MAD to 2.4 Å resolution. The structure consists of an α + β fold formed by an unusual homodimer, where the two core β‐sheets are interdigitated, containing strands alternating from both subunits. The decrease in solvent‐accessible surface area upon dimerization is unusually large (3960 Ų) for a protein of its size. The percentage of the total surface area buried in the interface (41.1%) is one of the largest observed in a nonredundant set of homodimers in the PDB and is above the mean for nearly all other types of homo‐oligomers. AF2331 has no sequence homologs, and no structure similar to AF2331 could be found in the PDB using the CE, TM‐align, DALI, or SSM packages. The protein has been identified in Pfam 23.0 as the archetype of a new superfamily and is topologically dissimilar to all other proteins with the “3‐Layer (BBA) Sandwich” fold in CATH. Therefore, we propose that AF2331 forms a novel α + β fold. AF2331 contains multiple negatively charged surface clusters and is located on the same operon as the basic protein AF2330. We hypothesize that AF2331 and AF2330 may form a charge‐stabilized complex in vivo, though the role of the negatively charged surface clusters is not clear.     

Mapping disease‐related missense mutations in the immunoglobulin‐like fold domain of lamin A/C reveals novel genotype–phenotype associations for laminopathies

Mutations in A‐type nuclear lamins cause laminopathies. However, genotype–phenotype correlations using the 340 missense mutations within the LMNA gene are unclear: partially due to the limited availability of three‐dimensional structure. The immunoglobulin (Ig)‐like fold domain has been solved, and using bioinformatics tools (including Polyphen‐2, Fold X, Parameter OPtimized Surfaces, and PocketPicker) we characterized 56 missense mutations for position, surface exposure, change in charge and effect on Ig‐like fold stability. We find that 21 of the 27 mutations associated with a skeletal muscle phenotype are distributed throughout the Ig‐like fold, are nonsurface exposed and predicted to disrupt overall stability of the Ig‐like fold domain. Intriguingly, the remaining 6 mutations clustered, had higher surface exposure, and did not affect stability. The majority of 9 lipodystrophy or 10 premature aging syndrome mutations also did not disrupt Ig‐like fold domain stability and were surface exposed and clustered in distinct regions that overlap predicted binding pockets. Although buried, the 10 cardiac mutations had no other consistent properties. Finally, most lipodystrophy and premature aging mutations resulted in a ‐1 net charge change, whereas skeletal muscle mutations caused no consistent net charge changes. Since premature aging, lipodystrophy and the subset of 6 skeletal muscle mutations cluster tightly in distinct, charged regions, they likely affect lamin A/C –protein/DNA/RNA interactions: providing a consistent genotype–phenotype relationship for mutations in this domain. Thus, this subgroup of skeletal muscle laminopathies that we term the ‘Skeletal muscle cluster’, may have a distinct pathological mechanism. These novel associations refine the ability to predict clinical features caused by certain LMNA missense mutations. Proteins 2014; 82:904–915. © 2013 Wiley Periodicals, Inc.