Mitochondrial-driven bicarbonate transport supports photosynthesis in a marine microalga

Abstract

The CO(2)-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO(3)(-) transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO(3)(-) to CO(2) for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO(2) generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO(2) concentration to a level above its chemical equilibrium that was diagnostic for active HCO(3)(-) transport. The transient rise in extracellular CO(2) occurred in the light and the dark and was resolved from concurrent respiratory CO(2) efflux using H(13)CO(3)(-) stable isotope techniques. H(13)CO(3)(-) pump-(13)CO(2) leak activity of the CCM was unaffected by 10 microM 3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O(2) evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished (13)CO(2) efflux in the dark and light. These results indicate that H(13)CO(3)(-) transport was supported by mitochondrial energy production in contrast to other algae and cyanobacteria in which it is supported by photosynthetic electron transport. This is the first report of a direct role for mitochondria in the energization and functioning of the CCM in a photosynthetic organism.

Figure 1

Measurement of ¹²CO₂ (———) and ¹³CO₂ (— — —) fluxes in the dark and light. a, An illuminated (1 mmol m⁻² s⁻¹, photosynthetically active radiation) cell suspension of N. gaditana was allowed to reach the CO₂ compensation point, and the light was turned off. Changes in ¹²CO₂ concentration were followed with time, and the light was then turned on. The asterisk indicates the transition from the slow to the fast phase of ¹²CO₂ decline. b, As in a except that bovine CA (40 μg mL⁻¹) was added to the darkened cell suspension during the rise in ¹²CO₂. c, K₂¹³CO₃ (100 μm) was added to reaction buffer (-cells) to determine the equilibrium ¹³CO₂ concentration at pH 8.0 and 25°C. d, As in a except that 100 μm K₂¹³CO₃ was added 2 min after darkening and both ¹²CO₂ and ¹³CO₂ concentrations were measured over time. The time courses are superimposed for comparison.

Figure 2

Measurement of ¹²CO₂ (———) and ¹⁶O₂ (. . . . .) fluxes in a cell suspension of N. gaditana in the light (L) and dark (D) and in the absence and presence of the HCO₃⁻ transport inhibitor DIDS (500 μm). The experimental procedure was essentially the same as that for Figure 1. The asterisk indicates the transition from the slow to the fast phase of ¹²CO₂ decline.

Figure 3

Measurement of ¹²CO₂ (———) and ¹³CO₂ (— — —) fluxes in the dark and light in the presence of 0 (a), 1.5 (b), 5.6 (c), and 130 (d) μm O₂. The plots obtained at 230 μm O₂ are shown in Figure 1d. The time courses are superimposed for comparison. The experimental procedure was essentially the same as that for Figure 1; off, light turned off; on, light turned on.

Figure 4

Measurement of ¹²CO₂ (———) and ¹³CO₂ (— — —) fluxes in the dark and light in the absence (a; control) and presence (b and c) of 250 μm KCN. KCN was added during the slow decline in CO₂ (b) or 2 min before darkening (c). The time courses are superimposed for comparison. The experimental procedure was essentially the same as that for Figure 1; off, lights turned off; on, lights turned on.

Figure 5

Effect of 250 μm KCN on the time course of photosynthetic O₂ evolution in the presence of various levels of external Ci (▪). For comparison, O₂ evolution in the absence of KCN at 0.1 mm Ci (●) is also shown.

Figure 6

Measurement of ¹²CO₂ (———), ¹³CO₂ (— — —), and ¹⁶O₂ (. . . . .) fluxes in the dark and light in the absence and presence of 10 μm DCMU. The time courses are superimposed for comparison. The experimental procedure was essentially the same as that for Figure 1.