High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K_i of approximately 4 μM. Uptake studies with [1,2-dimethyl-¹⁴C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K_m value of 4 ± 1 μM and a maximum rate of transport (V_max) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance ¹³C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

Validating the prediction accuracies of marker-assisted and genomic selection of Fusarium head blight resistance in wheat using an independent sample

Key message

Compared with independent validation, cross-validation simultaneously sampling genotypes and environments provided similar estimates of accuracy for genomic selection, but inflated estimates for marker-assisted selection.

Abstract

Estimates of prediction accuracy of marker-assisted (MAS) and genomic selection (GS) require validations. The main goal of our study was to compare the prediction accuracies of MAS and GS validated in an independent sample with results obtained from fivefold cross-validation using genomic and phenotypic data for Fusarium head blight resistance in wheat. In addition, the applicability of the reliability criterion, a concept originally developed in the context of classic animal breeding and GS, was explored for MAS. We observed that prediction accuracies of MAS were overestimated by 127% using cross-validation sampling genotype and environments in contrast to independent validation. In contrast, prediction accuracies of GS determined in independent samples are similar to those estimated with cross-validation sampling genotype and environments. This can be explained by small population differentiation between the training and validation sets in our study. For European wheat breeding, which is so far characterized by a slow temporal dynamic in allele frequencies, this assumption seems to be realistic. Thus, GS models used to improve European wheat populations are expected to possess a long-lasting validity. Since quantitative trait loci information can be exploited more precisely if the predicted genotype is more related to the training population, the reliability criterion is also a valuable tool to judge the level of prediction accuracy of individual genotypes in MAS.

     

Analyzing the Ecosystem Carbon Dynamics of Four European Coniferous Forests Using a Biogeochemistry Model

This paper provides the first steps toward a regional-scale analysis of carbon (C) budgets. We explore the ability of the ecosystem model BIOME-BGC to estimate the daily and annual C dynamics of four European coniferous forests and shifts in these dynamics in response to changing environmental conditions. We estimate uncertainties in the model results that arise from incomplete knowledge of site management history (for example, successional stage of forest). These uncertainties are especially relevant in regional-scale simulations, because this type of information is difficult to obtain. Although the model predicted daily C and water fluxes reasonably well at all sites, it seemed to have a better predictive capacity for the photosynthesis-related processes than for respiration. Leaf area index (LAI) was modeled accurately at two sites but overestimated at two others (as a result of poor long-term climate drivers and uncertainties in model parameterization). The overestimation of LAI (and consequently gross photosynthetic production (GPP)), in combination with reasonable estimates of the daily net ecosystem productivity (NEP) of those forests, also illustrates the problem with modeled respiration. The model results suggest that all four European forests have been net sinks of C at the rate of 100–300 gC/m²/y and that this C sequestration capacity would be 30%–70% lower without increasing nitrogen (N) deposition and carbon dioxide (CO₂) concentrations. The magnitude of the forest responses was dependent not only on the rate of changes in environmental factors, but also on site-specific conditions such as climate and soil depth. We estimated that the modeled C exchange at the study sites was reduced by 50%–100% when model simulations were performed for climax forests rather than regrowing forests. The estimates of water fluxes were less sensitive to different initializations of state variables or environmental change scenarios than C fluxes.     

Diversity of Pol IV Function Is Defined by Mutations at the Maize rmr7 Locus

Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses.     

Dimethylglycine Provides Salt and Temperature Stress Protection to Bacillus subtilis

Glycine betaine is a potent osmotic and thermal stress protectant of many microorganisms. Its synthesis from glycine results in the formation of the intermediates monomethylglycine (sarcosine) and dimethylglycine (DMG), and these compounds are also produced when it is catabolized. Bacillus subtilis does not produce sarcosine or DMG, and it cannot metabolize these compounds. Here we have studied the potential of sarcosine and DMG to protect B. subtilis against osmotic, heat, and cold stress. Sarcosine, a compatible solute that possesses considerable protein-stabilizing properties, did not serve as a stress protectant of B. subtilis. DMG, on the other hand, proved to be only moderately effective as an osmotic stress protectant, but it exhibited good heat stress-relieving and excellent cold stress-relieving properties. DMG is imported into B. subtilis cells primarily under osmotic and temperature stress conditions via OpuA, a member of the ABC family of transporters. Ligand-binding studies with the extracellular solute receptor (OpuAC) of the OpuA system showed that OpuAC possesses a moderate affinity for DMG, with a K_d value of approximate 172 μM; its K_d for glycine betaine is about 26 μM. Docking studies using the crystal structures of the OpuAC protein with the sulfur analog of DMG, dimethylsulfonioacetate, as a template suggest a model of how the DMG molecule can be stably accommodated within the aromatic cage of the OpuAC ligand-binding pocket. Collectively, our data show that the ability to acquire DMG from exogenous sources under stressful environmental conditions helps the B. subtilis cell to cope with growth-restricting osmotic and temperature challenges.     

Mapping QTLs with main and epistatic effects underlying grain yield and heading time in soft winter wheat

There is increasing awareness that epistasis plays a role for the determination of complex traits. This study employed an association mapping approach in a large panel of 455 diverse European elite soft winter wheat lines. The genotypes were evaluated in multi-environment trials and fingerprinted with SSR markers to dissect the underlying genetic architecture of grain yield and heading time. A linear mixed model was applied to assess marker-trait associations incorporating information of covariance among relatives. Our findings indicate that main effects dominate the control of grain yield in wheat. In contrast, the genetic architecture underlying heading time is controlled by main and epistatic effects. Consequently, for heading time it is important to consider epistatic effects towards an increased selection gain in marker-assisted breeding.     

Factors affecting body size and fat content in a digger wasp

Body size is one of the most important life history traits. In mass-provisioning solitary Hymenoptera, the maximum attainable adult size is not under the control of the larva but is limited by the amount of resources provided by the mother. I investigated the effect of the amount of different maternal resources and potentially interfering abiotic (temperature) and biotic (fungus infestation) factors on offspring body size and fat reserves in a solitary digger wasp, the European beewolf, Philanthus triangulum F. (Hymenoptera, Sphecidae). Females provide different resources for their progeny that might influence progeny size (egg, brood cell, and paralysed honey-bees as food). The number of bees provisioned explained the largest proportion of variation in cocoon length. With an increasing number of bees (one to four), progeny gained less weight per bee. Relative fat content increased with size. With a given number of bees, males were smaller than females. The duration of the feeding period was independent of the number of bees in a brood cell but decreased with increasing ambient temperatures (20, 25, 30°C). Cocoon size was influenced by temperature but the effect was not uniform. Cocoons from brood cells containing two and three bees were larger at 25°C than at 20°C; those at 30°C did not differ from those at either lower temperature. However, in brood cells containing one bee, cocoon length was independent of temperature. Sublethal levels of fungus infestation may have a small negative effect on cocoon size. Different temperatures during hibernation (8 vs 13°C) did not affect the size or fat content of emerging adults. These results on a mass-provisioning hunting wasp are compared with the well-studied herbivorous insects. Received: 12 April 1999 / Accepted: 3 December 1999