Biology subjects

Zimmer, D.

Publications and source records attributed to Zimmer, D..

6 recordsLinked to original sources

bioRxiv2023

Degradation of Fatty Acid Export Protein1 by Rhomboid-Like Protease11 Contributes to Cold Tolerance in Arabidopsis

Plants need to adapt to different stresses to optimize growth under unfavorable conditions. The abundance of the chloroplast envelope located Fatty Acid Export Protein1 (FAX1) decreases after the onset of low temperatures. However, it was unclear how FAX1 degradation occurs and whether altered FAX1 abundance contributes to cold tolerance in plants. The rapid cold-induced increase in rhomboid-like protease11 (RBL11) transcript, the physical interaction of RBL11 with FAX1, the specific FAX1 degradation after RBL11 expression, and the absence of cold-induced FAX1 degradation in rbl11 loss-of-function mutants suggest that this enzyme is responsible for FAX1 degradation. Proteomic analyses showed that rbl11 mutants have higher levels of FAX1 and other proteins involved in membrane lipid homeostasis, suggesting that RBL11 is a key element in the remodeling of membrane properties during cold. Consequently, in the cold, rbl11 mutants show a shift in lipid biosynthesis towards the eukaryotic pathway, which coincides with impaired cold tolerance. To demonstrate that cold sensitivity is due to increased FAX1 levels, FAX1 overexpressors were analyzed. rbl11 and FAX1 overexpressor mutants show superimposable phenotypic defects upon exposure to cold temperatures. Our results show that the cold-induced degradation of FAX1 by RBL11 is critical for Arabidopsis to survive cold and freezing periods. One sentence summaryDegradation of the inner envelope protein Fatty Acid Export1 via Rhomboid Like Protease11 represents a critical process to achieve cold and frost tolerance in Arabidopsis

plant biology↗

bioRxiv2022

Proteomics and constraint-based modelling reveal enzyme kinetic properties of Chlamydomonas reinhardtii on a genome scale

Biofuels produced from microalgae offer a promising solution for carbon neutral economy, and integration of turnover numbers into metabolic models can improve the design of metabolic engineering strategies towards achieving this aim. However, the coverage of enzyme turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic model Saccharomyces cerevisiae often used as a cell factory. Here we generated protein abundance data from Chlamydomonas reinhardtii cells grown in various experiments, covering between 2337 and 3708 proteins, and employed these data with constraint-based metabolic modeling approaches to estimate in vivo maximum apparent turnover numbers for this model organism. The gathered data allowed us to estimate maximum apparent turnover numbers for 568 reactions, of which 46 correspond to transporters that are otherwise difficult to characterize. The resulting, largest-to-date catalogue of proxies for in vivo turnover numbers increased the coverage for C. reinhardtii by more than 10-fold. We showed that incorporation of these in vivo turnover numbers into a protein-constrained metabolic model of C. reinhardtii improves the accuracy of predicted enzyme usage in comparison to predictions resulting from the integration on in vitro turnover numbers. Together, the integration of proteomics and physiological data allowed us to extend our knowledge of previously uncharacterized enzymes in the C. reinhardtii genome and subsequently increase predictive performance for biotechnological applications. Significance statementCurrent metabolic modelling approaches rely on the usage of in vitro turnover numbers (kcat) that provide limited information on enzymes operating in their native environment. This knowledge gap can be closed by data-integrative approaches to estimate in vivo kcat values that can improve metabolic modelling and design of metabolic engineering strategies. In this work, we assembled a high-quality proteomics data set containing 27 samples of various culture conditions and strains of Chlamydomonas reinhardtii. We used this resource to create the largest data set of estimates for in vivo turnover numbers to date. Subsequently, we showed that metabolic models parameterized with these estimates provide better predictions of enzyme abundance than those obtained by using in vitro turnover numbers.

systems biology↗

bioRxiv2021

Systems-wide Analysis Revealed Shared and Unique Responses to Moderate and Acute High Temperatures in the Green Alga Chlamydomonas reinhardtii

Different intensities of high temperatures affect the growth of photosynthetic cells in nature. To elucidate the underlying mechanisms, we cultivated the unicellular green alga Chlamydomonas reinhardtii under highly controlled photobioreactor conditions and revealed systems-wide shared and unique responses to 24-hour moderate (35{degrees}C) and acute (40{degrees}C) high temperatures and subsequent recovery at 25{degrees}C. We identified previously overlooked unique elements in response to moderate high temperature. Heat at 35{degrees}C transiently arrested the cell cycle followed by partial synchronization, up-regulated transcripts/proteins involved in gluconeogenesis/glyoxylate-cycle for carbon uptake, promoted growth, and increased starch accumulation. Heat at 40{degrees}C arrested the cell cycle, inhibited growth, resulting in carbon uptake over usage and increased starch accumulation. Both high temperatures induced photoprotection, while 40{degrees}C decreased photosynthetic efficiency, distorted thylakoid/pyrenoid ultrastructure, and affected the carbon concentrating mechanism. We demonstrated increased transcript/protein correlation during both heat treatments, suggesting reduced post-transcriptional regulation during heat may help coordinate heat tolerance activities efficiently. During recovery after both heat treatments, transcripts/proteins related to DNA synthesis increased while those involved in photosynthetic light reactions decreased. We propose down-regulating photosynthetic light reactions during DNA replication benefits cell cycle resumption by reducing ROS production. Our results provide potential targets to increase thermotolerance in algae and crops.

cell biology↗

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↗

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↗

bioRxiv2019

Differential degradation of RNA species by autophagy related pathways in plants

An important function of the plant vacuole is the recycling of the delivered proteins and RNA by autophagy. We provide the first plant vacuolar small RNome by isolation of intact vacuoles from Barley and Arabidopsis, subsequent RNA purification and Next Generation Sequencing. In these vacuolar sRNomes, all types of cellular RNAs were found including those of chloroplast origin, suggesting a bulk-type of RNA transfer to, and breakdown in vacuoles. ATG5 is a major representative of autophagy genes and the vacuolar RNA composition in corresponding knockout plants differed clearly from controls as most chloroplast derived RNA species were missing. Moreover, the read length distribution of RNAs found in ATG5 mutants differed to control samples, indicating altered RNA processing. In contrast, vacuolar RNA length and composition of plants lacking the vacuolar RNase2 (rns2-2), involved in cellular RNA homeostasis, showed minor alterations, only. Our data therefore suggests that mainly autophagy components are responsible for selective transport and targeting of different RNA species into the vacuole for degradation. In addition, mature miRNAs were detected in all vacuolar preparations, however in ATG5 mutants at much lower frequency, indicating a new biological role for vacuolar miRNAs apart from becoming degraded.

plant biology↗