Data from: Metals and other ligands balance carbon fixation and photorespiration in chloroplasts
Data files
Sep 03, 2024 version files 15.54 MB
-
CarboxyData.csv
22.32 KB
-
MalicEnzymeData.csv
8.12 MB
-
OxygenData.csv
3.36 MB
-
PyruvateData.csv
35.28 KB
-
README.md
4.25 KB
-
Tobacco6PG2PGAData.csv
9.19 KB
-
TobaccoNadp2PGAData.csv
13.25 KB
-
TobaccoNadp6PG2PGAData.csv
14.02 KB
-
TobaccoNadph2PGAData.csv
10.56 KB
-
TobaccoNadpNadph6PGOxyData.csv
3.96 MB
Abstract
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.
README: Photorespiratory Pathway Data
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 O2 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+, 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+, 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 λem/ex = 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 CO2 prior to the reaction. "High" indicates that 12 mM CO2, as NaHCO3, 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.
Methods
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 NaHCO3, 0.5 μL carbonic anhydrase (~2.5 units), 5 μL of iPGM (10 μM), 4 μL of MgCl2 (final concentration 20 mM) or MnCl2 (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 OD570nm 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 O2 optical oxygen and temperature meter (FSO2-4) monitored changes in O2 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% N2, 20.96% O2, and 0.04% CO2), (b) elevated CO2 content (78.96% N2, 20.96% O2, and 0.08% CO2), and (c) reduced O2 content (89% N2, 10.96% O2, and 0.04% CO2). Precision mass flow controllers (Apex Vacuum) calibrated against soap bubble flowmeters mixed pure N2, O2, and CO2 to the desired concentrations. A non-dispersive Infrared Gas Analyser (Li-cor) checked the CO2 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 NaHCO3, 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 MgCl2 (final concentration 20 mM) or MnCl2 (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 NaHCO3, either 15 mM MgCl2 or 3.75 mM MnCl2, 5 units ml−1 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+, 400 μM 6PG and/or NADP+ 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 g, 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 λem/ex = 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−1 as measured by a Bradford assay). To this mixture, we added 10 mM MgCl2 or 1.7 mM MnCl2 and 500 μM NADP+ 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 MgCl2 or 1.7 mM MnCl2, 30 mM NaHCO3, 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.