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Its in vivo activity is usually determined by 14CO2 incorporation into 3-phosphoglycerate (3PGA). However, the radiometric analysis of 3PGA does not distinguish carbon positions. Hence, RUBISCO activity that fixes carbon into the 1-C position of 3PGA and Calvin\u2013Benson\u2013Bassham (CBB) cycle activities that redistribute carbon into its 2-C and 3-C positions are not resolved. This study aims to develop technology that differentiates between these activities. In source fragmentation of gas chromatography-mass spectrometry (GC-MS) enables paired isotopologue distribution analyses of fragmented substructures and the complete metabolite structure. GC-MS measurements after dynamic photosynthetic 13CO2 labeling allowed quantification of the 13C fractional enrichment (E13C) and molar carbon assimilation rates (A13C) at carbon position 1-C of 3PGA by combining E13C from carbon positions 2,3-C2 and 1,2,3-C3 with quantification of 3PGA concentrations. We validated the procedure using two GC-time of flight-MS instruments, operated at nominal or high mass resolution, and tested the expected 3PGA positional labeling by in vivo glycolysis of positional labeled glucose isotopomers. Mutant analysis of the highly divergent GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASEs (GAPDH1 and 2) from Synechocystis sp. PCC 6803 revealed full inactivation of the CBB cycle with maintained RUBISCO activity in \u0394gapdh2 and a CBB cycle modulating role of GAPDH1 under fluctuating CO2 supply. RUBISCO activity in the CBB-deficient \u0394gapdh2 can re-assimilate CO2 released by catabolic pathways. We suggest that RUBISCO activity in Synechocystis can scavenge carbon lost through the pentose phosphate pathway or other cellular decarboxylation reactions.<\/jats:p>","DOI":"10.1093\/plphys\/kiaf020","type":"journal-article","created":{"date-parts":[[2025,1,21]],"date-time":"2025-01-21T16:19:04Z","timestamp":1737476344000},"source":"Crossref","is-referenced-by-count":5,"title":["Dynamic photosynthetic labeling and carbon-positional mass spectrometry monitor in vivo RUBISCO carbon assimilation rates"],"prefix":"10.1093","volume":"197","author":[{"given":"Yogeswari","family":"Rajarathinam","sequence":"first","affiliation":[{"name":"Max-Planck-Institute of Molecular Plant Physiology , Am M\u00fchlenberg 1, D-14476 Potsdam-Golm ,","place":["Germany"]},{"name":"Environmental and Biochemical Sciences, The James Hutton Institute , Invergowrie, Dundee DD2 5DA ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3249-0441","authenticated-orcid":false,"given":"Luisa","family":"Wittemeier","sequence":"additional","affiliation":[{"name":"Max-Planck-Institute of Molecular Plant Physiology , Am M\u00fchlenberg 1, D-14476 Potsdam-Golm ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4366-423X","authenticated-orcid":false,"given":"Kirstin","family":"Gutekunst","sequence":"additional","affiliation":[{"name":"Molecular Plant Physiology, Bioenergetics in Photoautotrophs, University Kassel , Heinrich-Plett-Stra\u00dfe 40, D-34132 Kassel ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2059-2061","authenticated-orcid":false,"given":"Martin","family":"Hagemann","sequence":"additional","affiliation":[{"name":"Plant Physiology Department, Institute of Biological Sciences, Rostock University , Albert-Einstein-Stra\u00dfe 3, D-18059 Rostock ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9675-4883","authenticated-orcid":false,"given":"Joachim","family":"Kopka","sequence":"additional","affiliation":[{"name":"Max-Planck-Institute of Molecular Plant Physiology , Am M\u00fchlenberg 1, D-14476 Potsdam-Golm ,","place":["Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,1,21]]},"reference":[{"issue":"5","key":"2025021115371317900_kiaf020-B1","doi-asserted-by":"publisher","first-page":"355","DOI":"10.1038\/nchembio.1786","article-title":"A pentose bisphosphate pathway for nucleoside degradation in Archaea","volume":"11","author":"Aono","year":"2015","journal-title":"Nat Chem Biol"},{"issue":"5643","key":"2025021115371317900_kiaf020-B2","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1126\/science.1086997","article-title":"A functional link between RubisCO-like protein of Bacillus and photosynthetic RubisCO","volume":"302","author":"Ashida","year":"2003","journal-title":"Science"},{"issue":"1","key":"2025021115371317900_kiaf020-B3","doi-asserted-by":"publisher","first-page":"2006.0008","DOI":"10.1038\/msb4100050","article-title":"Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection","volume":"2","author":"Baba","year":"2006","journal-title":"Mol Sys Biol"},{"issue":"2","key":"2025021115371317900_kiaf020-B4","doi-asserted-by":"publisher","first-page":"781","DOI":"10.1016\/S0021-9258(18)56368-7","article-title":"The path of carbon in photosynthesis: VIII. 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