Glutathione synthesis is essential for pollen germination in vitro

BACKGROUND: The antioxidant glutathione fulfills many important roles during plant development, growth and defense in the sporophyte, however the role of this important molecule in the gametophyte generation is largely unclear. Bioinformatic data indicate that critical control enzymes are negligibly transcribed in pollen and sperm cells. Therefore, we decided to investigate the role of glutathione synthesis for pollen germination in vitro in Arabidopsis thaliana accession Col-0 and in the glutathione deficient mutant pad2-1 and link it with glutathione status on the subcellular level. RESULTS: The depletion of glutathione by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, reduced pollen germination rates to 2-5% compared to 71% germination in wildtype controls. The application of reduced glutathione (GSH), together with BSO, restored pollen germination and glutathione contents to control values, demonstrating that inhibition of glutathione synthesis is responsible for the decrease of pollen germination in vitro. The addition of indole-3-acetic acid (IAA) to media containing BSO restored pollen germination to control values, which demonstrated that glutathione depletion in pollen grains triggered disturbances in auxin metabolism which led to inhibition of pollen germination. CONCLUSIONS: This study demonstrates that glutathione synthesis is essential for pollen germination in vitro and that glutathione depletion and auxin metabolism are linked in pollen germination and early elongation of the pollen tube, as IAA addition rescues glutathione deficient pollen

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

Comparison of Ultrastructural Alterations in Peripheral Artery Disease Skeletal Muscle

Peripheral artery disease (PAD) is characterized by obstructed hemodynamics and claudication reducing quality of life and muscle function. A myopathy has been shown to develop in PAD patients and characterization of changes in skeletal muscle needs further elucidation. PURPOSE: To assess myofibrillar ultrastructural changes between control and stage IV PAD patients. METHODS: Twenty-six participants (13 control:13 stage IV) were recruited to take part in this cross-sectional study. The mean(±SD) age, mass, height, and BMI were 53(±11) years, 81(±22) kg, 165(±15) cm, and 30(±1.5) kg/m2. Muscle samples were collected from the gastrocnemius and prepared for transmission electron microscopy. Relative mitochondria area, average mitochondrial size, number of mitochondria/250μm2, relative myofibril area, average m-line length, number of z-discs/250μm2, mitochondria/z-disc, relative lipid droplet area, average lipid droplet size, and number of lipid droplets/250μm2 were measured and averaged for each participant using two-individual micrographs. All variables were statistically assessed using an independent t-test or Mann-Whitney U at a significance value of pRESULTS:Relative mitochondrial area (U=11.534, p2 (t=5.343, p2 (t=-1.902, p=.07) was observed. M-line lengths were shorter for stage IV PAD patients than controls (U=11.543, p2 (U=.037, p=.848). Average mitochondria/z-disc was significantly greater in controls than in stage IV PAD patients (t=5.737, pCONCLUSION:The largest changes seen in PAD skeletal muscle ultrastructure are in the mitochondria number and total mitochondria area. This decrease in mitochondria may explain altered muscle function not accounted for by hemodynamic obstructions

Dynamic compartment specific changes in glutathione and ascorbate levels in Arabidopsis plants exposed to different light intensities

BACKGROUND: Excess light conditions induce the generation of reactive oxygen species (ROS) directly in the chloroplasts but also cause an accumulation and production of ROS in peroxisomes, cytosol and vacuoles. Antioxidants such as ascorbate and glutathione occur in all cell compartments where they detoxify ROS. In this study compartment specific changes in antioxidant levels and related enzymes were monitored among Arabidopsis wildtype plants and ascorbate and glutathione deficient mutants (vtc2-1 and pad2-1, respectively) exposed to different light intensities (50, 150 which was considered as control condition, 300, 700 and 1,500 μmol m(-2) s(-1)) for 4 h and 14 d. RESULTS: The results revealed that wildtype plants reacted to short term exposure to excess light conditions with the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol and an increased activity of catalase in the leaves. Long term exposure led to an accumulation of ascorbate and glutathione mainly in chloroplasts. In wildtype plants an accumulation of ascorbate and hydrogen peroxide (H(2)O(2)) could be observed in vacuoles when exposed to high light conditions. The pad2-1 mutant reacted to long term excess light exposure with an accumulation of ascorbate in peroxisomes whereas the vtc2-1 mutant reacted with an accumulation of glutathione in the chloroplasts (relative to the wildtype) and nuclei during long term high light conditions indicating an important role of these antioxidants in these cell compartments for the protection of the mutants against high light stress. CONCLUSION: The results obtained in this study demonstrate that the accumulation of ascorbate and glutathione in chloroplasts, peroxisomes and the cytosol is an important reaction of plants to short term high light stress. The accumulation of ascorbate and H(2)O(2) along the tonoplast and in vacuoles during these conditions indicates an important route for H(2)O(2) detoxification under these conditions

Marchantia polymorpha model reveals conserved infection mechanisms in the vascular wilt fungal pathogen Fusarium oxysporum

How co-evolution has shaped the interaction between plants andtheir associated microbes remains a central question in organis-mic interactions (Bonfante & Genre, 2010; Delaux & Schor-nack, 2021). Plants have evolved a sophisticated and multilayeredimmune system to ward off potential microbial invaders (Jones& Dangl, 2006; Boller & Felix, 2009). In addition, pathogenshave developed mechanisms allowing them to enter living plants,colonise their tissues and overcome their defence responses.Pathogenicity factors can be either broadly conserved or speciesspecific and include regulators of cell signalling, gene expressionor development, as well as secreted effector molecules that modu-late the host environment (Jongeet al., 2011; Turr aet al., 2014;Weiberget al., 2014; Prestiet al., 2015; Ryder & Talbot, 2015;van der Does & Rep, 2017).A particularly destructive group of plant pathogens are thosecausing vascular wilt diseases, which infect the roots and colonisethe highly protected and nutrient poor niche of the xylem(Yadeta & Thomma, 2013). The ascomycete fungusFusariumoxysporum(Fo) represents a species complex with worldwidedistribution that provokes devastating losses in more than 150different crops (Deanet al., 2012). Fo exhibits a hemibiotrophlifestyle with an initial biotrophic phase characterised by intercel-lular growth in the root cortex, followed by invasion of the vascu-lature and transition to the necrotrophic phase resulting inmaceration and death of the colonised host (Redkaret al., 2021).In the soil, Fo is able to locate roots by sensing secreted plant per-oxidases via its sex pheromone receptors and the cell wallintegrity mitogen activated protein kinase (MAPK) pathway(Turr aet al., 2015). Once inside the root, the fungus secretes asmall regulatory peptide that mimics plant Rapid ALkalinisationFactor (RALF) to induce host alkalisation, which in turn activatesa conserved MAPK cascade that promotes plant invasive growth(Masachiset al., 2016). Additional pathogenicity determinantsinclude transcriptional regulators, fungus/plant cell wall remod-elling components or secondary metabolites, among others(Michielse & Rep, 2009).Individual Fo isolates exhibit host-specific pathogenicity,which is determined by lineage-specific (LS) chromosomes thatencode distinct repertoires of effectors known as Secreted inXylem (Six) (Maet al., 2010; van Damet al., 2016). Some Six proteins appear to primarily target plant defence responses, butcan also be recognised as avirulence factors by specific host recep-tors (Houtermanet al., 2009; Tintoret al., 2020). In addition tothe pathogenic forms, the Fo species complex (FOSC) alsoincludes endophytic isolates such as Fo47, which was isolatedfrom a natural disease suppressive soil (Alabouvette, 1986; Wanget al., 2020). Fo47 colonises plant roots without causing wiltsymptoms and functions as a biological control agent againstpathogenic Fo strains. How vascular wilt fungi such as Fo gainedthe ability to associate with plant hosts and evolved endophyticand pathogenic lifestyles remains poorly understood.The bryophyteMarchantia polymorpha(Mp) belongs to theancient lineage of liverworts and has emerged as the primenonvascular plant model for studying the evolution of molecularplant–microbe interactions (Evo-MPMI), due to its low geneticredundancy, the simplicity of its gene families and an accessiblemolecular genetic toolbox (Ishizakiet al., 2008; Lockhart, 2015;Bowmanet al., 2017; Upsonet al., 2018; Gimenez-Ibanezet al.,2019). Importantly, Mp possesses receptor-like kinases (RLKs),nucleotide binding, leucine-rich repeat receptors (NLRs) and sal-icylic acid (SA) pathway genes similar to those mediatingimmune signalling in angiosperms (Xueet al., 2012; Bowmanet al., 2017), therefore allowing the study of plant–microbe inter-actions across evolutionarily distant land plant lineages such asliverworts and eudicots, which diverged>450 million years ago(Ma) (Carellaet al., 2018). A current shortcoming of Mp is thatonly few pathogen infection models have been developed forin vitropathogenicity assays. These include the fungiXylariacubensisandColletotrichum sp1, the oomycetePhytophthorapalmivoraand the Gram-negative bacteriumPseudomonassyringae(Nelsonet al., 2018; Carellaet al., 2019; Gimenez-Ibanezet al., 2019). A survey of the Mp microbiome identified anumber of fungal endophytes, some of which can also act aspathogens (Matsuiet al., 2019; Nelson & Shaw, 2019). Whetherroot-infecting vascular wilt fungi can colonise this land plant lin-eage, which is evolutionarily distant to eudicots and lacks bothtrue roots and xylem, is currently unknown.Here we established a new pathosystem between Fo and Mp.We find that Fo isolates that are either endophytic or pathogenicon different crops (tomato, banana, cotton) are all able tocolonise and macerate the thallus of this nonvascular plant. Infec-tion of Mp by Fo requires fungal core pathogenicity factors,whereas LS effectors are dispensable suggesting that this vascularwilt fungus employs conserved mechanisms during infection ofevolutionarily distant plant lineages. We further show that thefungal transition from biotrophic intercellular growth tonecrotrophic maceration and sporulation, which on angiospermsrelies on host-specific factors promoting xylem invasion, occursdirectly on the nonvascular plant Mp

The effects of ionizing radiation on the structure and antioxidative and metal binding capacity of the cell wall of microalga Chlorella sorokiniana

The impact of ionizing radiation on microorganisms such as microalgae is a topic of increasing importance for understanding the dynamics of aquatic ecosystems in response to environmental radiation, and for the development of efficient approaches for bioremediation of mining and nuclear power plants wastewaters. Currently, nothing is known about the effects of ionizing radiation on the microalgal cell wall, which represents the first line of defence against chemical and physical environmental stresses. Using various microscopy, spectroscopy and biochemical techniques we show that the unicellular alga Chlorella sorokiniana elicits a fast response to ionizing radiation. Within one day after irradiation with doses of 1–5 Gy, the fibrilar layer of the cell wall became thicker, the fraction of uronic acids was higher, and the capacity to remove the main reactive product of water radiolysis increased. In addition, the isolated cell wall fraction showed significant binding capacity for Cu2+, Mn2+, and Cr3+. The irradiation further increased the binding capacity for Cu2+, which appears to be mainly bound to glucosamine moieties within a chitosan-like polymer in the outer rigid layer of the wall. These results imply that the cell wall represents a dynamic structure that is involved in the protective response of microalgae to ionizing radiation. It appears that microalgae may exhibit a significant control of metal mobility in aquatic ecosystems via biosorption by the cell wall matrix

Subcellular compartmentation of glutathione in dicotyledonous plants

This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method

Immunocytochemical determination of the subcellular distribution of ascorbate in plants

Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress