Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding

Effects of exogenous glutathione on suspension callus cells of spruce [Picea abies (L.) Karst.]

Callus cells of Picea abies (L.) Karst, were exposed to different concentrations (50, 100, 500, 1000 μM) of reduced glutathione (GSH) for 48 hours. These physiologically relevant concentrations of glutathione caused changes in the investigated tissue depending on the concentration applied. Feeding of glutathione increased the cellular concentrations of thiols, decreased the rate of cell division, induced mitotic abnormalities, generated increased amounts of micronuclei and affected the cell ultrastructure. The glutathione system in the callus culture cells was measured by a quantitative image analysis method, using histochemical staining by monochlorobimane. This measurement showed an increase of thiols at the cellular level after GSH treatment. Whereas no distinct structural modifications were found in cells treated with 50 and 100 μM, the treatment with 500 and 1000 μM GSH had multiple effects on the investigated tissue in comparison to control cells. Damage observed in the electron microscope involved separation of the plasma membrane from the cell wall, swollen plastids and mitochondria, and heavily granulated chromatin in the nuclei. The investigation of the chromosomal aberrations showed an increased amount of chromosomal defects in the GSH treated cells. The chromosomal aberration types observed most frequently were defects in the form of sticky chromosomes and vagrant chromosomes indicating severe damages in the genetic material

Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato

Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the senescence-delaying hormone trans-zeatin and decreases in the senescence-inducing ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in the leaves. Thus, cwInv critically functions at the integration point of metabolic, hormonal, and stress signals, providing a novel strategy to overcome drought-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions.FPA and co-workers are funded by the Spanish MICINN-FEDER (projects AT2009-0038 and AGL2011-27996) and the European Commission (ROOTOPOWER Contract # 289365). TR and FPA were jointly funded by the Spanish–Austrian bilateral project AT2009-0038. AA was supported by post-doctoral fellowships from the Fundación Séneca (Comunidad Autónoma de la Región de Murcia) and the FWF (Austrian Science Fund), and currently by the JAE DOC Programme

A Genomewide Screen Reveals a Role of Mitochondria in Anaerobic Uptake of Sterols in Yeast

The mechanisms that govern intracellular transport of sterols in eukaryotic cells are not well understood. Saccharomyces cerevisiae is a facultative anaerobic organism that becomes auxotroph for sterols and unsaturated fatty acids in the absence of oxygen. To identify pathways that are required for uptake and transport of sterols, we performed a systematic screen of the yeast deletion mutant collection for genes that are required for growth under anaerobic conditions. Of the ∼4800 nonessential genes represented in the deletion collection, 37 were essential for growth under anaerobic conditions. These affect a wide range of cellular functions, including biosynthetic pathways for certain amino acids and cofactors, reprogramming of transcription and translation, mitochondrial function and biogenesis, and membrane trafficking. Thirty-three of these mutants failed to grow on lipid-supplemented media when combined with a mutation in HEM1, which mimics anaerobic conditions in the presence of oxygen. Uptake assays with radio- and fluorescently labeled cholesterol revealed that 17 of the 33 mutants strongly affect uptake and/or esterification of exogenously supplied cholesterol. Examination of the subcellular distribution of sterols in these uptake mutants by cell fractionation and fluorescence microscopy indicates that some of the mutants block incorporation of cholesterol into the plasma membrane, a presumably early step in sterol uptake. Unexpectedly, the largest class of uptake mutants is affected in mitochondrial functions, and many of the uptake mutants show electron-dense mitochondrial inclusions. These results indicate that a hitherto uncharacterized mitochondrial function is required for sterol uptake and/or transport under anaerobic conditions and are discussed in light of the fact that mitochondrial import of cholesterol is required for steroidogenesis in vertebrate cells