Biology subjects

Schroda, M.

Publications and source records attributed to Schroda, M..

25 records · Page 2Linked to original sources

bioRxiv2020

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

H2O2 has been recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end we engineered the hypersensitive H2O2 sensor, roGFP2-Tsa2{Delta}CR, as a genetic part for the Chlamydomonas Modular Cloning toolbox. Using previously generated parts, together with new ones, we constructed modules and devices that target the sensor to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and ER. The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H2O2 produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under non-physiological conditions. Our results thus imply the establishment of steep intracellular H2O2 gradients under normal physiological conditions and suggest that the cytosolic complement of H2O2 scavenging enzymes effectively limits H2O2 diffusion. Furthermore, in heat stressed cells, we show that cytosolic H2O2 levels closely mirror temperature up- and downshifts and are independent from PET. We anticipate that these sensors will greatly facilitate future investigations into H2O2 biology in algal and plant cells.

plant biology↗

bioRxiv2020

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the rings lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

plant biology↗

bioRxiv2020

Functional and molecular early enteric biomarkers for Parkinson's disease in mice and men

Parkinsons disease (PD) usually has a late clinical onset. The lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use an -synuclein-overexpressing transgenic (Th-1-SNCA-A30P) mouse model of PD to identify appropriate candidate markers in the gut for early stages of PD before hallmark symptoms begin to manifest. A30P mice did not show alterations in gait parameters at 2 months of age, and these mice were therefore defined as pre-symptomatic A30P mice (psA30P). We discovered early functional motility changes in the gut and early molecular dysregulations in the myenteric plexus of psA30P mice by comparative protein and miRNA profiling and cell culture experiments. We found that the proteins neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are potential biomarkers of early PD that may facilitate timely treatment and/or prevention of PD in men.

neuroscience↗

bioRxiv2020

Overexpression of sedoheptulose-1,7-bisphosphatase enhances photosynthesis in Chlamydomonas reinhardtii and has no effect on the abundance of other Calvin-Benson cycle enzymes

The productivity of plants and microalgae needs to be increased to feed the growing world population and to promote the development of a low-carbon economy. This goal can be achieved by improving photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7 bisphosphatase (SBP1) up to 3-fold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the nuclear transgene represented [~]0.3% of total cell protein. Photosynthetic rate and growth were significantly increased in SBP1-overexpressing lines under high-light and high-CO2 conditions. Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This analysis also revealed that the eleven CBC enzymes represent 11.9% of total cell protein in Chlamydomonas. Here the range of concentrations of CBC enzymes turned out to be much larger than estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates are often but not always higher than the binding site concentrations of the enzymes for which they act as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates for overexpression in subsequent engineering steps.

plant biology↗

bioRxiv2020

Vernalization alters sugar beet (Beta vulgaris) sink and source identities and reverses phloem translocation from taproots to shoots

During vegetative growth, biennial sugar beets maintain a steep gradient between the shoot (source) and the sucrose-storing taproot (sink). To shift from vegetative to generative growth, they require a chilling phase, called vernalization. Here, we studied sugar beet sink-source dynamics upon cold temperature-induced vernalization and revealed a pre-flowering taproot sink to source reversal. This transition is induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in long distance transport system, the phloem. As a key process for this transition, vacuolar sucrose importers and exporters, BvTST2;1 and BvSUT4, are oppositely regulated, leading to re-mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that the now deciphered processes are a prerequisite for bolting. Our data may thus serve dissecting metabolic and developmental triggers for bolting, which are potential targets for genome editing or breeding approaches.

plant biology↗

bioRxiv2019

Identification of chloroplast envelope proteins with critical importance for cold acclimation

The ability of plants to cope with cold temperatures relies on their photosynthetic activity. This already demonstrates that the chloroplast is of utmost importance for cold acclimation and acquisition of freezing tolerance. During cold acclimation, the properties of the chloroplast change markedly. To provide the most comprehensive view of the protein repertoire of chloroplast envelope, we analysed this membrane system in Arabidopsis thaliana using MS-based proteomics. Profiling chloroplast envelope membranes was achieved by a cross comparison of protein intensities across plastid and the enriched membrane fraction both under normal and cold conditions. Multivariable logistic regression models the probabilities for the classification problem to address envelop localization. In total, we identified 38 envelope membrane intrinsic or associated proteins exhibiting altered abundance after cold acclimation. These proteins comprise several solute carries, such as the ATP/ADP antiporter NTT2 (substantially increased abundance) or the maltose exporter MEX1 (substantially decreased abundance). Remarkably, analysis of the frost recovery of ntt loss-of-function and mex1 overexpressor mutants confirmed that the comparative proteome is well suited to identify novel key factors involved in cold acclimation and acquisition of freezing tolerance. Moreover, for proteins with known physiological function we propose scenarios explaining their possible role in cold acclimation. Furthermore, spatial proteomics introduces a novel layer of complexity and enabled the identification of proteins differentially localized at the envelope membrane under the changing environmental regime.

plant biology↗

bioRxiv2018

Hetero-oligomeric CPN60 resembles highly symmetric group I chaperonin structure revealed by Cryo-EM

The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as the model system, we revealed that chloroplast chaperonin is consisted of CPN60, CPN60{beta}1, and CPN60{beta}2, and co-chaperonin is composed of three subunits CPN20, CPN11 and CPN23 in vivo. CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only CPN11/20/23-CPN60{beta}1{beta}2 pair can fully replace GroES/GroEL in E. coli at stringent growth condition. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60:CPN60{beta}1:CPN60{beta}2. The cryo-EM structures of endogenous CPN60{beta}1{beta}2/ADP and CPN60{beta}1{beta}2/co-chaperonin/ADP were solved at resolutions of 4.06 [A] and 3.82[A], respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. The chloroplast co-chaperonin CPN11/20/23 formed seven GroES-like domains through hetero-oligomerization which symmetrically interact with CPN60{beta}1{beta}2. Our structures also reveal an uneven distribution of roof-like structures in the dome-shaped CPN11/20/23 and potentially diversified surface properties in the folding cavity of CPN60{beta}1{beta}2 that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

biochemistry↗