Variation in Adult Plant Phenotypes and Partitioning among Seed and Stem-Borne Roots across Brachypodium distachyon Accessions to Exploit in Breeding Cereals for Well-Watered and Drought Environments

Seedling roots enable plant establishment. Their small phenotypes are measured routinely. Adult root systems are relevant to yield and efficiency, but phenotyping is challenging. Root length exceeds the volume of most pots. Field studies measure partial adult root systems through coring or use seedling roots as adult surrogates. Here, we phenotyped 79 diverse lines of the small grass model Brachypodium distachyon to adults in 50-cm-long tubes of soil with irrigation; a subset of 16 lines was droughted. Variation was large (total biomass, ×8; total root length [TRL], ×10; and root mass ratio, ×6), repeatable, and attributable to genetic factors (heritabilities ranged from approximately 50% for root growth to 82% for partitioning phenotypes). Lines were dissected into seed-borne tissues (stem and primary seminal axile roots) and stem-borne tissues (tillers and coleoptile and leaf node axile roots) plus branch roots. All lines developed one seminal root that varied, with branch roots, from 31% to 90% of TRL in the well-watered condition. With drought, 100% of TRL was seminal, regardless of line because nodal roots were almost always inhibited in drying topsoil. Irrigation stimulated nodal roots depending on genotype. Shoot size and tillers correlated positively with roots with irrigation, but partitioning depended on genotype and was plastic with drought. Adult root systems of B. distachyon have genetic variation to exploit to increase cereal yields through genes associated with partitioning among roots and their responsiveness to irrigation. Whole-plant phenotypes could enhance gain for droughted environments because root and shoot traits are coselected.Adult plant root systems are relevant to the size and efficiency of seed yield. They supply water and nutrients for the plant to acquire biomass, which is positively correlated to the harvest index (allocation to seed grain), and the stages of flowering and grain development. Modeling in wheat (Triticum aestivum) suggested that an extra 10 mm of water absorbed by such adult root systems during grain filling resulted in an increase of approximately 500 kg grain ha⁻¹ (Manschadi et al., 2006). This was 25% above the average annual yield of wheat in rain-fed environments of Australia. This number was remarkably close to experimental data obtained in the field in Australia (Kirkegaard et al., 2007). Together, these modeling and field experiments have shown that adult root systems are critical for water absorption and grain yield in cereals, such as wheat, emphasizing the importance of characterizing adult root systems to identify phenotypes for productivity improvements.Most root phenotypes, however, have been described for seedling roots. Seedling roots are essential for plant establishment, and hence, the plant’s potential to set seed. For technical reasons, seedlings are more often screened than adult plants because of the ease of handling smaller plants and the high throughput. Seedling-stage phenotyping may also improve overall reproducibility of results because often, growth media are soil free. Seedling soil-free root phenotyping conditions are well suited to dissecting fine and sensitive mechanisms, such as lateral root initiation (Casimiro et al., 2003; Péret et al., 2009a, 2009b). A number of genes underlying root processes have been identified or characterized using seedlings, notably with the dicotyledonous models Arabidopsis (Arabidopsis thaliana; Mouchel et al., 2004; Fitz Gerald et al., 2006; Yokawa et al., 2013) and Medicago truncatula (Laffont et al., 2010) and the cereals maize (Zea mays; Hochholdinger et al., 2001) and rice (Oryza sativa; Inukai et al., 2005; Kitomi et al., 2008).Extrapolation from seedling to adult root systems presents major questions (Hochholdinger and Zimmermann, 2008; Chochois et al., 2012; Rich and Watt, 2013). Are phenotypes in seedling roots present in adult roots given developmental events associated with aging? Is expression of phenotypes correlated in seedling and adult roots if time compounds effects of growth rates and growth conditions on roots? Watt et al. (2013) showed in wheat seedlings that root traits in the laboratory and field correlated positively but that neither correlated with adult root traits in the field. Factors between seedling and adult roots seemed to be differences in developmental stage and the time that growing roots experience the environment.Seedling and adult root differences may be larger in grasses than dicotyledons. Grass root systems have two developmental components: seed-borne (seminal) roots, of which a number emerge at germination and continue to grow and branch throughout the plant life, and stem-borne (nodal or adventitious) roots, which emerge from around the three-leaf stage and continue to emerge, grow, and branch throughout the plant life. Phenotypes and traits of adult root systems of grasses, which include the major cereal crops wheat, rice, and maize, are difficult to predict in seedling screens and ideally identified from adult root systems first (Gamuyao et al., 2012).Phenotyping of adult roots is possible in the field using trenches (Maeght et al., 2013) or coring (Wasson et al., 2014). A portion of the root system is captured with these methods. Alternatively, entire adult root systems can be contained within pots dug into the ground before sowing. These need to be large; field wheat roots, for example, can reach depths greater than 1.5 m depending on genotype and environment. This method prevents root-root interactions that occur under normal field sowing of a plant canopy and is also a compromise.A solution to the problem of phenotyping adult cereal root systems is a model for monocotyledon grasses: Brachypodium distachyon. B. distachyon is a small-stature grass with a small genome that is fully sequenced (Vogel et al., 2010). It has molecular tools equivalent to those available in Arabidopsis (Draper et al., 2001; Brkljacic et al., 2011; Mur et al., 2011). The root system of B. distachyon reference line Bd21 is more similar to wheat than other model and crop grasses (Watt et al., 2009). It has a seed-borne primary seminal root (PSR) that emerges from the embryo at seed germination and multiple stem-borne coleoptile node axile roots (CNRs) and leaf node axile roots (LNRs), also known as crown roots or adventitious roots, that emerge at about three leaves through to grain development. Branch roots emerge from all root types. There are no known anatomical differences between root types of wheat and B. distachyon (Watt et al., 2009). In a recent study, we report postflowering root growth in B. distachyon line Bd21-3, showing that this model can be used to answer questions relevant to the adult root systems of grasses (Chochois et al., 2012).In this study, we used B. distachyon to identify adult plant phenotypes related to the partitioning among seed-borne and stem-borne shoots and roots for the genetic improvement of well-watered and droughted cereals (Fig. 1; Krassovsky, 1926; Navara et al., 1994), nitrogen, phosphorus (Tennant, 1976; Brady et al., 1995), oxygen (Wiengweera and Greenway, 2004), soil hardness (Acuna et al., 2007), and microorganisms (Sivasithamparam et al., 1978). Of note is the study by Krassovsky (1926), which was the first, to our knowledge, to show differences in function related to water. Krassovsky (1926) showed that seminal roots of wheat absorbed almost 2 times the water as nodal roots per unit dry weight but that nodal roots absorbed a more diluted nutrient solution than seminal roots. Krassovsky (1926) also showed by removing seminal or nodal roots as they emerged that “seminal roots serve the main stem, while nodal roots serve the tillers” (Krassovsky, 1926). Volkmar (1997) showed, more recently, in wheat that nodal and seminal roots may sense and respond to drought differently. In millet (Pennisetum glaucum) and sorghum (Sorghum bicolor), Rostamza et al. (2013) found that millet was able to grow nodal roots in a dryer soil than sorghum, possibly because of shoot and root vigor.Open in a separate windowFigure 1.B. distachyon plant scanned at the fourth leaf stage, with the root and shoot phenotypes studied indicated. Supplemental Table S1.

PARP inhibitors and metastatic castration-resistant prostate cancer: uture directions and pitfalls

PARP inhibitors (PARPi) gained major interest among prostate cancer researchers in the last few years, thanks to the outstanding results coming from the PROfound an TRITON2 studies. Following that, PARPi gained approval also in metastatic, castration-resistant prostate cancer (mCRPC) with mutations in homologous repair (HR) – related genes. Nevertheless, some questions still remain unanswered concerning the management of drug resistance and PARPi-sensitivity in patients harboring alterations in various DNA damage response (DDR) related genes, not only BRCA1 and BRCA2.In this perspective article we focus on the key issues concerning PARPi in mCRPC, specifically those related to drug sensitivity and resistance mechanisms, exploring the possible role of combination therapeutic approaches and trying to depict potential future addresses in translational oncology research.

Perspective Article (max: 1200 words)The DNA damage repair (DDR) pathway gained major interest between cancer researchers since 2005, when emerging studies demonstrated that the simultaneous inhibition of both Poly(ADP-ribose) polymerase 1 (PARP1) and tumor suppressors Breast Related Cancer Antigens 1 and 2 (BRCA1 and BRCA2) generates excessive DNA instability and, ultimately, leads to cellular death. This process, called synthetic lethal theory, constituted the rationale for the development of drugs targeting PARP1 in BRCA1/2 deficient clones, the PARP inhibitors (PARPi) [1, 2].In normal conditions, PARP1 plays a key role as regulator of multiple cellular processes, including DDR. When a DNA damage occurs, the activation of PARP1 results in the recruitment of several DNA repair factors, including BRCA1 and BRCA2, leading to the restoration of single-strand (SSBs) and double-strand DNA breaks (DSBs) [1,2]^. Particularly, BRCA1 and BRCA2 act downstream the PARP1 cascade in one of the two major pathways for DSBs repair, largely error free: the homologous repair (HR). Another crucial mechanism, which sees the synergic contribution of PARP1, BRCA1 and BRCA 2, is the stabilization of replication fork during the S phase of the cell cycle [2]. As a consequence of that, heterozygous germline mutations in DDR genes, especially BRCA1 and BRCA2, dramatically increase the risk of developing multiple neoplasms (e.g. breast, ovarian, prostate and pancreatic cancers )². In addition, somatic and germline mutations in one of these genes confer a strong sensitivity to DNA-damaging agents (e.g. platinum salts): these fundamental observations led researchers to successfully study and test pharmacological inhibition of the DDR pathway, using PARPi [2].Of note, it has been calculated that approximately 12% of metastatic, castration-resistant prostate cancer (mCRPC) patients harbor germline DDR mutations, while 20–25% harbor somatic DDR mutations. Overall, it is estimated that in almost 22.7% of mCRPC patients could be identified mutations in DDR-related genes, making them a considerable number of people who could take an advantage from PARPi administration [3].In 2014, the U.S. Food and Drug Administration (FDA) granted approval to Olaparib as the first PARPi viable for women suffering from BRCA 1–2 mutated metastatic ovarian cancer both for cases previously treated with three or more lines of chemotherapy, and also as maintenance therapy following platinum-based chemotherapy [2]. Since that, following the consistent results described by subsequent clinical trials, Olaparib and other PARPi (e.g. Rucaparib, Niraparib) gained approval for different clinical settings in ovarian cancer and for BRCA-mutated breast, pancreatic and prostate cancer [2].In 2020, thanks to the outstanding results of the PROFound trial, the FDA approved the administration of Olaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) progressing after therapy with enzalutamide or abiraterone and harboring mutations in HR-related genes [4]. Later the same year, the European Medicines Agency (EMA) recommended Olaparib in the same setting, with a slight but substantial difference: the main requirement was the identification of a BRCA 1 and BRCA 2 mutation (somatic or germline) in prostate cancer patients who have progressed to a prior therapy that included a new hormonal agent [5].Similarly, Rucaparib received the FDA accelerated approval after the publication of the TRITON2 study, that showed consistent overall response rate (ORR) and Prostate Specific Antigen (PSA) response rate values in patients with BRCA 1 and BRCA2 alterations [6].Nevertheless, it is well known that DDR mechanisms, including homologous repair (HR), are characterized by the interplay of a huge number of enzymes, co-factors, and molecules, not only BRCA1 and BRCA2 [2,5]. Specifically, HR requires the intervention of co-factors as PALB2 (Partner And Localizer Of BRCA2) and RAD51 (RAD51 Recombinase) to perform an accurate repair of double strand DNA breaks. In addition, BRCA1 and BRCA2 exhibit a crucial role during the S phase of the cell cycle, as protectors of the replication fork from the degradation activity carried out by nucleases. This is why, although PARPi seem to be more effective against BRCA1 and 2 mutations, data extrapolated from clinical trials suggest a benefit also for people harboring alterations in others genes, such as PALB2, RAD51 and ATM (Ataxia-Telangectasia Mutated) [2]. The PROFound trial, considered as a milestone, enlightened this aspect and its possible implications in prostate cancer: administering Olaparib to the whole cohort of HR-deficient patients could extend the survival benefit to a significant number of people, albeit the subgroup of BRCA1 and BRCA2 mutated cohort might have generated an overestimation of this effect in that trial [7]. Further studies need to be carried out in order to perform a correct prognostic and predictive gene-signature based stratification of patients.One of major concerns related to anti-cancer drugs, particularly targeted therapies, is drug-resistance. Even PARPi, although frequently characterized by initial good responses, ultimately loose their effectiveness, leading to disease relapse [2]. The reason is that cancerous cells learn how to escape from the pharmacological attack of PARPi via several mechanisms: upregulation of drug efflux pumps; mutations of the drug target; recovery of BRCA1 and BRCA2 function; re-establishment of replication fork stability [2,8]. The deep knowledge of these mechanisms could lead to overcome drug resistance: the most appealing hypothesis to get through this barrier appears to combine PARPi with agents affecting HR from other sides, such as Vascular Endothelial Growth Factor (VEGF) inhibitors, for which some encouraging data have been published in a cohort of ovarian cancer patients [2]. An interesting observation is also that HR deficient cancers might exhibit a high tumor mutational burden, often associated with an improved sensitivity to immunotherapy. Thus, clinical trials are now investigating the combination of PARPi and immune check-point inhibitors (ICIs) in mCRPC [9].Furthermore, several trials are ongoing to evaluate the efficacy of the combination of PARPi and new hormone agents (i.e. Abiraterone acetate, Enzalutamide) for metastatic prostate cancer, both in the hormone-sensitive and castration-resistant phases.Unfortunately, most of data concerning combination therapies were extrapolated from preliminary analyses of clinical trials, with many open issues still remaining. Firstly, drug safety: as previously stated in a phase I/II clinical trial, the addition of ICIs to PARPi seems to be well tolerated with no significant increase of severe adverse effects; at the same time, the administration of PARPi plus Abiraterone in mCRPC patients was investigated in a randomized, double-blind, placebo controlled phase II clinical trial, obtaining promising results in term of safety and also efficacy [2,10]. Another major concern regards the need to identify reliable biomarkers predictive of drug response, and this must be one of the addresses of future researches [1,2]. The last issue involves health care costs of such combinations therapies, again emphasizing the importance to perform a thorough stratification of mCRPC patients. [2]. These might be some branches for future researches, to explore where and when to combine PARPi with other agents, and in which patients subgroup [1,2,9].We have now several weapons in our hands, ready to be used, the most important represented by genomic analyses techniques [2]. In addition, following that principle of synthetic lethality, we need to hit cellular DNA repairing system from many sides, employing old and new drugs. The only way to cope with this huge amount of data is to team up with different professional figures (e.g. biotechnologists, pharmacologists, biostatisticians), constructing a cooperative network system. Only by doing this we will make it up to the mountain.

Bar-Coded Pyrosequencing of 16S rRNA Gene Amplicons Reveals Changes in Ileal Porcine Bacterial Communities Due to High Dietary Zinc Intake

Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.Zinc oxide (ZnO) is used as a feed additive for diarrhea prophylaxis in piglets (23). However, the mode of action of ZnO is not fully understood. Besides its effects on the host (10, 30, 31), high dietary zinc levels may affect the diversity of intestinal microbial communities (2, 11, 20). The prevention of postweaning diarrhea in piglets due to high dietary ZnO intake may not be directly related to a reduction of pathogenic E. coli (8) but, rather, to the diversity of the coliform community (15). Studies on the impact of high ZnO levels on the porcine ileal bacterial community are scarce but nevertheless important, as bacterial diarrhea is initiated in the small intestine (9, 17). The small intestine is a very complex habitat with many different factors shaping the bacterial community. Studies on the ecophysiology (22) and maturation of the porcine ileal microbiota (13, 27) indicate a drastic impact directly after weaning and a gradual decline of modifications during the following 2 weeks. Thus, the time point for analysis chosen in this study (14 days postweaning) does reflect a more stable period of the ileal porcine microbiota. In this study, we used bar-coded pyrosequencing of 16S rRNA genes to gain further insight into the mode of action of pharmacological levels of ZnO in the gastrointestinal tract of young pigs.Total DNA was extracted from the ileal digesta of 40- to 42-day-old piglets using a commercial kit (Qiagen stool kit; Qiagen, Hilden, Germany) and PCR amplified with unique bar-coded primer sets targeting the V1-to-V3 and the V6-to-V8 hypervariable regions (see the supplemental material for detailed methods). The rationale behind this approach was derived from the fact that no single “universal” primer pair can completely cover a complex bacterial habitat (4, 24, 32, 33). Furthermore, these studies also show that in silico information on the coverage of selected primer sets diverges from empirical results, and hence, two hypervariable regions were chosen in this study to maximize the detection of phylogenetically diverse bacterial groups.Equimolar dilutions of all samples were combined into one master sample. Pyrosequencing was performed by Agowa (Berlin, Germany) on a Roche genome sequencer FLX system using a Titanium series PicoTiterPlate. The resulting data files were uploaded to the MG-RAST server (http://metagenomics.nmpdr.org/) (19) and processed with its SEED software tool using the RDP database (5) as the reference database. After automated sequence analysis, all sequences with less than five identical reads per sample were deleted in order to increase the confidence of sequence reads and reduce bias from possible sequencing errors (12, 16). Thus, 0.43% of all sequences were not considered (1,882 of 433,302 sequences). These sequences were assigned to a total of 238 genera, of which most only occurred in a few samples (see the supplemental material). Furthermore, all unclassified sequences were removed (8.7%; 41,467 of 474,769 sequences). Due to the use of the RDP reference database, the SEED software incorrectly assigned the majority of unclassified sequences as unclassified Deferribacterales (83%; 34,393 sequences), which were actually identified as 16S soybean or wheat chloroplasts by BLAST or as cyanobacterial chloroplasts by the RDP II seqmatch tool.The pyrosequencing results for the two primer combinations were merged by taking only sequences from the primer combination that yielded the higher number of reads for a specific sequence assignment in a sample. The remaining reads were used to calculate the relative contribution of assigned sequences to total sequence reads in a sample.The Firmicutes phylum dominated the small intestinal bacterial communities in both the control group and the group with high dietary ZnO intake, with 98.3% and 97.0% of total sequence reads, respectively. No significant influence of high dietary ZnO intake was found for the main phyla Proteobacteria (0.92% versus 1.84%), Actinobacteria (0.61% versus 0.75%), Bacteroidetes (0.15% versus 0.17%), and Fusobacteria (0.09% versus 0.12%).On the order level, a total of 20 bacterial orders were detected (data not shown). Lactobacillales dominated bacterial communities in the control and high-dietary-ZnO-intake groups, with 83.37% and 93.24% of total reads. Lactic acid bacteria are well known to dominate the bacterial community in the ileum of piglets (11, 22). No significant difference between the control group and the group with high dietary ZnO intake was observed on the order level, although high dietary ZnO intake led to a strong numerical decrease for Clostridiales (14.4 ± 24.0% [mean ± standard deviation] versus 2.8 ± 1.7%), as well as to numerical increases for Pseudomonadales (0.3 ± 0.3% versus 0.6 ± 0.6%) and Enterobacteriales (0.2 ± 0.2% versus 0.5 ± 0.6%).On the genus level, a total of 103 genera were detected. Table ​Table11 summarizes the main 31 genera which exceeded 0.05% of total reads (see the supplemental material for a complete list). Lactobacilli clearly dominated the bacterial communities in both trial groups, but they also were numerically lower due to high dietary ZnO intake.

TABLE 1.

Bacterial genera in the ileum of piglets fed diets supplemented with 200 or 3,000 ppm ZnO

Arabidopsis thaliana overexpressing glycolate oxidase in chloroplasts: H2O2-induced changes in primary metabolic pathways

Reactive oxygen species (ROS) represent both toxic by-products of aerobic metabolism as well as signaling molecules in processes like growth regulation and defense pathways. The study of signaling and oxidative-damage effects can be separated in plants expressing glycolate oxidase in the plastids (GO plants), where the production of H₂O₂ in the chloroplasts is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions. Thus, GO plants represent an ideal non-invasive model to study events related to the perception and responses to H₂O₂ accumulation. Metabolic profiling of GO plants indicated that under high light a sustained production of H₂O₂ imposes coordinate changes on central metabolic pathways. The overall metabolic scenario is consistent with decreased carbon assimilation, which results in lower abundance of glycolytic and tricarboxylic acid cycle intermediates, while simultaneously amino acid metabolism routes are specifically modulated. The GO plants, although retarded in growth and flowering, can complete their life cycle indicating that the reconfiguration of the central metabolic pathways is part of a response to survive and thus, to adapt to stress conditions imposed by the accumulation of H₂O₂ during the light period.Key words: Arabidopsis thaliana, H₂O₂, oxidative stress, reactive oxygen species, signalingReactive oxygen species (ROS) are key molecules in the regulation of plant development, stress responses and programmed cell death. Depending on the identity of ROS species or its subcellular production site, different cellular responses are provoked.¹ To assess the effects of metabolically generated H₂O₂ in chloroplasts, we have recently generated Arabidopsis plants in which the peroxisomal GO was targeted to chloroplasts.² The GO overexpressing plants (GO plants) show retardation in growth and flowering time, features also observed in catalase, ascorbate peroxidase and MnSOD deficient mutants.^3–5 The analysis of GO plants indicated that H₂O₂ is responsible for the observed phenotype. GO plants represent an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation can be modulated by growing the plants under different ambient conditions. By this, growth under low light or high CO₂ concentrations minimizes the oxygenase activity of RubisCO and thus the flux through GO whereas the exposition to high light intensities enhances photorespiration and thus the flux through GO.Here, we explored the impact of H₂O₂ production on the primary metabolism of GO plants by assessing the relative levels of various metabolites by gas chromatography coupled to mass spectrometry (GC-MS)⁶ in rosettes of plants grown at low light (30 µmol quanta m⁻² s⁻¹) and after exposing the plants for 7 h to high light (600 µmol quanta m⁻² s⁻¹). The results obtained for the GO5 line are shown in After 1 h at 30 µEAfter 7 h at 600 µEAlanine0.88 ± 0.052.83 ± 0.68Asparagine1.39 ± 0.123.64 ± 0.21Aspartate0.88 ± 0.031.65 ± 0.10GABA1.14 ± 0.051.13 ± 0.05Glutamate0.97 ± 0.041.51 ± 0.07Glutamine1.06 ± 0.111.87 ± 0.06Glycine1.23 ± 0.070.30 ± 0.02Isoleucine3.52 ± 0.403.00 ± 0.15Leucine1.36 ± 0.220.57 ± 0.06Lysine1.49 ± 0.130.38 ± 0.02Methionine0.96 ± 0.054.54 ± 0.51Phenylalanine0.95 ± 0.030.94 ± 0.04Proline1.32 ± 0.221.60 ± 0.13Serine1.05 ± 0.041.49 ± 0.15Threonine4.74 ± 0.175.51 ± 0.34Valine0.91 ± 0.130.29 ± 0.02Citrate/Isocitrate0.65 ± 0.020.64 ± 0.022-oxoglutarate0.95 ± 0.110.76 ± 0.05Succinate0.78 ± 0.040.72 ± 0.02Fumarate0.64 ± 0.030.31 ± 0.01Malate0.74 ± 0.030.60 ± 0.02Pyruvate1.19 ± 0.280.79 ± 0.04Ascorbate1.13 ± 0.142.44 ± 0.45Galactonate-γ-lactone1.81 ± 0.401.62 ± 0.28Fructose1.20 ± 0.130.37 ± 0.01Glucose1.38 ± 0.170.30 ± 0.01Mannose0.90 ± 0.271.34 ± 0.28Sucrose1.04 ± 0.070.49 ± 0.02Fructose-6P0.82 ± 0.151.20 ± 0.15Glucose-6P0.87 ± 0.061.25 ± 0.183-PGA1.13 ± 0.110.35 ± 0.02DHAP1.38 ± 0.091.26 ± 0.08Glycerate0.99 ± 0.040.67 ± 0.01Glycerol1.07 ± 0.041.12 ± 0.05Shikimate1.18 ± 0.040.35 ± 0.01Salicylic acid1.04 ± 0.180.66 ± 0.18Open in a separate windowPlants were grown at 30 µmol m⁻² sec⁻¹ (30 µE). The samples were collected 1 h after the onset of the light period and after 7 h of exposure to 600 µmol m⁻² sec⁻¹ (600 µE), respectively. The values are relative to the respective wild-type (each metabolite = 1) and represent means ± SE of four determinations of eight plants. (*) indicates the value is significantly different from the respective wild-type as determined by the Student''s t test (p < 0.05).At the beginning of the light period in low light conditions, some significant deviations in the levels of metabolites tested were observed in GO plants when compared to the wild-type (2 the transgenic GO activity is sufficient to induce a characteristic metabolic phenotype (Fig. 1). The levels of the tricarboxylic acid (TCA) cycle intermediates, citrate/isocitrate, succinate, fumarate and malate were lower in the GO plants (7 In consequence, OAA might not freely enter the TCA cycle and is redirected to the synthesis of Lys, Thr and Ile, which accumulate in the GO plants (Open in a separate windowFigure 1Simplified scheme of the primary metabolism showing the qualitative variations in metabolite abundance in GO plants obtained by GC-MS analysis (2 Blue boxes indicate a significant increase in the content of the particular metabolite compared to the wild-type, while red boxes indicate a significant decrease. Metabolites without boxes have not been determined. The arrows do not always indicate single steps. Adapted from Baxter et al., 2007.High light treatment induced massive changes in the metabolic profile of GO plants (Fig. 1). The OAA-derived amino acids Asp, Asn, Thr, Ile and Met as well as the 2-oxoglutarate-derived amino acids Glu and Gln accumulated. On the contrary, the levels of the Pyr-derived amino acids Val and Leu and the OAA-derived amino acid Lys decreased. A rational explanation for these metabolic changes is difficult to assess, but these changes could be a consequence of a metabolic reconfiguration in response to high light leading to required physiological functions and thus ensuring continued cellular function and survival, e.g., production of secondary metabolites to mitigate photooxidative damage. The higher levels of Glu observed in the GO plants could be attributed to alternative pathways of glyoxylate metabolism that may occur during photorespiration.⁸ It has been shown earlier that isocitrate derived from glyoxylate and succinate is decarboxylated by cytosolic isocitrate dehydrogenase producing 2-oxoglutarate and further glutamate.⁸In GO plants grown under low light conditions (minimized photorespiratory conditions), the levels of Gly were similar to those of the wild-type whereas, after exposure to high light (photorespiratory conditions), the Gly levels were extremely low, indicating that the GO activity diverts a significant portion of flux from the photorespiratory pathway (7 and also the levels of the lipoic acid-containing subunits of the pyruvate- and 2-oxoglutarate dehydrogenases were shown to be significantly reduced under oxidative stress conditions.^9,10 Similarly, the contents of the soluble sugars sucrose, fructose and glucose and those of 3-PGA and glycerate were lower. In addition, the GO plants showed an impairment in the accumulation of starch under high light conditions, a feature that was not observed if the plants were grown under non-photorespiratory conditions.²Together, these results indicate that the low photosynthetic carbon assimilation in the GO plants exposed to high light is most probably due to enhanced photoinhibition,² the repression of genes encoding photosynthetic components by H₂O₂,^11–13 and the direct damage or inhibition of enzyme activities involved in CO₂ assimilation and energy metabolism by H₂O₂.^7,10,14,15 Moreover, Scarpeci and Valle¹³ showed that in plants treated with the superoxid anion radical producing methylviologen (MV) most of the genes involved in phosphorylytic starch degradation, e.g., the trioseP/Pi translocator and genes involved in starch and sucrose synthesis were repressed, while genes involved in hydrolytic starch breakdown and those involved in sucrose degradation were induced. In line with this, the contents of carbohydrates were also lower in MV-treated plants. Together, these observations can also explain the lower growth rates of the GO plants in conditions where the oxygenase activity of RubisCO becomes important and thus, the flux through GO increases.²The levels of shikimate were lower in GO plants (2^,16 and the low levels of substrates available, as anthocyanins are ultimately synthesized from photosynthates and the GO plants showed a diminished photosynthetic performance.²As expected, the levels of ascorbate and its precursor, galactonate-γ-lactone, were enhanced in the GO plants clearly showing the activation of the cellular antioxidant machinery (10 described the metabolic response to oxidative stress of heterotrophic Arabidopsis cells treated with menadione, which also generates superoxide anion radicals. This oxidative stress was shown to induce metabolic inhibition of flux through the TCA cycle and sectors of amino acid metabolism together with a diversion of carbon into the oxidative pentose phosphate pathway.Signaling and oxidative-damage effects are difficult to separate by manipulating the enzymes of antioxidant systems. In this regard, the GO plants represent a challenging inducible model that avoid acclimatory and adaptative effects. Moreover, it is possible to control the H₂O₂ production in the chloroplasts of GO plants without inducing oxidative damage by changing the conditions of growth.² Further exploration of metabolic changes imposed by different ROS at the cellular and whole organ levels will allow to address many intriguing questions on how plants can rearrange metabolism to cope with oxidative stresses.     

Environmental Isolates of Burkholderia pseudomallei in Ceará State,Northeastern Brazil

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. ​(Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. ​(Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table ​(Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth

Evolution and Function of the Plant Cell Wall Synthesis-Related Glycosyltransferase Family 8

Carbohydrate-active enzyme glycosyltransferase family 8 (GT8) includes the plant galacturonosyltransferase1-related gene family of proven and putative α-galacturonosyltransferase (GAUT) and GAUT-like (GATL) genes. We computationally identified and investigated this family in 15 fully sequenced plant and green algal genomes and in the National Center for Biotechnology Information nonredundant protein database to determine the phylogenetic relatedness of the GAUTs and GATLs to other GT8 family members. The GT8 proteins fall into three well-delineated major classes. In addition to GAUTs and GATLs, known or predicted to be involved in plant cell wall biosynthesis, class I also includes a lower plant-specific GAUT and GATL-related (GATR) subfamily, two metazoan subfamilies, and proteins from other eukaryotes and cyanobacteria. Class II includes galactinol synthases and plant glycogenin-like starch initiation proteins that are not known to be directly involved in cell wall synthesis, as well as proteins from fungi, metazoans, viruses, and bacteria. Class III consists almost entirely of bacterial proteins that are lipooligo/polysaccharide α-galactosyltransferases and α-glucosyltransferases. Sequence motifs conserved across all GT8 subfamilies and those specific to plant cell wall-related GT8 subfamilies were identified and mapped onto a predicted GAUT1 protein structure. The tertiary structure prediction identified sequence motifs likely to represent key amino acids involved in catalysis, substrate binding, protein-protein interactions, and structural elements required for GAUT1 function. The results show that the GAUTs, GATLs, and GATRs have a different evolutionary origin than other plant GT8 genes, were likely acquired from an ancient cyanobacterium (Synechococcus) progenitor, and separate into unique subclades that may indicate functional specialization.Plant cell walls are composed of three principal types of polysaccharides: cellulose, hemicellulose, and pectin. Studying the biosynthesis and degradation of these biopolymers is important because cell walls have multiple roles in plants, including providing structural support to cells and defense against pathogens, serving as cell-specific developmental and differentiation markers, and mediating or facilitating cell-cell communication. In addition to their important roles within plants, cell walls also have many economic uses in human and animal nutrition and as sources of natural textile fibers, paper and wood products, and components of fine chemicals and medicinal products. The study of the biosynthesis and biodegradation of plant cell walls has become even more significant because cell walls are the major components of biomass (Mohnen et al., 2008), which is the most promising renewable source for the production of biofuels and biomaterials (Ragauskas et al., 2006; Pauly and Keegstra, 2008). Analyses of fully sequenced plant genomes have revealed that they encode hundreds or even thousands of carbohydrate-active enzymes (CAZy; Henrissat et al., 2001; Yokoyama and Nishitani, 2004; Geisler-Lee et al., 2006). Most of these CAZy enzymes (Cantarel et al., 2009) are glycosyltransferases (GTs) or glycoside hydrolases, which are key players in plant cell wall biosynthesis and modification (Cosgrove, 2005).The CAZy database is classified into 290 protein families (www.cazy.org; release of September 2008), of which 92 are GT families (Cantarel et al., 2009). A number of the GT families have been previously characterized to be involved in plant cell wall biosynthesis. For example, the GT2 family is known to include cellulose synthases and some hemicellulose backbone synthases (Lerouxel et al., 2006), such as mannan synthases (Dhugga et al., 2004; Liepman et al., 2005), putative xyloglucan synthases (Cocuron et al., 2007), and mixed linkage glucan synthases (Burton et al., 2006). With respect to the synthesis of xylan, a type of hemicellulose, four Arabidopsis (Arabidopsis thaliana) proteins from the GT43 family, irregular xylem 9 (IRX9), IRX14, IRX9-L, and IRX14-L, and two proteins from the GT47 family, IRX10 and IRX10-L, are candidates (York and O''Neill, 2008) for glucuronoxylan backbone synthases (Brown et al., 2007, 2009; Lee et al., 2007a; Peña et al., 2007; Wu et al., 2009). In addition, three proteins have been implicated in the synthesis of an oligosaccharide thought to act either as a primer or terminator in xylan synthesis (Peña et al., 2007): two from the GT8 family (IRX8/GAUT12 [Persson et al., 2007] and PARVUS/GATL1 [Brown et al., 2007; Lee et al., 2007b]) and one from the GT47 family (FRA8/IRX7 [Zhong et al., 2005]).The GT families involved in the biosynthesis of pectins have been relatively less studied until recently. In 2006, a gene in CAZy family GT8 was shown to encode a functional homogalacturonan α-galacturonosyltransferase, GAUT1 (Sterling et al., 2006). GAUT1 belongs to a 25-member gene family in Arabidopsis, the GAUT1-related gene family, that includes two distinct but closely related families, the galacturonosyltransferase (GAUT) genes and the galacturonosyltransferase-like (GATL) genes (Sterling et al., 2006). Another GAUT gene, GAUT8/QUA1, has been suggested to be involved in pectin and/or xylan synthesis, based on the phenotypes of plant lines carrying mutations in this gene (Bouton et al., 2002; Orfila et al., 2005). It has further been suggested that multiple members of the GT8 family are galacturonosyltransferases involved in pectin and/or xylan biosynthesis (Mohnen, 2008; Caffall and Mohnen, 2009; Caffall et al., 2009).Aside from the 25 GAUT and GATL genes, Arabidopsis has 16 other family GT8 genes, according to the CAZy database, which do not seem to have the conserved sequence motifs found in GAUTs and GATLs: HxxGxxKPW and GLG (Sterling et al., 2006). Eight of these 16 genes are annotated as galactinol synthase (GolS) by The Arabidopsis Information Resource (TAIR; www.arabidopsis.org), and three of these AtGolS enzymes have been implicated in the synthesis of raffinose family oligosaccharides that are associated with stress tolerance (Taji et al., 2002). The other eight Arabidopsis GT8 genes are annotated as plant glycogenin-like starch initiation proteins (PGSIPs) in TAIR. PGSIPs have been proposed to be involved in the synthesis of primers necessary for starch biosynthesis (Chatterjee et al., 2005). Hence, the GT8 family is a protein family consisting of enzymes with very distinct proven and proposed functions. Indeed, a suggestion has been made to split the GT8 family into two groups (Sterling et al., 2006), namely, the cell wall biosynthesis-related genes (GAUTs and GATLs) and the non-cell wall synthesis-related genes (GolSs and PGSIPs).We are interested in further defining the functions of the GAUT and GATL proteins in plants, in particular their role(s) in plant cell wall synthesis. The apparent disparate functions of the GT8 family (i.e. the GAUTs and GATLs as proven and putative plant cell wall polysaccharide biosynthetic α-galacturonosyltransferases, the eukaryotic GolSs as α-galactosyltransferases that synthesize the first step in the synthesis of the oligosaccharides stachyose and raffinose, the putative PGSIPs, and the large bacterial GT8 family of diverse α-glucosyltransferases and α-galactosyltransferases involved in lipopolysaccharide and lipooligosaccharide synthesis) indicate that the GT8 family members are involved in several unique types of glycoconjugate and glycan biosynthetic processes (Yin et al., 2010). This observation led us to ask whether any of the GT8 family members are sufficiently closely related to GAUT and GATL genes to be informative regarding GAUT or GATL biosynthetic function(s) and/or mechanism(s).To investigate the relatedness of the members of the GT8 gene family, we carried out a detailed phylogenetic analysis of the entire GT8 family in 15 completely sequenced plant and green algal genomes (AbbreviationCladeSpeciesGenome PublishedDownloaded frommpcGreen algaeMicromonas pusilla CCMP1545Worden et al. (2009)JGI version 2.0mprGreen algaeMicromonas strain RCC299Worden et al. (2009)JGI version 2.0olGreen algaeOstreococcus lucimarinusPalenik et al. (2007)JGI version 1.0otGreen algaeOstreococcus tauriDerelle et al. (2006)JGI version 1.0crGreen algaeChlamydomonas reinhardtiiMerchant et al. (2007)JGI version 3.0vcGreen algaeVolvox carteri f. nagariensisNoJGI version 1.0ppMossPhyscomitrella patens ssp. patensRensing et al. (2008)JGI version 1.1smSpike mossSelaginella moellendorffiiNoJGI version 1.0ptDicotPopulus trichocarpaTuskan et al. (2006)JGI version 1.1atDicotArabidopsis thalianaArabidopsis Genome Initiative (2000)TAIR version 9.0vvDicotVitis viniferaJaillon et al. (2007)http://www.genoscope.cns.fr/gmDicotGlycine maxSchmutz et al. (2010)JGI version 1.0osMonocotOryza sativaGoff et al. (2002); Yu et al. (2002)TIGR version 6.1sbMonocotSorghum bicolorPaterson et al. (2009)JGI version 1.0bdMonocotBrachypodium distachyonVogel et al. (2010)JGI version 1.0Open in a separate window     

Growth of Arthrobacter sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen

Arthrobacter sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.Nitroglycerin (NG) is manufactured widely for use as an explosive and a pharmaceutical vasodilator. It has been found as a contaminant in soil and groundwater (7, 9). Due to NG''s health effects as well as its highly explosive nature, NG contamination in soils and groundwater poses a concern that requires remedial action (3). Natural attenuation and in situ bioremediation have been used for remediation in soils contaminated with certain other explosives (16), but the mineralization of NG in soil and groundwater has not been reported.To date, no pure cultures able to grow on NG as the sole carbon, energy, and nitrogen source have been isolated. Accashian et al. (1) observed growth associated with the degradation of NG under aerobic conditions by a mixed culture originating from activated sludge. The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3, 9, 11, 20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3, 6, 9, 11, 20), and the enzymes involved in denitration have been characterized in some detail (4, 8, 15, 21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8, 9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8, 9), and Agrobacterium radiobacter (20).Here we describe the isolation of bacteria able to degrade NG as the sole source of carbon, nitrogen, and energy. The inoculum for selective enrichment was soil historically contaminated with NG obtained at a facility that formerly manufactured explosives located in the northeastern United States. The enrichment medium consisted of minimal medium prepared as previously described (17) supplemented with NG (0.26 mM), which was synthesized as previously described (18). During enrichment, samples of the inoculum (optical density at 600 nm [OD₆₀₀] ∼ 0.03) were diluted 1/16 in fresh enrichment medium every 2 to 3 weeks. Isolates were obtained by dilution to extinction in NG-supplemented minimal medium. Cultures were grown under aerobic conditions in minimal medium at pH 7.2 and 23°C or in tryptic soy agar (TSA; 1/4 strength).Early stages of enrichment cultures required extended incubation with lag phases of over 200 h and exhibited slow degradation of NG (less than 1 μmol substrate/mg protein/h). After a number of transfers over 8 months, the degradation rates increased substantially (2.2 μmol substrate/mg protein/h). A pure culture capable of growth on NG was identified based on 16S rRNA gene analysis (504 bp) as an Arthrobacter species with 99.5% similarity to Arthrobacter pascens (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU246730","term_id":"289474572","term_text":"GU246730"}}GU246730). Purity of the cultures was confirmed microscopically and by formation of a single colony type on TSA plates. 16S gene sequencing and identification were done by MIDI Labs (Newark, DE) and SeqWright DNA Technology Services (Houston, TX). The Arthrobacter cells stained primarily as Gram-negative rods with a small number of Gram-positive cocci (data not shown); Gram variability is also a characteristic of the closely related Arthrobacter globiformis (2, 19). The optimum growth temperature is 30°C, and the optimum pH is 7.2. Higher pH values were not investigated because NG begins to undergo hydrolysis above pH 7.5 (data not shown). The isolated culture can grow on glycerol, acetate, succinate, citrate, and lactate, with nitrite as the nitrogen source. Previous authors described an Arthrobacter species able to use NG as a nitrogen source in the presence of additional sources of carbon. However, only dinitroesters were formed, and complete mineralization was not achieved (9).To determine the degradation pathway, cultures of the isolated strain (5 ml of inoculum grown on NG to an OD₆₀₀ of 0.3) were grown in minimal medium (100 ml) supplemented with NG at a final concentration of 0.27 mM. Inoculated bottles and abiotic controls were continuously mixed, and NG, 1,2DNG, 1,3DNG, 1MNG, 2MNG, nitrite, nitrate, CO₂, total protein, and optical density were measured at appropriate intervals. Nitroesters were analyzed with an Agilent high-performance liquid chromatograph (HPLC) equipped with an LC-18 column (250 by 4.6 mm, 5 μm; Supelco) and a UV detector at a wavelength of 214 nm (13). Methanol-water (50%, vol/vol) was used as the mobile phase at a flow rate of 1 ml/min. Nitrite and nitrate were analyzed with an ion chromatograph (IC) equipped with an IonPac AS14A anion-exchange column (Dionex, CA) at a flow rate of 1 ml/min. Carbon dioxide production was measured with a Micro Oxymax respirometer (Columbus Instruments, OH), and total protein was quantified using the Micro BCA protein assay kit (Pierce Biotechnology, IL) according to manufacturer''s instructions. During the degradation of NG the 1,2DNG concentration was relatively high at 46 and 72 h (Fig. ​(Fig.1).1). 1,3DNG, detected only at time zero, resulted from trace impurities in the NG stock solution. Trace amounts of 1MNG appeared transiently, and trace amounts of 2MNG accumulated and did not disappear. Traces of nitrite at time zero were from the inoculum. The concentration of NG in the abiotic control did not change during the experiment (data not shown).Open in a separate windowFIG. 1.Growth of strain JBH1 on NG. ×, NG; ▵, 1,2DNG; ⋄, 1MNG; □, 2MNG; ○, protein.Results from the experiment described above were used to calculate nitrogen and carbon mass balances (Tables ​(Tables11 and ​and2).2). Nitrogen content in protein was approximated using the formula C₅H₇O₂N (14). Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6, 9, 20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4, 8, 21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5, 12) or from oxidation of nitrite. Cultures supplemented with glycerol or other carbon sources assimilated all of the nitrite (data not shown).

TABLE 1.

Nitrogen mass balance

Peptidoglycan Fine Structure of the Radiotolerant Bacterium Deinococcus radiodurans Sark

Peptidoglycan from Deinococcus radiodurans was analyzed by high-performance liquid chromatography and mass spectrometry. The monomeric subunit was: N-acetylglucosamine–N-acetylmuramic acid–l-Ala–d-Glu-(γ)–l-Orn-[(δ)Gly-Gly]–d-Ala–d-Ala. Cross-linkage was mediated by (Gly)₂ bridges, and glycan strands were terminated in (1→6)anhydro-muramic acid residues. Structural relations with the phylogenetically close Thermus thermophilus are discussed.The gram-positive bacterium Deinococcus radiodurans is remarkable because of its extreme resistance to ionizing radiation (14). Phylogenetically the closest relatives of Deinococcus are the extreme thermophiles of the genus Thermus (4, 11). In 16S rRNA phylogenetic trees, the genera Thermus and Deinococcus group together as one of the older branches in bacterial evolution (11). Both microorganisms have complex cell envelopes with outer membranes, S-layers, and ornithine-Gly-containing mureins (7, 12, 19, 20, 22, 23). However, Deinococcus and Thermus differ in their response to the Gram reaction, having positive and negative reactions, respectively (4, 14). The murein structure for Thermus thermophilus HB8 has been recently elucidated (19). Here we report the murein structure of Deinococcus radiodurans with similar detail.D. radiodurans Sark (23) was used in the present study. Cultures were grown in Luria-Bertani medium (13) at 30°C with aeration. Murein was purified and subjected to amino acid and high-performance liquid chromatography (HPLC) analyses as previously described (6, 9, 10, 19). For further analysis muropeptides were purified, lyophilized, and desalted as reported elsewhere (6, 19). Purified muropeptides were subjected to plasma desorption linear time-of-flight mass spectrometry (PDMS) as described previously (1, 5, 16, 19). Positive and negative ion mass spectra were obtained on a short linear ²⁵²californium time-of-flight instrument (BioIon AB, Uppsala, Sweden). The acceleration voltage was between 17 and 19 kV, and spectra were accumulated for 1 to 10 million fission events. Calibration of the mass spectra was done in the positive ion mode with H⁺ and Na⁺ ions and in the negative ion mode with H⁻ and CN⁻ ions. Calculated m/z values are based on average masses.Amino acid analysis of muramidase (Cellosyl; Hoechst, Frankfurt am Main, Germany)-digested sacculi (50 μg) revealed Glu, Orn, Ala, and Gly as the only amino acids in the muramidase-solubilized material. Less than 3% of the total Orn remained in the muramidase-insoluble fraction, indicating an essentially complete solubilization of murein.Muramidase-digested murein samples (200 μg) were analyzed by HPLC as described in reference 19. The muropeptide pattern (Fig. ​(Fig.1)1) was relatively simple, with five dominating components (DR5 and DR10 to DR13 [Fig. 1]). The muropeptides resolved by HPLC were collected, desalted, and subjected to PDMS. The results are presented in Table ​Table11 compared with the m/z values calculated for best-matching muropeptides made up of N-acetylglucosamine (GlucNAc), N-acetylmuramic acid (MurNAc), and the amino acids detected in the murein. The more likely structures are shown in Fig. ​Fig.1.1. According to the m/z values, muropeptides DR1 to DR7 and DR9 were monomers; DR8, DR10, and DR11 were dimers; and DR12 and DR13 were trimers. The best-fitting structures for DR3 to DR8, DR11, and DR13 coincided with muropeptides previously characterized in T. thermophilus HB8 (19) and had identical retention times in comparative HPLC runs. The minor muropeptide DR7 (Fig. ​(Fig.1)1) was the only one detected with a d-Ala–d-Ala dipeptide and most likely represents the basic monomeric subunit. The composition of the major cross-linked species DR11 and DR13 confirmed that cross-linking is mediated by (Gly)₂ bridges, as proposed previously (20). Open in a separate windowFIG. 1HPLC muropeptide elution patterns of murein purified from D. radiodurans. Muramidase-digested murein samples were subjected to HPLC analysis, and the A₂₀₄ of the eluate was recorded. The most likely structures for each muroeptide as deduced by PDMS are shown. The position of residues in brackets is the most likely one as deduced from the structures of other muropeptides but could not be formally demonstrated. R = GlucNac–MurNac–l-Ala–d-Glu-(γ)→.

TABLE 1

Calculated and measured m/z values for the molecular ions of the major muropeptides from D. radiodurans

Cell Biology of Mitotic Recombination

Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.Homologous recombination (HR) is defined as the homology-directed exchange of genetic information between two DNA molecules (Fig. 1). Mitotic recombination is often initiated by single-stranded DNA (ssDNA), which can arise by several avenues (Mehta and Haber 2014). They include the processing of DNA double-strand breaks by 5′ to 3′ resection, during replication of damaged DNA, or during excision repair (Symington 2014). The ssDNA is bound by replication protein A (RPA) to control its accessibility to the Rad51 recombinase (Sung 1994, 1997a; Sugiyama et al. 1997; Morrical 2014). The barrier to Rad51-catalyzed recombination imposed by RPA can be overcome by a number of mediators, such as BRCA2 and Rad52, which serve to replace RPA with Rad51 on ssDNA, and the Rad51 paralogs Rad55-Rad57 (RAD51B-RAD51C-XRCC2-XRCC3) and the Psy3-Csm2-Shu1-Shu2 complex (SHU) (RAD51D-XRCC2-SWS1), which stabilize Rad51 filaments on ssDNA (see Sung 1997b; Sigurdsson et al. 2001; Martin et al. 2006; Bernstein et al. 2011; Liu et al. 2011; Qing et al. 2011; Amunugama et al. 2013; Zelensky et al. 2014). The Rad51 nucleoprotein filament catalyzes the invasion into a homologous duplex to produce a displacement loop (D-loop) (Fig. 1). At this stage, additional antirecombination functions are exerted by Srs2 (FBH1, PARI), which dissociates Rad51 filaments from ssDNA, and Mph1 (FANCM), which disassembles D-loops (see Daley et al. 2014). Upon Rad51-catalyzed strand invasion, the ATP-dependent DNA translocase Rad54 enables the invading 3′ end to be extended by DNA polymerases to copy genetic information from the intact duplex (Li and Heyer 2009). Ligation of the products often leads to joint molecules (JMs), such as single- or double-Holliday junctions (s/dHJs) or hemicatenanes (HCs), which must be processed to allow separation of the sister chromatids during mitosis. JMs can be dissolved by the Sgs1-Top3-Rmi1 complex (STR) (BTR, BLM-TOP3α-RMI1-RMI2) (see Bizard and Hickson 2014) or resolved by structure-selective nucleases, such as Mus81-Mms4 (MUS81-EME1), Slx1-Slx4, and Yen1 (GEN1) (see Wyatt and West 2014). Mitotic cells favor recombination events that lead to noncrossover events likely to avoid potentially detrimental consequences of loss of heterozygosity and translocations.Open in a separate windowFigure 1.Primary pathways for homology-dependent double-strand break (DSB) repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invade an intact homologous duplex (in red) to prime DNA synthesis. For DSBs that are repaired by the classical double-strand break repair (DSBR) model, the displaced strand from the donor duplex pairs with the 3′ single-stranded DNA (ssDNA) tail at the other side of the break, which primes a second round of DNA synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double-Holliday junction (dHJ) intermediate is generated. The dHJ can be either dissolved by branch migration (indicated by arrows) into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage (indicated by triangles) to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. Alternatively to the double-strand break repair (DSBR) pathway, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB. After fill-in synthesis and ligation, this pathway generates NCO products and is referred to as synthesis-dependent strand annealing (SDSA).

Table 1.

Evolutionary conservation of homologous recombination proteins between Saccharomyces cerevisiae and Homo sapiens

On the Extent of Tyrosine Phosphorylation in Chloroplasts

Reanalysis of published mass spectrometry data on Tyr-phosphorylated chloroplast proteins indicates that the majority of peptide spectrum matches reporting Tyr phosphorylation are ambiguous.Tyr phosphorylation is a controversial issue in plant phosphoproteomics, ever since early analyses reported up to 5% Tyr phosphorylation in Arabidopsis (Arabidopsis thaliana), despite the lack of a classical Tyr kinase in the Arabidopsis genome (Sugiyama et al., 2008; de la Fuente van Bentem and Hirt, 2009). The same controversy extends to the phosphorylation of chloroplast proteins. In the past 20 years, several indications for Tyr phosphorylation in chloroplasts were reported, and Rubisco is annotated as Tyr phosphorylated protein (www.arabidopsis.org). Initially, Tullberg et al. (1998) found the protein Tyr kinase inhibitor genistein to inhibit the phosphorylation of thylakoid membrane proteins. Supported by the observed stability of some thylakoid phosphoproteins against acid and base hydrolysis, a characteristic property of phospho-Tyr, the authors argue that Tyr phosphorylation of thylakoid membrane proteins is vital for short-term acclimation responses. Similar biochemical properties were observed for autophosphorylation of the chloroplast sensor kinase CSK (Puthiyaveetil et al., 2008). Support for Tyr phosphorylation came from the cross-reactivity of thylakoid membrane proteins and Calvin cycle enzymes (e.g. Rubisco) with phospho-Tyr-specific antibodies (Forsberg and Allen, 2001; Fedina et al., 2008; Ghelis et al., 2008). With the same set of methods, no Tyr phosphorylation was observed in mitochondrial proteins (Forsberg and Allen, 2001).The above reported data are indirect hints for Tyr phosphorylation, and none of the applied methods is sufficiently specific to serve as solid evidence. For example, all phospho-Tyr-specific antibodies have significant cross reactivity with phospho-Ser and phospho-Thr when these have an aromatic amino acid in the +1 position (Zerweck et al., 2009). Using phospho-Tyr-specific antibodies, Forsberg and Allen (2001) found genistein inhibition of light-harvesting complex II phosphorylation with a 50% inhibition of initial activity of around 15 µm. Surprisingly, the same inhibition kinetics were observed with phospho-Thr-specific antibodies, suggesting a lack of specificity of either genistein or the phospho-amino acid antibodies, or both. So far, direct proof for Tyr phosphorylation in chloroplasts by phospho-amino acid analyses is missing. However, mass spectrometry-based phosphoproteomics experiments with plant cell extracts reported phospho-Tyr-containing peptides in chloroplasts but surprisingly not in abundant thylakoid membrane proteins or Calvin cycle enzymes. A recent meta-analysis collated data from 27 published studies and several internal data sets, resulting in a cumulative data set with 5% Tyr-phosphorylated peptides in the entire data set and 12% to 19% in the mitochondria (van Wijk et al., 2014). In this data set, almost 30% of the plastid phosphoproteins are flagged as Tyr phosphorylated (90 proteins from around 300; see supplemental table 5B in van Wijk et al., 2014), standing in stark contrast to dedicated plastid phosphoproteome analyses that identified less than 1% Tyr phosphorylation in the cellular phosphoproteome and none in chloroplast proteins (Reiland et al., 2009).Many of the phospho-Tyr-containing peptides were identified in analyses that applied multistage activation to elevate fragment ion intensity in spectra dominated by the neutral loss of phosphoric acid from phospho-Ser and/or phospho-Thr, sometimes in combination with searches for the phospho-Tyr-specific immonium ion at mass-to-charge ratio 216.0426 (see table 1 in van Wijk et al., 2014). In one instance, phospho-Tyr-specific antibodies were used to enrich Tyr phosphorylated proteins from Arabidopsis full cell extracts (Mithoe et al., 2012). Remarkably, there is almost no overlap in phospho-Tyr peptide identification between the different studies (Mithoe et al., 2012; Wu et al., 2013; van Wijk et al., 2014). Although this could be the result of diverse acquisition methods, enrichment strategies, and data interpretation software in different analyses (Bodenmiller et al., 2007), the low reproducibility and the discrepancies in phospho-Tyr detection among different analyses require further attention, because both are characteristic for incorrect peptide spectrum matches. This is a specific problem here, because false discovery rates (FDRs) accumulate in cumulated data sets.Therefore, we decided to assess the quality of matches to Tyr phosphorylated peptides by a dedicated reanalysis of the original data and benchmarked the robustness of peptide identification by using different software tools for spectra interpretation. Different tools use different scoring schemes to calculate identification probabilities from the fragment ion spectrum; however, they all use basic rules for spectrum matching, such as consecutive b- or y-ion series, matching of the highest intensity peaks to peptide fragments, and identification of matches to plausible derivatives of the major fragments such as losses of ammonia or water. Because of the differences in scoring the identified fragment ions, software tools may interpret spectra differently. However, since the basic rules for peptide matching apply to all identifications, it is clear that robust and reliable identifications are made by different tools that agree on the same interpretation for a spectrum. A specific aspect in the interpretation of phosphopeptide spectra is the assignment of the exact modification site. Common database matching software is usually unsuitable to distinguish modifications at closely spaced amino acids, and it is insufficiently explicit when spectra do not allow distinguishing between alternatives. To circumvent this problem, specialized software tools were developed that score spectra for alternative phosphorylation sites within the peptide sequence by searching for specific fragment ions supporting one or another phosphorylation site (MacLean et al., 2008; Martin et al., 2010).We extracted from the different data sets Tyr-phosphorylated chloroplast proteins and extracted the spectrum information in the form of a MASCOT generic file (mgf) from either PhosphAT (van Wijk et al., 2014) or PRIDE (Mithoe et al., 2012). This resulted in 139 spectra identifying 53 unique peptides representing putative Tyr phosphorylation sites in 53 chloroplast proteins (Supplemental Table S1A; Supplemental Data Set S1). This set of spectra was reanalyzed with MASCOT to assess the significance of the identifications and two alternative software tools established for database searches: PEAKS, a database matching software with a de novo sequencing option (Ma et al., 2003); and Proteome Discoverer with the search engine SEQUEST (Thermo Scientific). With the original search parameters of dynamic phosphorylation of Ser, Thr, and Tyr, dynamic oxidation of Met, fixed carbamidomethylation of Cys, and maximum of two missed cleavages at mass tolerances for precursor and fragment ion matching of 20 ppm/0.5 D (Wu et al., 2013), 50 ppm/0.8 D (internal data sets in van Wijk et al., 2014), and 10 ppm/0.9 D (Mithoe et al., 2012), 11 out of 53 unique peptides were identified with the reported amino acid sequence above the MASCOT significance threshold of P < 0.05, while 42 mgf matchings were reported as insignificant or gave rise to an unrelated peptide identification (www.matrixscience.com; Supplemental Table S1B). The lack of significance correlates with the relaxed search parameters and the many degrees of freedom allowed for peptide matching. With a variation of the above mass tolerance settings, PEAKS identified five (9%) and Proteome Discoverer identified 11 (21%) out of 53 peptides from the data set at a fully relaxed FDR, of which three (6%) identifications by PEAKS and one (2%) by Proteome Discoverer were significant (van Wijk et al., 2014)Presented are those peptides that were identified at least once with one of the alternative tools at one of the indicated mass tolerance settings: precursor tolerance/tandem mass spectrometry/ion match tolerance 50 ppm/0.8 D, 20 ppm/0.5 D, or 10 ppm/0.9 D. We reported all identifications irrespective of the score. Identifications considered significant are labeled with asterisks. Provided is the FDR at which the identification was made. Proteome Discoverer has two FDR settings: below 1% (stringent) or below 5% (relaxed). All matches above a 5% FDR threshold are considered insignificant. The PEAKS FDR is calculated individually for every peptide. Dashes indicate that the peptide was not identified.

A conifer genome spruces up plant phylogenomics

The Norway spruce genome provides key insights into the evolution of plant genomes, leading to testable new hypotheses about conifer, gymnosperm, and vascular plant evolution.In the past year a burst of plant genome sequences have been published, providing enhanced phylogenetic coverage of green plants (Figure ​(Figure1)1) and inclusion of new agricultural, ecological, and evolutionary models. Collectively, these sequences are revealing some extraordinary structural and evolutionary attributes in plant genomes. Perhaps most surprising is the exceptionally high frequency of whole-genome duplication (WGD): nearly every genome that has been analyzed has borne the signature of one or more WGDs, with particularly notable events having occurred in the common ancestors of seed plants, of angiosperms, and of core eudicots (the latter ''WGD'' represents two WGDs in close succession) [1,2]. Given this tendency for plant genomes to duplicate and then return to an essentially diploid genetic system (an example is the cotton genomes, which have accumulated the effects of perhaps 15 WGDs [3]), the conservation of genomes in terms of gene number, chromosomal organization, and gene content is astonishing. From the publication of the first plant genome, Arabidopsis thaliana [4], the number of inferred genes has been between 25,000 and 30,000, with many gene families shared across all land plants, although the number of members and patterns of expansion and contraction vary. Furthermore, conserved synteny has been detected across the genomes of diverse angiosperms, despite WGDs, diploidization, and millions of years of evolution.Open in a separate windowFigure 1Simplified phylogeny of land plants, showing major clades and their component lineages. Asterisks indicate species (or lineage) for which whole-genome sequence (or sequences) is (are) available. Increases and decreases in genome size are shown by arrows.Despite the proliferation of genome sequences available for angiosperms, genome-level data for both ferns (and their relatives, collectively termed monilophytes; Figure ​Figure1)1) and gymnosperms have been conspicuously lacking - until recently, with the publication of the genome sequence of the gymnosperm Norway spruce (Picea abies) [5]. The large genome sizes for both monilophytes and gymnosperms have discouraged attempts at genome sequencing and assembly, whereas the smaller genome size of angiosperms has resulted in more genome sequences being available (Table ​(Table1)1) [6]. Because of this limited phylogenetic sample, our understanding of the timing and phylogenetic positions of WGDs, the core number of plant genes, possible conserved syntenic regions, and patterns of expansion and contraction of gene families across both tracheophytes (vascular plants) and across all land plants is imperfect. This sampling problem is particularly acute in analyses of the genes and genomes of seed plants; many hundreds of genes are present in angiosperms that are not present in mosses or lycophytes, but whether these genes arose in the common ancestor of seed plants or of angiosperms cannot be determined without a gymnosperm genome sequence. The Norway spruce genome therefore offers tremendous power, not only for understanding the structure and evolution of conifer genomes, but also as a reference for interpreting gene and genome evolution in angiosperms.

Table 1

Genome sizes in land plants

Antimicrobial Activity of Simulated Solar Disinfection against Bacterial,Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m⁻²) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log₁₀ after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log₁₀ after 6 h) was obtained only in the presence of riboflavin and 250 W m⁻² irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B₂) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 10⁶/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m⁻²) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m⁻² alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table ​Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table ​(Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)