The Chlamydomonas reinhardtii cia3 mutant lacking a thylakoid lumen-localized carbonic anhydrase is limited by CO2 supply to rubisco and not photosystem II function in vivo

Abstract

The Chlamydomonas reinhardtii cia3 mutant has a phenotype indicating that it requires high-CO(2) levels for effective photosynthesis and growth. It was initially proposed that this mutant was defective in a carbonic anhydrase (CA) that was a key component of the photosynthetic CO(2)-concentrating mechanism (CCM). However, more recent identification of the genetic lesion as a defect in a lumenal CA associated with photosystem II (PSII) has raised questions about the role of this CA in either the CCM or PSII function. To resolve the role of this lumenal CA, we re-examined the physiology of the cia3 mutant. We confirmed and extended previous gas exchange analyses by using membrane-inlet mass spectrometry to monitor(16)O(2),(18)O(2), and CO(2) fluxes in vivo. The results demonstrate that PSII electron transport is not limited in the cia3 mutant at low inorganic carbon (Ci). We also measured metabolite pools sizes and showed that the RuBP pool does not fall to abnormally low levels at low Ci as might be expected by a photosynthetic electron transport or ATP generation limitation. Overall, the results demonstrate that under low Ci conditions, the mutant lacks the ability to supply Rubisco with adequate CO(2) for effective CO(2) fixation and is not limited directly by any aspect of PSII function. We conclude that the thylakoid CA is primarily required for the proper functioning of the CCM at low Ci by providing an ample supply of CO(2) for Rubisco.

Figure 1.

Rates of gross O₂ evolution (▪), gross O₂ uptake (○), net O₂ evolution (▵), and net CO₂ uptake (▾) in response to external Ci concentration. Data are expressed as a percentage of the gross O₂ evolution rate at 250 μm Ci (wild type = 2.6 μmol mg^-1 Chl min^-1, cia3 = 1.6 μmol mg^-1 Chl min^-1). Cultures were concentrated by centrifugation and resuspended to a final concentration of 15 μg Chl mL^-1 in 20 mm HEPES, pH 7.0, and 30 μg mL^-1 CA. Starting conditions were 400 μm Ci, 300 μmol photons m^-2 s^-1, and 20°C. Measurements were initiated by switching on the light and allowing the cells to draw down the external Ci and were continued until the Ci compensation point was reached. The rates at various Ci concentrations were collected from the continuous draw-down experiments, n = 3.

Figure 2.

PSII activity (measured as gross O₂ evolution rate) divided by net CO₂ uptake (A) or net O₂ evolution (B) rate for wild-type (○) or mutant (▪) cells in response to external Ci concentration. Data are derived from Figure 1.

Figure 3.

Photosynthetic rates and metabolite pools in response to external Ci concentration for wild-type (○) or mutant (▪) cells. Cultures were concentrated by centrifugation and were resuspended to a final concentration of 15 μg Chl mL^-1 in 20 mm HEPES pH 7.0 and 30 μg mL^-1 CA. Ci draw-down experiments were conducted similar to those shown in Figure 1, and cell samples were killed by rapidly drawing them into a syringe containing trifluoroacetic acid (TFA; 10% final concentration) at a particular Ci concentration. A number of samples at different Ci levels could be collected from a single Ci drawn-down experiment. All measurements were made at 300 μmol m^-2 s^-1 light and 20°C.

Figure 4.

RuBP pool size versus photosynthetic rate (expressed as net O₂ evolution) for wild-type (○) or mutant (▪) cells. Data are taken from the values presented in Figure 3.

Figure 5.

Oxygen sensitivity of the CO₂ compensation point (A) and the Φ_PSII (C) and Φ_PSI (D) for wild-type (white columns) or cai3 mutant (black columns) cells. The Φ_PSI/Φ_PSII is presented in B. Cultures were concentrated onto a glass fiber filter by gentle centrifugation and were measured in the gas phase at 2% (Low) and 21% (High) O₂, 300 mol m^-2 s^-1 light, and 20°C in the presence of CA.

Figure 6.

Light response of photosynthesis and the Φ_PSI and Φ_PSII in wild-type (○) and mutant (▪) cells. Cultures were centrifuged and resuspended in 20 mm HEPES, pH 7.0, to a final concentration of 15 μg Chl mL^-1. Ci levels were maintained around 2 mm Ci, no CA was added, and the measurement temperature was 20°C, n = 3.