Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars
Plant phenotyping to date typically comprises morphological and physiological profiling in a high-throughput manner. A powerful method that allows for subcellular characterization of organelle stoichiometric/functional characteristics is still missing. Organelle abundance and crosstalk in cell dynam...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-05-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fpls.2019.00638/full |
| _version_ | 1831561283346890752 |
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| author | Sebastian Schneider Dominik Harant Gert Bachmann Thomas Nägele Thomas Nägele Ingeborg Lang Stefanie Wienkoop |
| author_facet | Sebastian Schneider Dominik Harant Gert Bachmann Thomas Nägele Thomas Nägele Ingeborg Lang Stefanie Wienkoop |
| author_sort | Sebastian Schneider |
| collection | DOAJ |
| description | Plant phenotyping to date typically comprises morphological and physiological profiling in a high-throughput manner. A powerful method that allows for subcellular characterization of organelle stoichiometric/functional characteristics is still missing. Organelle abundance and crosstalk in cell dynamics and signaling plays an important role for understanding crop growth and stress adaptations. However, microscopy cannot be considered a high-throughput technology. The aim of the present study was to develop an approach that enables the estimation of organelle functional stoichiometry and to determine differential subcellular dynamics within and across cultivars in a high-throughput manner. A combination of subcellular non-aqueous fractionation and liquid chromatography mass spectrometry was applied to assign membrane-marker proteins to cell compartmental abundances and functions of Pisum sativum leaves. Based on specific subcellular affiliation, proteotypic marker peptides of the chloroplast, mitochondria and vacuole membranes were selected and synthesized as heavy isotope labeled standards. The rapid and unbiased Mass Western approach for accurate stoichiometry and targeted absolute protein quantification allowed for a proportional organelle abundances measure linked to their functional properties. A 3D Confocal Laser Scanning Microscopy approach was developed to evaluate the Mass Western. Two P. sativum cultivars of varying morphology and physiology were compared. The Mass Western assay enabled a cultivar specific discrimination of the chloroplast to mitochondria to vacuole relations. |
| first_indexed | 2024-12-17T07:37:14Z |
| format | Article |
| id | doaj.art-a84252c00b994ef48d8ee4776a0bd54a |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-17T07:37:14Z |
| publishDate | 2019-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-a84252c00b994ef48d8ee4776a0bd54a2022-12-21T21:58:18ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2019-05-011010.3389/fpls.2019.00638442963Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum CultivarsSebastian Schneider0Dominik Harant1Gert Bachmann2Thomas Nägele3Thomas Nägele4Ingeborg Lang5Stefanie Wienkoop6Division of Molecular Systems Biology, Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaCore Facility Cell Imaging and Ultrastructure Research, University of Vienna, Vienna, AustriaDivision of Molecular Systems Biology, Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaDivision of Molecular Systems Biology, Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaDepartment Biology I, Plant Evolutionary Cell Biology, Ludwig-Maximilians Universität, Munich, GermanyCore Facility Cell Imaging and Ultrastructure Research, University of Vienna, Vienna, AustriaDivision of Molecular Systems Biology, Department of Ecogenomics and Systems Biology, University of Vienna, Vienna, AustriaPlant phenotyping to date typically comprises morphological and physiological profiling in a high-throughput manner. A powerful method that allows for subcellular characterization of organelle stoichiometric/functional characteristics is still missing. Organelle abundance and crosstalk in cell dynamics and signaling plays an important role for understanding crop growth and stress adaptations. However, microscopy cannot be considered a high-throughput technology. The aim of the present study was to develop an approach that enables the estimation of organelle functional stoichiometry and to determine differential subcellular dynamics within and across cultivars in a high-throughput manner. A combination of subcellular non-aqueous fractionation and liquid chromatography mass spectrometry was applied to assign membrane-marker proteins to cell compartmental abundances and functions of Pisum sativum leaves. Based on specific subcellular affiliation, proteotypic marker peptides of the chloroplast, mitochondria and vacuole membranes were selected and synthesized as heavy isotope labeled standards. The rapid and unbiased Mass Western approach for accurate stoichiometry and targeted absolute protein quantification allowed for a proportional organelle abundances measure linked to their functional properties. A 3D Confocal Laser Scanning Microscopy approach was developed to evaluate the Mass Western. Two P. sativum cultivars of varying morphology and physiology were compared. The Mass Western assay enabled a cultivar specific discrimination of the chloroplast to mitochondria to vacuole relations.https://www.frontiersin.org/article/10.3389/fpls.2019.00638/fullorganelle marker peptidesorganelle stoichiometryfield peaMass Western3D Confocal Laser Scanning Microscopy |
| spellingShingle | Sebastian Schneider Dominik Harant Gert Bachmann Thomas Nägele Thomas Nägele Ingeborg Lang Stefanie Wienkoop Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars Frontiers in Plant Science organelle marker peptides organelle stoichiometry field pea Mass Western 3D Confocal Laser Scanning Microscopy |
| title | Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars |
| title_full | Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars |
| title_fullStr | Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars |
| title_full_unstemmed | Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars |
| title_short | Subcellular Phenotyping: Using Proteomics to Quantitatively Link Subcellular Leaf Protein and Organelle Distribution Analyses of Pisum sativum Cultivars |
| title_sort | subcellular phenotyping using proteomics to quantitatively link subcellular leaf protein and organelle distribution analyses of pisum sativum cultivars |
| topic | organelle marker peptides organelle stoichiometry field pea Mass Western 3D Confocal Laser Scanning Microscopy |
| url | https://www.frontiersin.org/article/10.3389/fpls.2019.00638/full |
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