Carboxylation and Oxygenation Kinetics and Large Subunit (rbcL) DNA Sequences for Rubisco From Two Ecotypes of Plantago lanceolata L. That Are Native to Sites Differing in Atmospheric CO2 Levels

Abstract

Rubisco, the most prevalent protein on Earth, catalysers both a reaction that initiates C₃ carbon fixation, and a reaction that initiates photorespiration, which stimulates protein synthesis. Regulation of the balance between these reactions under atmospheric CO₂ fluctuations remains poorly understood. We have hypothesised that vascular plants maintain organic carbon-to-nitrogen homoeostasis by adjusting the relative activities of magnesium and manganese in chloroplasts to balance carbon fixation and nitrate assimilation rates. The following examined the influence of magnesium and manganese on carboxylation and oxygenation for rubisco purified from two ecotypes of Plantago lanceolata L.: one adapted to the elevated CO₂ atmospheres that occur near a natural CO₂ spring and the other adapted to more typical CO₂ atmospheres that occur nearby. The plastid DNA coding for the large unit of rubisco was similar in both ecotypes. The kinetics of rubiscos from the two ecotypes differed more when associated with manganese than magnesium. Specificity for CO₂ over O₂ (S_c/o) for rubisco from both ecotypes was higher when the enzymes were bound to magnesium than manganese. Differences in the responses of rubisco from P. lanceolata to the metals may account for the adaptation of this species to different CO₂ environments.

Keywords: Plantago lanceolata; adaptation to CO2‐enriched environments; magnesium versus manganese; rbcL DNA sequences; rubisco kinetics; specificity for CO2 over O2.

Figure 1

Rates of RuBP carboxylation and oxygenation in turnovers per second by rubisco purified from two ecotypes of Plantago lanceolata L. One ecotype (Spring) collected near a CO₂ spring had experienced high CO₂ for many generations, and the other ecotype collected nearby (Ambient) had experienced ambient CO₂ atmospheres. The enzyme was bound to Mg²⁺ or Mn²⁺ and exposed to 21% O₂ and 0.04% or 0.10% CO₂. Shown are the mean ± SE, n = 4–10.

Figure 2

Tukey test comparing RuBP carboxylation and oxygenation rates under various combinations of metals (Mg²⁺ vs. Mn²⁺), Plantago lanceolata ecotypes (Spr vs. Amb), CO₂ concentrations (0.04% vs. 0.1%), and O₂ concentrations (21% vs. 30%). Shown are mean difference ± SE, n = 4–10. Labels in blue designate that only carboxylation differed, labels in red designate that only oxygenation differed, labels in purple designate that both carboxylation and oxygenation differed, and labels in black designate that neither carboxylation nor oxygenation differed. For example, in the top line, carboxylation of the Mn²⁺‐treated rubisco from the Spring ecotype tested at 0.1% CO₂ and 21% O₂ was greater than carboxylation of the Mg²⁺‐treated rubisco, but oxygenation did not differ significantly. For additional statistical analyses, see Supporting Information S1: Tables S1 and S2. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Influence of Mg²⁺ or Mn²⁺ (mean ± credible interval, n = 4–20) on the kinetics of rubisco purified from two ecotypes of Plantago lanceolata L.: one (Spring) collected near a CO₂ spring has experienced high CO₂ for many generations; the other collected nearby (Ambient) has experienced ambient CO₂. V _cmax is the maximum velocity of carboxylation, V _omax is the maximum velocity of oxygenation, K _c is the Michaelis constant of rubisco for CO₂, K _o is the Michaelis constant of rubisco for O₂, S _c/o is the specificity of rubisco for CO₂ over O₂, and V _cmax/V _omax is the ratio of the maximum velocities. Also displayed are the 2‐way ANOVAs for the influence of Ecotype, Metal, or their interaction on the kinetic parameters for rubiscos purified from the two ecotypes in the presence of Mg²⁺ or Mn²⁺. For additional statistical analysis, see Supporting Information S1: Table S3. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

cpDNA map of rbcL from the (A) CO₂‐spring and (B) ambient ecotypes. The orange and green arrows indicate open reading frames (ORFs). The restriction enzymes shown are unique and dual cutters. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Portions of two published crystal structures of spinach rubisco showing the substrate, metal ion, and nearby residues of interest. (A) Rubisco bound to Mg²⁺ and CAP (PDB: 8RUC) (Andersson 1996). (B) Rubisco bound to Ca²⁺ and RuBP (PDB: 1RXO) (Taylor and Andersson 1997). KCX denotes the catalytic carbamylated lysine and CAP denotes 2‐carboxyarabinitol‐1,5‐diphosphate. [Color figure can be viewed at wileyonlinelibrary.com]