Cyanide-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration during postharvest ripening of tomato fruit

Abstract

Tomato (Lycopersicon esculentum) mitochondria contain both alternative oxidase (AOX) and uncoupling protein as energy-dissipating systems that can decrease the efficiency of oxidative phosphorylation. We followed the cyanide (CN)-resistant, ATP-synthesis-sustained, and uncoupling-protein-sustained respiration of isolated mitochondria, as well as the immunologically detectable levels of uncoupling protein and AOX, during tomato fruit ripening from the mature green stage to the red stage. The AOX protein level and CN-resistant respiration of isolated mitochondria decreased with ripening from the green to the red stage. The ATP-synthesis-sustained respiration followed the same behavior. In contrast, the level of uncoupling protein and the total uncoupling-protein-sustained respiration of isolated mitochondria decreased from only the yellow stage on. We observed an acute inhibition of the CN-resistant respiration by linoleic acid in the micromolar range. These results suggest that the two energy-dissipating systems could have different roles during the ripening process.

Figure 1

Respiratory network in plant mitochondria. This scheme shows the pathways of electron transport from succinate as the oxidizable substrate and three ways in which the proton electrochemical gradient (ΔμH⁺) is used. Inhibitors of the different complexes are shown. NADH oxidation in plant mitochondria and other ways of using ΔμH⁺ (e.g. transport of ions and metabolites) are not shown. olig, Oligomycin; Q, ubiquinone.

Figure 2

A and B, Determination of ATP-synthesis (plus H⁺ leak)-sustained respiration (defined as [BHAM plus BSA and GTP]-resistant respiration) in states 3 (A) and 4 (B). State 4 (B) was measured after ADP exhaustion. Mitochondria (mito) were incubated in a standard reaction medium containing 0.5% BSA, 1 mm GTP, and 2 mm ADP (for state 3), as described in Methods. Two millimolar BHAM, 0.17 mm ADP, and 1.5 mm KCN were added. The numbers on the traces refer to the O₂ consumption rates in nmol min⁻¹ mg⁻¹ protein. The respiration of green tomato mitochondria is shown as an example. C, ATP-synthesis (plus H⁺ leak)-sustained respiration ([BHAM plus BSA and GTP]-resistant respiration) in state (st.) 3 (▪), state 4 (•), and state 4 plus oligomycin (♦) in the four stages of tomato fruit ripeness (G, green; Y, yellow; O, orange; and R, red). Respiratory rates are in nmol O₂ min⁻¹ mg⁻¹ protein. The data are presented as the means ± sd of four independent experiments. The respiratory control values (RC) obtained from the average values for states-3 and -4 respiration at each stage of ripening also appear.

Figure 3

Determination of AOX-sustained respiration (defined as [KCN plus BSA and GTP]-resistant respiration) in state 3 (A) and state 4 (B) in the presence and absence of DTT and pyruvate (Pyr). Mitochondria (mito) were incubated in a standard reaction medium in the presence of 0.5% BSA, 1 mm GTP, and 2 mm ADP (state 3) or 2.5 μg oligomycin mL⁻¹ incubation medium (state 4), as described in Methods; 1.5 mm KCN and 2 mm BHAM were added. The numbers on the traces refer to the O₂-consumption rates in nmol min⁻¹ mg⁻¹ protein. The respiration of green tomato mitochondria is shown as an example. C, CN-resistant respiration (BSA plus GTP) in the absence (•) and presence (▪) of DTT and pyruvate in the four stages of tomato fruit ripeness (G, green; Y, yellow; O, orange; and R, red). The effect of 3.9 μm LA in the presence of DTT and pyruvate (minus BSA minus GTP) is shown (♦). Respiratory rates are in nmol O₂ min⁻¹ mg⁻¹ in states 3 and 4 from four to six independent experiments.

Figure 4

A, Determination of PUMP-sustained respiration. Mitochondria (mito) were incubated in a standard reaction medium in the presence of 2.5 μg oligomycin mg⁻¹ protein and 2 mm BHAM, as described in Methods; 10 μm LA, 0.5% BSA, 1 mm GTP, and 1 μm FCCP were added. The numbers on the traces refer to the O₂-consumption rates in nmol min⁻¹ mg⁻¹ protein. The numbers in parentheses refer to the slopes used to calculate the total PUMP activity and stimulation by LA shown in B. The respiration of green tomato mitochondria is shown as an example. B, PUMP-sustained respiration in the four stages of ripeness (G, green; Y, yellow; O, orange; and R, red). ▪, Total PUMP activity plus H⁺ leak (respiration after the addition of LA; slope 2 from A); ▴, endogenous PUMP activity plus H⁺ leak in the absence of LA (respiration before the addition of LA; slope 1 from A); •, H⁺ leak (respiration after the addition of BSA and GTP; slope 3 from A). Respiratory rates are in nmol O₂ min⁻¹ mg⁻¹ protein. The data are presented as the means ± sd from three to four independent experiments.

Figure 5

Immunoblot analysis of tomato total mitochondrial proteins from fruits at different stages of ripeness (G, green; Y, yellow; O, orange; and R, red). Proteins were electrophoresed, transferred to nitrocellulose, and reacted with monoclonal antibodies against the S. guttatum AOX (left) or with polyclonal antibodies against the potato PUMP (right), as described in Methods. The total mitochondrial protein load was 150 μg for AOX detection and 30 μg for PUMP detection. The molecular mass markers appear on the left.