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Rubisco supplies pyruvate for the 2-C-methyl-d-erythritol-4-phosphate pathway

Nature Plants volume 10, pages 1453–1463 (2024)Cite this article

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Abstract

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco) produces pyruvate in the chloroplast through β-elimination of the aci-carbanion intermediate1. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid and branched-chain amino acid biosynthesis in photosynthetically active tissue. 13C labelling studies of intact Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate to serve as a precursor. Low oxygen stimulates Rubisco carboxylase activity and increases pyruvate production and flux through the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis2,3. Metabolome analysis of mutants defective in phosphoenolpyruvate or pyruvate import and biochemical characterization of isolated chloroplasts further support Rubisco as the main source of pyruvate in chloroplasts. Seedlings incorporated exogenous,13C-labelled pyruvate into MEP pathway intermediates, while adult plants did not, underscoring the developmental transition in pyruvate sourcing. Rubisco β-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin–Benson–Bassham cycle. These insights solve the “pyruvate paradox”4, improve the fit of metabolic models for central metabolism and connect the MEP pathway directly to carbon assimilation.

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Fig. 1: Metabolism of pyruvate in plant cells and the Rubisco β-elimination mechanism.
Fig. 2: Role of Rubisco in supplying pyruvate in illuminated chloroplasts.
Fig. 3: Changes to flux in C3 central metabolites and the MEP pathway under low-oxygen conditions.
Fig. 4: Role of transporters in supplying pyruvate to the chloroplast in Arabidopsis leaf tissue.

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Data availability

All study data are included in the Letter and/or its supplementary and extended information. The isotopic labelling data displayed in Figs. 2 and 3 are compiled in Supplementary Dataset 4. The raw MS data supporting Figs. 1–4 and Extended Data Figs. 1–10 are available under identifier MSV000095073 at the MassIVE data repository (https://massive.ucsd.edu/). Source data are provided with this paper.

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Acknowledgements

This study was funded by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (no. RGPIN-2017-06400) and a John Evans Leadership Fund grant from the Canadian Foundation for Innovation (no. 36131). This work was also supported by a grant from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (grant no. DE-FG02-91ER20021). T.D.S. received partial salary support from Michigan AgBioResearch. Rubisco was isolated from N. tabacum by A. Johnson; we thank L. Gregory for help with the Rubisco assay. We also acknowledge help from the MSU Mass Spectrometry and Metabolomics Core Facility, and we thank the MSU Institute for Cyber-Enabled Research for providing a high-performance computing cluster and services and J. Young for making INCA accessible. We also acknowledge a generous NSERC CGSD graduate scholarship supporting M.E.B. and thank UTM staff members B. Pitton of the Teaching Greenhouse, P. Duggan of the Academic Machine Shop and Stores Supervisor M. Malcolm (POE) for their excellent technical and logistical support.

Author information

Author notes

  1. Matthew E. Bergman

    Present address: Department of Biochemistry, Purdue University, West Lafayette, IN, USA

Authors and Affiliations

  1. Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

    Sonia E. Evans, Matthew E. Bergman, Scott A. Ford & Michael A. Phillips

  2. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI, USA

    Yuan Xu & Thomas D. Sharkey

  3. Department of Biology, University of Toronto Mississauga, Mississauga, Ontario, Canada

    Yingxia Liu & Michael A. Phillips

  4. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA

    Thomas D. Sharkey

  5. Plant Resilience Institute, Michigan State University, East Lansing, MI, USA

    Thomas D. Sharkey

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Contributions

S.E.E. labelled plants and performed the MS analysis. S.E.E. and M.E.B. performed data analysis. S.A.F. analysed fatty acid methyl esters and amino acids. Y.L. prepared plants for experiments and genotyped mutants. Y.X. quantified Rubisco product profiles and carried out the mass flux analysis. T.D.S. edited the manuscript and analysed Rubisco-catalysed pyruvate production. M.A.P. wrote the manuscript and directed the research.

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Correspondence to Michael A. Phillips.

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Extended data

Extended Data Fig. 1 Enzyme activity of plastid-localized ENO and PGM from isolated chloroplasts of Arabidopsis.

Crude protein extracts were obtained from intact chloroplasts isolated from 7-day-old seedlings and rosette-staged leaves (42 days) and incubated with 2 mM 3-PGA or 1 mM 2-PGA to assess the relative activity of PGM and ENO glycolytic enzymes. a, Assay showing lower activity of plastidic ENO in rosette leaves when compared to seedlings. b, Coupled enzyme activity of PGM and ENO shows low conversion of 3-PGA to PEP in rosette leaves (ENO P = 0.0002; PGM + ENO P = 0.0026). c, Representative chromatograms depicting PEP product formation after one hour incubation with 2-PGA. All chromatograms are shown on the same scale. d, Intact chloroplasts isolated from 7-day-old seedlings. Bar = 10 μm. The experiment was repeated once with identical results. Error bars represent standard deviation of measurements (n = 3). Asterisks indicate significant differences (** P < 0.001; *** P < 0.0001) as determined by Student’s two-sided t-test.

Extended Data Fig. 2 13C label incorporation and absolute concentrations of photorespiratory intermediates in Arabidopsis leaves under normal (21%) and low (1%) oxygen conditions.

Details on administration of 13C under physiological conditions, extraction of metabolites, and analysis by liquid chromatography – tandem mass spectrometry are given in the Methods section. For absolute quantification, the data are shown as box plots, with the central line indicating the median, upper and lower bounds representing quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively, and the whiskers denote the minima and maxima of the data points. Mean values of n = 18–25 individual plants are shown as yellow diamonds (Glycerate P = 0.0022). Asterisks indicate significant differences at * P < 0.01, based on Student’s two-sided t-test.

Extended Data Fig. 3 Adaptation of Arabidopsis plants to low oxygen conditions.

a, Representative total carbon assimilation (A) during adaptation of plants to normal (21%) or low (1%) oxygen conditions in a dynamic flow cuvette connected to a LI-COR 6800 photosynthesis system. The flat line indicates a photosynthetic steady state prior to initiation of 13CO2 labelling, indicated by the arrow. b, Metabolite concentrations were monitored in time course plants for any changes in metabolite pool size during the labelling experiment. The low R2 values indicate metabolite pool size is independent of duration of the labelling experiment. Triose-P, glyceraldehyde-3-phosphate + dihydroxyacetone phosphate; DXP, 1-deoxyxylulose-5-phosphate; MEcDP, 2-C-methylerythritol-2,4-cyclodiphoshate; IDP/DMADP, isopentenyl and dimethylallyl diphosphate.

Extended Data Fig. 4 13C labelling incorporation and absolute concentrations of glycolytic and Calvin-Benson-Bassham cycle intermediates under normal (21%) and low (1%) oxygen conditions.

For absolute quantification, the data are visualized as box plots, with the central line indicating the median, upper and lower bounds represent quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively, and the whiskers denote the minima and maxima of the data points. Mean values (yellow diamonds) show averages (n = 18–25) (PEP P = 0.0012). Asterisks indicate significant differences at * P < 0.01, based on Student’s two-sided t-test. G6P, glucose-6-phosphate; F6P, fructose-5-phosphate; S7P, sedoheptulose-7-phosphate; Xu5P, xylulose-5-phosphate; PEP, phosphoenolpyruvate.

Extended Data Fig. 5 13CO2 in vivo labelling of Arabidopsis seedlings.

F = fractional 13C labelling of the carbon atoms in the indicated central metabolite pool. Seedlings were labelled under normal (21%) or low (1%) oxygen conditions to observe the effect of stimulating Rubisco carboxylase activity on seedling metabolism. Seedlings grown for 7 d on solid media were adapted in a controlled environment dynamic flow cuvette (lower right). After 1 h of monitoring with a LI-COR 6800 system to verify the seedlings had reached a photosynthetic steady state, the air supply (normal or low oxygen) was switched to one containing 400 μL ∙ L−1 13CO2. The seedlings were incubated in the 13CO2 containing atmosphere for 30 min and then flash frozen for lyophilization and metabolite extraction. Details of the analysis of central metabolites and calculation of label incorporation are provided in online methods. Bar graphs show means of 3–6 individual plates while error bars show standard deviation (Serine P = 0.0008). The individual data points are overlain as dots. Asterisks indicate statistical significance, based on Student’s two-sided t-test (* = P < 0.01). Otherwise, no statistically significant difference was observed. DXP, 1-deoxy-d-xylulose-5-phosphate; MEcDP, 2-C-methylerythritol-2,4-cyclodiphosphate; IDP/DMADP, isopentenyl and dimethylallyl diphosphate; 3-PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; S7P, sedoheptulose-7-phosphate; Xu5P, xylulose-5-phosphate; R5P, ribose-5-phosphate.

Extended Data Fig. 6 Incorporation of [2-13C] pyruvate into intermediates of the 2-C-methylerythritol-4-phosphate (MEP) pathway in Arabidopsis seedlings (7-day old) vs. rosettes (42-day old).

Following incubation with [2-13C] pyruvate, 13C was detected in MEP pathway intermediates in seedlings but not in rosette leaves. a, Label incorporation into 1-deoxy-d-xylulose-5-phosphate (DXP). b, Label incorporation into 2-C-methylerythritol-2,4,-cyclodiphosphate (MEcDP); c, Label incorporation into isopentenyl and dimethylallyl diphosphate (IDP/DMADP). IDP and DMADP, which are the products of both the MEP pathway in the chloroplast and the mevalonate pathway in the cytosol, displayed measurable label incorporation in both seedlings and rosette leaves. d, Detection of exogenously supplied [2-13C] pyruvate in extracts of seedlings or rosette leaves. Seedlings were grown in ¾ MS media supplemented (or not) with 5 mM 2-13C pyruvate for 7 days while leaves of rosette plants were infiltrated for 24 hours with MS media supplemented with labelled pyruvate at the same concentration (or MS media only for controls). Pooled leaves or seedlings were flash-frozen and extracted for LCMS/MS and GCMS analysis. Percent labelling incorporation into each metabolite was calculated using the equation (\(1/N\)) \({\sum }_{i}^{N}Mi\times i\), where N is the number of carbon atoms in the molecule and Mi is the fractional abundance of the ith isotopologue. Error bars indicate standard deviation of measurement (n = 3-4). Asterisks indicate significant differences (** P < 0.001; *** P < 0.0001) as determined by Student’s two-sided t-test. n.s., Not statistically significant.

Extended Data Fig. 7 Genotyping of mutants deficient in PPT1 (cue1) and BASS2 (bass2-1 and bass2-2).

a, b Genotyping of bass2-1 and bass2-2 mutants using gene specific primers (BASS2 LP and RP or full-length (FL) primers) and T-DNA left border primers (LBb1.3). Amplification was performed using genomic DNA. c, RT-PCR analysis of PPT1 and BASS2 in cue1, bass2-1 and bass2-2 mutant plants. The cue1 mutant lacks full-length transcripts for PPT1, and bass2 mutant lines similarly lack full-length transcripts for BASS2. d, The Arabidopsis thaliana dihydroxy-acid dehydratase gene (DHAD; AT3G23940) was used as a control to confirm cDNA quality. M, DNA marker. e, Uncropped gel images shown in a-d. PCR and gel analyses in a-d were repeated at least once with identical results.

Extended Data Fig. 8 Roles of transporters in supplying pyruvate and phosphoenolpyruvate for amino acid biosynthesis in the chloroplast.

Concentrations of aromatic (phenylalanine, tryptophan, and tyrosine) and branched chain amino acids (isoleucine, leucine and valine) were analyzed by liquid chromatography – tandem mass spectrometry in leaf extracts from wild-type (WT), cue1, and bass2 plants. Raw peak areas were normalized to the internal standard N-methylglucamine and compared to external standard calibration curves derived from authentic standards. The data are displayed as box plots, with the central line indicating the median and upper and lower bounds representing quartile 3 (75th percentile) and quartile 1 (25th percentile), respectively. Whiskers denote the minima and maxima of the data points. Yellow diamonds indicate mean values (n = 6-7 individual plants). Asterisk indicates significant differences (* P < 0.01, ** P < 0.001, *** P < 0.0001) based on Student’s two-sided t-test.

Extended Data Fig. 9 Fatty acid methyl ester (FAME) analysis in Arabidopsis wild-type (WT), cue1, and bass2 mutant plants.

a, Representative chromatogram showing elution profile of FAMEs by gas chromatography – mass spectrometry. b, Total fatty acid content of each genotype expressed as summed peak areas of FAMEs relative to the internal standards (IS), triheptadecanoin (C17) and dotriacontane (n = 4–6). There were no statistical differences in total fatty acid content between groups (P < 0.01), but content of some individual components were lower in cue1 compared to WT (see Supplementary Dataset 2 for details).

Extended Data Fig. 10 CO2 assimilation (A) and intercellular CO2 concentration (Ci) in Arabidopsis wild-type (WT), bass2 and cue1 mutant plants.

A/Ci curves were fit based on the Farquhar-von Caemmerer-Berry (FvCB) model of photosynthesis using the plantecophys package on R46. Estimates of Vcmax (maximum Rubisco carboxylation rate) and Jmax (the maximum rate of electron transport) are presented as mean ± SD from individual fit (n = 3-4 plants). Ac represents photosynthesis limited by rubisco, while Aj indicates limitation due to RuBP-regeneration.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1–5.

Reporting Summary

Supplementary Data

Supplementary Datasets 1–4.

Source data

Source Data Fig. 1

ChemDraw file for the Rubisco mechanism shown in Fig. 1b.

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Evans, S.E., Xu, Y., Bergman, M.E. et al. Rubisco supplies pyruvate for the 2-C-methyl-d-erythritol-4-phosphate pathway. Nat. Plants 10, 1453–1463 (2024). https://doi.org/10.1038/s41477-024-01791-z

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