Periodic Reporting for period 1 - SMFP (Forward and reverse genetic approaches to understanding sphingolipid metabolism and functions in plants using the model bryophyte Physcomitrella patens)
Reporting period: 2021-09-01 to 2023-08-31
Sphingolipids are ubiquitous and essential components of eukaryotic cells. Analytical profiling of their content in different biological systems is limited by difficulties in extracting them, and detection and quantification is limited by their large, amphipathic, and complex structures. Studies of the precise functions of sphingolipids in vivo is also challenging, here due to lethality of mutants generated for study using standard knock-out mutagenesis, and well as pleiotropic phenotypes of non-lethal mutants, or RNAi lines.
The metabolism and functions of sphingolipids is fundamental knowledge that we need for understanding and interpreting broader biological processes. This includes further basic, foundational research as well as applied investigation into, for example, programmed cell death in plants that is associated with accumulation of simple sphingolipids (ceramides and long chain bases (LCBs)), or human metabolic disorders caused specifically by defects in sphingolipid metabolism (e.g. Tay-Sachs disease, Gaucher disease, and Niemann-Pick disease).
With this action, I set out to (1) better understand sphingolipid metabolism in plants by developing optimized analytical approaches. I joined the Department of Plant Biochemistry led by Prof. Ivo Feussner in Göttingen, as they have a state-of-the-art analytical platform and specialized knowledge and decades of experience working with plant lipids. We sought to specifically develop better methods for analyzing glycosyl inositol phosphorylceramides (GIPCs) the most challenging class of sphingolipids to analyze, but also those which are most abundant in plant tissues. A second objective was to better understand the functions of sphingolipids. Here, I anticipated that using a model system with the simplest and most easily-manipulated developmental patterning and organ structure would facilitate interpretation of complex phenotypes. To that end, I used the model moss Physcomitrium patens (formerly Physcomitrella patens). I set out to optimize CRISPR/Cas9 tools to obtain collections of higher-order mutants in gene families required for GIPC biosynthesis, and also optimize our approach to specifically obtain knock-down genotypes where knock-outs would be lethal. Finally, I set up a forward-genetic screen in P. patens, selecting mutants that were resistant to programmed cell death caused by LCB accumulation after treatment with a fungal inhibitor of sphingolipid biosynthesis. This screen was designed to improve our understanding of the link between sphingolipid content and programmed cell death in plants.
The concrete research output of this action includes improved methods for analysis of sphingolipids, improved methods for genetic manipulation of P. patens and its further development as a model system, and mutant phenotypes that pinpoint functions of GIPCs in plants. These serve the broader research community and have also locally served to establish a highly-productive, specialized work cluster in my host lab in Göttingen. The specialized skill set I have obtained through training with the analytical platform unique to my host lab will support my next career step and help me obtain an independent position.
The metabolism and functions of sphingolipids is fundamental knowledge that we need for understanding and interpreting broader biological processes. This includes further basic, foundational research as well as applied investigation into, for example, programmed cell death in plants that is associated with accumulation of simple sphingolipids (ceramides and long chain bases (LCBs)), or human metabolic disorders caused specifically by defects in sphingolipid metabolism (e.g. Tay-Sachs disease, Gaucher disease, and Niemann-Pick disease).
With this action, I set out to (1) better understand sphingolipid metabolism in plants by developing optimized analytical approaches. I joined the Department of Plant Biochemistry led by Prof. Ivo Feussner in Göttingen, as they have a state-of-the-art analytical platform and specialized knowledge and decades of experience working with plant lipids. We sought to specifically develop better methods for analyzing glycosyl inositol phosphorylceramides (GIPCs) the most challenging class of sphingolipids to analyze, but also those which are most abundant in plant tissues. A second objective was to better understand the functions of sphingolipids. Here, I anticipated that using a model system with the simplest and most easily-manipulated developmental patterning and organ structure would facilitate interpretation of complex phenotypes. To that end, I used the model moss Physcomitrium patens (formerly Physcomitrella patens). I set out to optimize CRISPR/Cas9 tools to obtain collections of higher-order mutants in gene families required for GIPC biosynthesis, and also optimize our approach to specifically obtain knock-down genotypes where knock-outs would be lethal. Finally, I set up a forward-genetic screen in P. patens, selecting mutants that were resistant to programmed cell death caused by LCB accumulation after treatment with a fungal inhibitor of sphingolipid biosynthesis. This screen was designed to improve our understanding of the link between sphingolipid content and programmed cell death in plants.
The concrete research output of this action includes improved methods for analysis of sphingolipids, improved methods for genetic manipulation of P. patens and its further development as a model system, and mutant phenotypes that pinpoint functions of GIPCs in plants. These serve the broader research community and have also locally served to establish a highly-productive, specialized work cluster in my host lab in Göttingen. The specialized skill set I have obtained through training with the analytical platform unique to my host lab will support my next career step and help me obtain an independent position.
For the first objective, improving our analytical approaches, we made substantial progress. We tried, compared, and selected optimal extraction techniques for sphingolipids. We tested and selected chemical treatments to enrich for sphingolipids and reduce background/non-specific signals in our MS analysis. We compared and selected methods for lipid quantification and signal normalization, and performed structural validation of unusual and/or novel lipid species using both positive and negative ionization, product ion scans, and GC-MS analysis of some hydrolysed lipid moieties.
For the second objective, improving our understanding of the functions of sphingolipids, we were successful in developing genetic tools and obtaining a vast collection of useful mutants in P. patens, sepcifically deficient in INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS), SPHINGOMYELIN SYNTHASE-LIKE (SMS), INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE (IPUT), and GOLGI NUCLEOTIDE SUGAR TRANSPORTER (GONST) activity. The mutants displayed a spectrum of unique biochemical phenotypes, and growth phenotypes including stunting, reduced cell division, plasmodesmatal structural defects, and abnormalities in cell wall formation. Results are reported in our pre-print, https://doi.org/10.1101/2023.09.20.558677(opens in new window) while the manuscript is currently in peer-review. Two additional publications reporting on our findings are in the final stages of experimentation in the lab.
Our pipeline used protoplasted cells for UV-mutagenesis. We tried different methods for selecting mutants for our screen, and finally used resistance to the mycotoxin fumonisin B1 (FB1). The screen was preformed twice, once in the Gransden 2004 genetic background, and a second time in Gransden 2017. This was due to fertility defects in the Gransden 2004 strain that prohibited us from mapping the mutant loci responsible for FB1 resistance. The biochemical phenotypes of the mutants were investigated, however, we did not progress to mapping the gene loci of interest. This was primarily due to the work required for WP1 of this grant, and the unexpected directions this project took.
For the second objective, improving our understanding of the functions of sphingolipids, we were successful in developing genetic tools and obtaining a vast collection of useful mutants in P. patens, sepcifically deficient in INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS), SPHINGOMYELIN SYNTHASE-LIKE (SMS), INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE (IPUT), and GOLGI NUCLEOTIDE SUGAR TRANSPORTER (GONST) activity. The mutants displayed a spectrum of unique biochemical phenotypes, and growth phenotypes including stunting, reduced cell division, plasmodesmatal structural defects, and abnormalities in cell wall formation. Results are reported in our pre-print, https://doi.org/10.1101/2023.09.20.558677(opens in new window) while the manuscript is currently in peer-review. Two additional publications reporting on our findings are in the final stages of experimentation in the lab.
Our pipeline used protoplasted cells for UV-mutagenesis. We tried different methods for selecting mutants for our screen, and finally used resistance to the mycotoxin fumonisin B1 (FB1). The screen was preformed twice, once in the Gransden 2004 genetic background, and a second time in Gransden 2017. This was due to fertility defects in the Gransden 2004 strain that prohibited us from mapping the mutant loci responsible for FB1 resistance. The biochemical phenotypes of the mutants were investigated, however, we did not progress to mapping the gene loci of interest. This was primarily due to the work required for WP1 of this grant, and the unexpected directions this project took.
Progress beyond the state of the art includes a methodological improvement to our approach for generating and selecting mutants of interest in plants, especially mutants that have formerly been difficult to study due to pleiotropy and non-viability. Mutagenesis and mutant analysis are keystones of molecular genetic research, therefore this is broadly useful in the plant sciences. This in turn supports agricultural and pharmaceutical sciences as these rely on fundamental plant research.
The results this work improved our understanding of the functions of sphingolipids. A better understanding of the functions of cell membranes and their components is valuable for our understanding of basic cell functions. Improvements to foundational knowledge such as this are critical to spur new innovation and approaches in agricultural research and in predicting and controlling plant-pathogen interactions.
The results this work improved our understanding of the functions of sphingolipids. A better understanding of the functions of cell membranes and their components is valuable for our understanding of basic cell functions. Improvements to foundational knowledge such as this are critical to spur new innovation and approaches in agricultural research and in predicting and controlling plant-pathogen interactions.