Adaptation of plants to low-oxygen stress

Plants are obligate aerobic organisms and, therefore, need oxygen for survival. However, unlike animals, plants do not possess an active oxygen transport system to supply their organs with oxygen. Hence, oxygen can fall to low levels inside plant tissues if the diffusion of oxygen cannot keep pace with the rate of oxygen consumption. As a consequence, plants have developed special mechanisms to save oxygen. Studies show that plants actively regulate their respiration and metabolism in relation to the internal oxygen concentration. Nevertheless, the sequences of metabolic events leading to this adaptation are not yet known. Therefore, an experiment was developed to treat potato tuber slices with 4% oxygen (v/v) and analyze the metabolic response in a time dependent manner. The low-oxygen treatment led to a rapid inhibition of respiration and a general metabolic depression at different sites, while fermentation was activated at a later point in time. Regulatory sites have been identified in glycolysis and in the tricarboxylic acid (TCA) cycle. The experiments also revealed cytosolic pyruvate kinase (PKc) as important control site in glycolysis. PKc is crucial under low-oxygen conditions as it provides pyruvate for respiration and for fermentation. To further investigate the role of PKc under low-oxygen conditions transgenic potato tubers with decreased expression of PKc mediated by RNA interference were treated with 4% oxygen, and a comprehensive metabolic profile was performed. Indeed, the results indicate that PKc regulates the availability of pyruvate for fermentation, thereby influencing the metabolic performance under low-oxygen. Whereas potato tuber discs are an easy system to manipulate the surrounding oxygen concentration, there are only limited tools available for genetic manipulation. Therefore, Arabidopsis thaliana was used as model system for reverse genetic studies. Transgenic Arabidopsis plants carrying a T-DNA insertion in the catalytic subunit of mitochondrial NAD-dependent isocitrate dehydrogenase (IDH) were used to investigate the importance of the mitochondrial alpha-ketoglutarate provision for the reorganization of the TCA cycle under low-oxygen. The idhv mutant showed an improved low-oxygen tolerance accompanied by specific alterations of hypoxic metabolism compared to wild type, thus, suggesting that mitochondrial alpha-ketoglutarate production through IDH is dispensable under low-oxygen conditions in Arabidopsis. Moreover, the experiments showed that an increased activity of extramitochondrial pathways for 2-oxoglutarate production is beneficial for plant survival under low-oxygen. In addition to the modifications in primary metabolism for an improved survival under low-oxygen, changes in the redox state are also common characteristics of hypoxia. NADPH-dependent thioredoxin reductases (NTRs) modulate the activity of redox-regulated enzymes depending on the cellular redox-state. To explore the role of the NTR system under low-oxygen, a knockout of the plastidial NADPH-dependent thioredoxin reductase (NTRC) and a double knockout of the extraplastidial NADPH-dependent thioredoxin reductase A (NTRA) and NADPH-dependent thioredoxin reductase B (NTRB) in Arabidopsis thaliana were treated with hypoxia, and the relevant redox related parameters were measured. The results show opposed effects of the low-oxygen treatment for the ntrc and the ntrantrb mutant. Whereas the ntrantrb mutant revealed an increased resistance to hypoxia, the ntrc mutant displayed the opposite behavior. Apparently, the plastidial and extraplastidial NTR systems play different roles in the adaptation to low-oxygen, although the underlying reasons for this phenomenon are not yet fully understood. A further area of plant metabolism being affected by low-oxygen is the cellular energy status. With falling oxygen concentrations inside the cell the production of ATP through respiration decreases and the energy status declines. This in turn affects the biosynthesis pathways and, ultimately, the plant growth which needs to be adjusted to the energy deficit. A possible regulator that connects energy homeostasis with plant growth is the sucrose non-fermenting-1-related protein kinase (SnRK1). Transgenic Arabidopsis plants with beta-estradiol inducible transcriptional silencing of the regulatory SNF4 subunit of SnRK1 were used to study the function of SnRK1 under low-oxygen. The transgenic plants displayed a lower anoxic survival rate, a decrease in hypoxia marker genes expression and alterations in primary metabolism compared to wild type. Altogether, these results suggest an important role of SnRK1 in the low-oxygen response in Arabidopsis thaliana

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways

Evaluation of Indirect Fluorescent Antibody Assays Compared to Rapid Influenza Diagnostic Tests for the Detection of Pandemic Influenza A (H1N1) pdm09

Performance of indirect fluorescent antibody (IFA) assays and rapid influenza diagnostic tests (RIDT) during the 2009 H1N1 pandemic was evaluated, along with the relative effects of age and illness severity on test accuracy. Clinicians and laboratories submitted specimens on patients with respiratory illness to public health from April to mid October 2009 for polymerase chain reaction (PCR) testing as part of pandemic H1N1 surveillance efforts in Orange County, CA; IFA and RIDT were performed in clinical settings. Sensitivity and specificity for detection of the 2009 pandemic H1N1 strain, now officially named influenza A(H1N1)pdm09, were calculated for 638 specimens. Overall, approximately 30% of IFA tests and RIDTs tested by PCR were falsely negative (sensitivity 71% and 69%, respectively). Sensitivity of RIDT ranged from 45% to 84% depending on severity and age of patients. In hospitalized children, sensitivity of IFA (75%) was similar to RIDT (84%). Specificity of tests performed on hospitalized children was 94% for IFA and 80% for RIDT. Overall sensitivity of RIDT in this study was comparable to previously published studies on pandemic H1N1 influenza and sensitivity of IFA was similar to what has been reported in children for seasonal influenza. Both diagnostic tests produced a high number of false negatives and should not be used to rule out influenza infection

Performance of the QuickVue Influenza A+B Rapid Test for Pandemic H1N1 (2009) Virus Infection in Adults

To investigate the diagnostic accuracy of the QuickVue® Influenza A+B rapid test we conducted a prospective observational study in which this rapid test was compared with a real-time reverse transcription polymerase chain reaction (RT-PCR) for pandemic influenza A H1N1 (2009) infection in Austrian adults. The sensitivity, specificity, and positive and negative predictive values of the QuickVue test compared with the RT-PCR were 26% (95% CI 18–35), 98% (95% CI 92–100), 94% (95% CI 80–99) and 50% (95% CI 42–58), respectively. The prevalence of pandemic H1N1 (2009) virus infection among the 209 patients included in the study was 57%. Our data suggest that a positive QuickVue test provides considerable information for the diagnosis of pandemic influenza A H1N1 (2009) virus infection in young adults but that a negative QuickVue test result should, if relevant for patient management or public health measures, be verified using PCR

Filopodyan: An open-source pipeline for the analysis of filopodia

Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/ VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time.J.L. Gallop and V. Urbančič are supported by the Wellcome Trust (WT095829AIA). J. Mason and B. Richier are supported by the European Research Council (281971). C.E. Holt is supported by the Wellcome Trust (program grant 085314) and the European Research Council (advanced grant 322817). The Gurdon Institute is funded by the Wellcome Trust (203144) and Cancer Research UK (C6946/A24843)