DOI: 10.5061/dryad.gb5mkkwwn
title: Data from: Metals and other ligands balance carbon fixation and photorespiration in chloroplasts

description: The behavior of many plant enzymes depends on the metals and other ligands
 to which they are bound. A previous study demonstrated that tobacco
 Rubisco binds almost equally to magnesium and manganese and rapidly
 exchanges one metal for the other. The present study characterizes the
 kinetics of Rubisco and the plastidial malic enzyme when bound to either
 metal. When Rubisco purified from five C3 species was bound to
 magnesium rather than manganese, the specificity for CO2 over O2,
 (Sc/o) increased by 25% and the ratio of the maximum velocities of
 carboxylation / oxygenation (Vcmax/Vomax) increased by 39%. For the
 recombinant plastidial malic enzyme, the forward reaction (malate
 decarboxylation) was 30% slower and the reverse reaction (pyruvate
 carboxylation) was three times faster when bound to manganese rather than
 magnesium. Adding 6-phosphoglycerate and NADP+ inhibited
 carboxylation and oxygenation when Rubisco was bound to magnesium and
 stimulated oxygenation when it was bound to manganese. Conditions that
 favored RuBP oxygenation stimulated Rubisco to convert as much as 15% of
 the total RuBP consumed into pyruvate. These results are consistent with a
 stromal biochemical pathway in which (1) Rubisco when associated with
 manganese converts a substantial amount of RuBP into pyruvate, (2) malic
 enzyme when associated with manganese carboxylates a substantial portion
 of this pyruvate into malate, and (3) chloroplasts export additional
 malate into the cytoplasm where it generates NADH for assimilating nitrate
 into amino acids. Thus, plants may regulate the activities of magnesium
 and manganese in leaves to balance organic carbon and organic nitrogen as
 atmospheric CO2 fluctuates.
description: Carboxylation/2PGA measurements: We added an assay buffer
 containing 20 mM Tris–HCl, 1 mM EDTA, 100 mM Triethanolamine at pH 7.8 to
 one 5 mL tube; added 1 or 2 μL of Rubisco (0.5–1 μM), 10 μL of 250 mM
 NaHCO<sub>3</sub>, 0.5 μL carbonic anhydrase (~2.5 units),
 5 μL of iPGM (10 μM), 4 μL of MgCl<sub>2</sub> (final
 concentration 20 mM) or MnCl<sub>2</sub> (5 mM); mixed
 everything thoroughly and allowed the mixture to sit for 5 minutes. The
 final assay volume in the tube was 1750 μL. We started the reaction by
 adding 45 μL of 10 mM RuBP to the tube, split the mixture into five tubes,
 and stopped the reaction after 1, 2, 3, 4, or 5 mins by adding 0.5 N HCl
 to each tube. We added KOH to each reaction tube to adjust the pH to about
 pH 7.8 and added to the tubes equal amounts of freshly mixed 2PGA
 colorimetric cocktail that we prepared from a 2-phosphoglycerate assay kit
 (ab174097, Abcam), mixed all tubes well, and moved the tubes to an opaque
 box. We measured OD<sub>570nm</sub> after 40 min for each of
 the tubes and calculated the 2PGA concentration in each reaction tube
 based on a calibration curve determined by adding specified quantities of
 a 1 mM 2PGA standard solution that the assay kit provided.
 Oxygenation measurements:  A needle-type
 micro-optode OXF50-OI (PyroScience GmbH) on a FireSting
 O<sub>2</sub> optical oxygen and temperature meter (FSO2-4)
 monitored changes in O<sub>2</sub> concentration in a
 spectrophotometer 3.5 mL quartz cuvette (Millipore Sigma) during our
 Rubisco oxygenation experiments. We conducted these experiments under
 three sets of conditions at 25°C: (a) ambient air (79%
 N<sub>2</sub>, 20.96% O<sub>2</sub>, and 0.04%
 CO<sub>2</sub>), (b) elevated
 CO<sub>2</sub> content (78.96% N<sub>2</sub>,
 20.96% O<sub>2</sub>, and 0.08% CO<sub>2</sub>),
 and (c) reduced O<sub>2</sub> content (89%
 N<sub>2</sub>, 10.96% O<sub>2</sub>, and 0.04%
 CO<sub>2</sub>). Precision mass flow controllers (Apex Vacuum)
 calibrated against soap bubble flowmeters mixed pure
 N<sub>2</sub>, O<sub>2</sub>, and
 CO<sub>2</sub> to the desired concentrations. A non-dispersive
 Infrared Gas Analyser (Li-cor) checked the
 CO<sub>2</sub> concentration. We conducted a two-point
 calibration of the oxygen optode in both air and air-saturated water. The
 final assay volume in each tube was 3000 μL. Before the
 oxygenation assay, we activated Rubisco as follows: we added assay buffer
 (20 mM Tris–HCl, 1 mM EDTA, 10 mM NaHCO<sub>3</sub>, 100 mM
 triethanolamine, pH 7.8) to a covered, temperature-controlled, 4 mL quartz
 cuvette; added 10 μL of Rubisco (0.5–1 μM), 50 μL of iPGM (10 μM), 5 μL
 carbonic anhydrase (~25 units), and 40 μL of
 MgCl<sub>2</sub> (final concentration 20 mM) or
 MnCl<sub>2</sub> (5 mM) and mixed the contents of the cuvette
 thoroughly. We allowed the mixture to sit for 5 minutes before inserting
 the oxygen sensor and sparging it for 1 minute with the gas mixture
 corresponding to the experiment's conditions. After about 30 s, once
 the oxygen sensor reading stabilized, we started the reaction by adding
 100 μL of 10 mM RuBP (pre-equilibrated with the same gas mixture) and
 began collecting data Pyruvate measurements: We mixed a
 solution containing 12.5 mM NaHCO<sub>3</sub>, either 15 mM
 MgCl<sub>2</sub> or 3.75 mM MnCl<sub>2</sub>,
 5 units ml<sup>−1</sup> carbonic anhydrase, and 8% purified
 Rubisco solution with an assay buffer (20 mM Tris–HCl, 1 mM EDTA, 100 mM
 Triethanolamine, pH 7.8) and allowed it to sit for 5 minutes to activate
 Rubisco. For ambient experiments, we subsequently purged it for 5 minutes
 with ambient air. For experiments on the influence of 6PG and/or
 NADP<sup>+</sup>, 400 μM 6PG and/or
 NADP<sup>+</sup> was added, respectively. We started all
 experiments by adding 8 μM RuBP, removed samples immediately and after 3,
 6, 9, and 12 minutes, and stopped the experiment by heating to 80°C. We
 centrifuged the samples for 3 minutes in a tabletop microcentrifuge at 21
 100 <em>g</em>, extracted the liquid, and used a commercial
 kit to assess pyruvate via pyruvate oxidase generation of hydrogen
 peroxide that reacted with nonfluorescent Amplex Red at a 1:1
 stoichiometry and formed the fluorescent product resorufin. We measured
 resorufin with a Horiba Fluorolog at
 λ<sub>em/ex</sub> = 585/530 nm. Malic
 enzyme measurements:  For malic enzyme malate oxidative
 decarboxylation reactions, we added a buffer (50 mM MOPS pH 7.3, 0.25 M
 NaCl, 0.01% (v/v) Triton X-100 and 1 mM DTT) to a quartz cuvette along
 with purified malic enzyme (3.5–5.0 μg ml<sup>−1</sup> as
 measured by a Bradford assay). To this mixture, we added 10 mM
 MgCl<sub>2</sub> or 1.7 mM MnCl<sub>2</sub> and
 500 μM NADP<sup>+</sup> and started the reaction by adding
 10 mM malate as a final concentration. For pyruvate
 reductive carboxylation reactions, we used the same buffer and protein. To
 this mixture, we added 10 mM MgCl<sub>2</sub> or 1.7 mM
 MnCl<sub>2</sub>, 30 mM NaHCO<sub>3</sub>, and
 100 μM NADPH and started the reaction by adding 50 mM pyruvate (final
 concentration). In both cases, we monitored the
 reaction on a UV/Vis spectrophotometer at 1-minute intervals.
description: # Photorespiratory Pathway Data
 [https://doi.org/10.5061/dryad.gb5mkkwwn](https://doi.org/10.5061/dryad.gb5mkkwwn) This dataset contains data from experiments measuring the activity of Rubisco and malic enzyme. ## Description of the data and file structure All data are in CSV format. Oxygenation data (OxygenData.csv and TobaccoNadpNadph6PGOxyData.csv) were measured on a FireSting O<sub>2</sub> optical oxygen and temperature sensor using a needle-type micro-optode as described in the paper. The columns are as follows: time: Time in seconds, measured from the start of the experiment.  For experiments with non-ambient oxygen levels, negative times indicate a period prior to starting the experiment for measuring background. oxygen: Measured level of dissolved oxygen in mg/L. id: Unique identifier for each experiment. date (TobaccoNadpNadph6PGOxyData.csv only): Date on which the experiment was performed, for pairing purposes. plant: Either the plant species from which Rubisco was purified, or "Blank" to indicate a blank run. metal: Either Mg or Mn to indicate the metal ion present in solution. o2, co2: Oxygen and carbon dioxide concentrations, in percent, present in the gas mixture with which the solution was sparged prior to the experiment. nadp, nadph, sixpg (TobaccoNadpNadph6PGOxyData.csv only): Concentrations of NADP<sup>+</sup>, NADPH, and 6PG, respectively, in mM, present in solution. rubiscoConc: Rubisco concentration, in M, present in solution. 2-PGA data (CarboxyData.csv and all files ending in 2PGAData.csv) were measured via a commercially available 2-PGA assay kit on a UV/vis spectrophotometer, as described in the paper. The columns are as follows: time: Time in seconds that the reaction was allowed to proceed before stopping it. conc: Calculated concentration of 2-PGA, in M. rConc: Concentration of Rubisco, in M, present in solution. plant: The plant species from which Rubisco was purified. run: Unique identifier for each run. metal: Either Mg or Mn to indicate the metal ion present in solution. nadp, nadph, sixpg (where present): Concentrations of NADP<sup>+</sup>, NADPH, and 6PG, respectively, in mM, present in solution. Pyruvate data (PyruvateData.csv) were measured using a commercially available pyruvate assay kit with a Horiba Fluorolog at λ<sub>em/ex</sub> = 585/530 nm as described in the paper. The columns are as follows: peak: Emission at 585nm as measured by the fluorimeter. time: Time in minutes that the reaction was allowed to proceed before stopping it. For calibration runs, this is instead the concentration of pyruvate in the standard used, as a multiple of 10 mM. metal: Either Mg or Mn to indicate the metal ion present in solution, or "cal" for a calibration run. co2: "Amb" indicates that the mixture was purged with air containing ambient CO<sub>2</sub> prior to the reaction. "High" indicates that 12 mM CO<sub>2</sub>, as NaHCO<sub>3</sub>, was allowed to remain in the solution during the reaction. sixpg: "6PG" indicates that 0.4 mM 6PG was added prior to the reaction. calId: id of the calibration run used for each kit. id: Unique identifier for each run. A negative id indicates a calibration run. rubiscoConc: Concentration of Rubisco, in M, present in solution. Malic enzyme data (MalicEnzymeData.csv) were measured on a UV/vis spectrophotometer, as described in the paper. The columns are as follows: wavelength: Wavelength at which the absorbance was measured. absorbance: Measured absorbance on the spectrophotometer. time: -2 indicates a measurement of the protein and buffer only. -1 indicates a measurement of the mixture immediately before pyruvate or malate is added to start the reaction. Any nonnegative number indicates the time in minutes after the reaction was started. id: Unique identifier for each run. meConc: Concentration of malic enzyme, in μM, present in solution. metal: Either Mg or Mn to indicate the metal ion present in solution. reaction: "fwd" indicates the malate oxidative decarboxylation ("forward") reaction. "rev" indicates the pyruvate reductive carboxylation ("reverse") reaction. ## Sharing/Access information All data in this dataset are original data from our lab. At present, they are not available from any other source.