RESUMEN
The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.
Asunto(s)
Eritritol , Eritritol/análogos & derivados , Populus , Fosfatos de Azúcar , Transferasas , Populus/genética , Populus/metabolismo , Populus/enzimología , Eritritol/metabolismo , Fosfatos de Azúcar/metabolismo , Transferasas/metabolismo , Transferasas/genética , Hemiterpenos/metabolismo , Butadienos/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Pentanos/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Computational biology is a diverse research field that has gained increasing importance over the last two decades. Broadly, it aims to apply computational approaches to advance our understanding of biological systems. This can take place on multiple levels, for example, by creating computational models of specific biological systems, by developing algorithms that assist in the analysis of experimental data, or by investigating fundamental biological design principles through modelling. The articles in this special issue highlight and review four such distinct applications of computational biology.
Asunto(s)
Biología Computacional , Biología Computacional/métodos , Humanos , Algoritmos , Modelos BiológicosRESUMEN
Thioredoxin, glutaredoxin and peroxiredoxin systems play central roles in redox regulation, signaling and metabolism in cells. In these systems, reducing equivalents from NAD(P)H are transferred by coupled thiol-disulfide exchange reactions to redoxins which then reduce a wide array of targets. However, the characterization of redoxin activity has been unclear, with redoxins regarded as enzymes in some studies and redox metabolites in others. Consequently, redoxin activities have been quantified by enzyme kinetic parameters in vitro, and redox potentials or redox ratios within cells. By analyzing all the reactions within these systems, computational models showed that many kinetic properties attributed to redoxins were due to system-level effects. Models of cellular redoxin networks have also been used to estimate intracellular hydrogen peroxide levels, analyze redox signaling and couple omic and kinetic data to understand the regulation of these networks in disease. Computational modeling has emerged as a powerful complementary tool to traditional redoxin enzyme kinetic and cellular assays that integrates data from a number of sources into a single quantitative framework to accelerate the analysis of redoxin systems.
Asunto(s)
Glutarredoxinas , Oxidación-Reducción , Peroxirredoxinas , Tiorredoxinas , Tiorredoxinas/metabolismo , Humanos , Glutarredoxinas/metabolismo , Peroxirredoxinas/metabolismo , Peroxirredoxinas/química , Simulación por Computador , Cinética , Modelos Biológicos , Animales , Catálisis , Transducción de SeñalRESUMEN
A critical feature of the cellular antioxidant response is the induction of gene expression by redox-sensitive transcription factors. In many cells, activating these transcription factors is a dynamic process involving multiple redox steps, but it is unclear how these dynamics should be measured. Here, we show how the dynamic profile of the Schizosaccharomyces pombe Pap1 transcription factor is quantifiable by three parameters: signal amplitude, signal time and signal duration. In response to increasing hydrogen peroxide concentrations, the Pap1 amplitude decreased while the signal time and duration showed saturable increases. In co-response plots, these parameters showed a complex, non-linear relationship to the mRNA levels of four Pap1-regulated genes. We also demonstrate that hydrogen peroxide and tert-butyl hydroperoxide trigger quantifiably distinct Pap1 activation profiles and transcriptional responses. Based on these findings, we propose that different oxidants and oxidant concentrations modulate the Pap1 dynamic profile, leading to specific transcriptional responses. We further show how the effect of combination and pre-exposure stresses on Pap1 activation dynamics can be quantified using this approach. This method is therefore a valuable addition to the redox signalling toolbox that may illuminate the role of dynamics in determining appropriate responses to oxidative stress.