Respiratory metabolism of illuminated leaves depends on CO2 and O2 conditions

Abstract

Day respiration is the process by which nonphotorespiratory CO2 is produced by illuminated leaves. The biological function of day respiratory metabolism is a major conundrum of plant photosynthesis research: because the rate of CO2 evolution is partly inhibited in the light, it is viewed as either detrimental to plant carbon balance or necessary for photosynthesis operation (e.g., in providing cytoplasmic ATP for sucrose synthesis). Systematic variations in the rate of day respiration under contrasting environmental conditions have been used to elucidate the metabolic rationale of respiration in the light. Using isotopic techniques, we show that both glycolysis and the tricarboxylic acid cycle activities are inversely related to the ambient CO2/O2 ratio: day respiratory metabolism is enhanced under high photorespiratory (low CO2) conditions. Such a relationship also correlates with the dihydroxyacetone phosphate/Glc-6-P ratio, suggesting that photosynthetic products exert a control on day respiration. Thus, day respiration is normally inhibited by phosphoryl (ATP/ADP) and reductive (NADH/NAD) poise but is up-regulated by photorespiration. Such an effect may be related to the need for NH2 transfers during the recovery of photorespiratory cycle intermediates.

Fig. 1.

Steady isotope discrimination and carbon isotope composition of CO₂. (A) Carbon isotope discrimination (Δ_obs) associated with photosynthesis of detached leaves [at 21°C and 400 μmol·m⁻²·s⁻¹ photosynthetic photon flux density (PPFD)] fed with either ¹³C-1- or ¹³C-2-enriched Pyr under four CO₂/O₂ conditions: 140, 400, or 1,000 μl·liter⁻¹ CO₂ in 21% O₂ or 400 μl·liter⁻¹ CO₂ in 2% O₂. (B) Carbon isotope composition (δ¹³C) of respired CO₂ in darkness after the corresponding light periods. When leaves experienced a light period under 2% O₂, the carbon isotope composition was measured in either 21% (indicated as 400 μl·liter⁻¹, 2–21%) or 2% O₂ (indicated as 400 μl·liter⁻¹, 2–2%). Each value is the mean ± SE of three measurements. The control δ¹³C value of respired CO₂ was −22.1 ± 0.5‰.

Fig. 2.

Decarboxylation rates and inhibition of decarboxylation by light calculated from data of Fig. 1, using the method of ref. . (A and B) The decarboxylation rates by PDH (empty bars) and the TCA cycle (filled bars) are given in the light (A, denoted as r_light) and in the dark (B, denoted as r_night). (C) Inhibition by light (calculated as 1 − r_light/r_night, in %) is indicated. Conditions experienced by leaves during the light period are indicated on the x axis as in Fig. 1: 140, 400, and 1,000 μl·liter⁻¹ CO₂ in 21% O₂ and 400 μl·liter⁻¹ CO₂ in 2% O₂. The δ¹³C values of dark-respired CO₂ obtained in 2% O₂ (Fig. 1B Right) were used to calculate the inhibition value after a light period in 2% O₂.

Fig. 3.

Isotopomics array representation of ¹³C abundance in the carbon atom positions of major metabolites in detached leaves incubated with ¹³C-substrates for 2 h at 21°C, 21% O₂, and 400 μmol·m⁻²·s⁻¹ PPFD. CO₂ mole fraction was 140 μl·liter⁻¹, 400 μl·liter⁻¹, and 1,000 μl·liter⁻¹. At t = 2 h, leaves were immediately frozen in liquid nitrogen for perchloric acid extraction. Perchloric extracts were analyzed for positional ¹³C abundances by NMR. Each column is a separate set of experimental conditions. Cit, citrate; Ido/Gal, uncertain d-hexofuranose belonging to the idose-galactose group; Obt, oxobutyrate; SF and SG, fructosyl and glucosyl moieties of sucrose, respectively. Red and green cells indicate ¹³C abundances above and below the natural abundance (which is 1.1%). Below-natural abundance cells appear dark green because the ¹³C abundance is still very close to 1.1%.

Fig. 4.

¹³C abundance in Glu (black bars), Succ (light gray bars), and Fum (dark gray bars) relative to that in Mal. Values are from the data of Fig. 3. The three different CO₂ mole fractions (in μmol·mol⁻¹) used in the experiment are indicated on the x axis. (Inset) Data of Fig. 3 replotted to show the relationship between the positional ¹³C abundance (in percentage of ¹³C) in Mal C-2 (▾), Fum C-2/3 (●), and Glu C-2 (□) and the quantity of metabolite (in μmol per gram of fresh weight). Short dashed lines indicate exponential decay (Fum and Mal) and linear (Glu) regressions; both are significant: F = 7.26 (P < 0.005) and F = 16.38 (P < 0.003), respectively.

Fig. 5.

Relationship between the inhibition of the TCA cycle in the light (in %; data from Fig. 2) and the DHAP to inorganic phosphate (P_i) (○) or Glc-6-phosphate (●) ratio. Phosphorylated compounds were measured by ³¹P NMR on the same samples used for ¹³C NMR after ¹³C labeling. Lines stand for linear regressions. The regression with DHAP/Glc-6-P is significant (F = 61.7, P < 0.08).