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. 2021 Nov 3;187(3):1387-1398.
doi: 10.1093/plphys/kiab351.

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas

Ashwani K Rai  1 , Timothy Chen  1 , James V Moroney  1
Affiliations

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas

Ashwani K Rai et al. Plant Physiol. .

Abstract

Chlamydomonas reinhardtii can grow photosynthetically using CO2 or in the dark using acetate as the carbon source. In the light in air, the CO2 concentrating mechanism (CCM) of C. reinhardtii accumulates CO2, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO2 concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO2 (0.04% CO2 [v/v] in air). These strains were not able to grow at very low CO2 (<0.02% CO2 [v/v] in air), and their ability to accumulate inorganic carbon (Ci = CO2 + HCO3-) was reduced. At low CO2 concentrations, the CCM is needed to both deliver Ci to Rubisco and to minimize the leak of CO2 generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO2 released from respiration and photorespiration as well as the CO2 leaked from the chloroplast to HCO3- thus "recapturing" this potentially lost CO2.

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Figures

Figure 1
Figure 1
CAH4/5 protein levels are strongly affected by CO2 levels. The left panel is an immunoblot probed with anti-CAH4 antibodies showing the CAH4/5 levels in D66 grown on minimal media in light at high CO2 or ambient CO2. Cells were grown in MIN media for 72 h in high CO2 conditions before incubating them under the respective CO2 conditions. The right panel is an SDS–PAGE gel of the same samples stained with Coomassie Blue.
Figure 2
Figure 2
CAH4/5 protein expression is under the control of CIA5. The left panel is an immunoblot probed with anti-CAH4 antibodies showing CAH4/5 levels in cia5 grown on MIN media in light at high CO2 or ambient CO2 compared to D66 grown at ambient CO2. Cells were grown in MIN media for 72 h in high CO2 conditions before incubating them under the respective CO2 conditions. The right panel is an SDS–PAGE gel of the same samples stained with Coomassie Blue.
Figure 3
Figure 3
Relative mRNA and protein expression of CAH4/5 in D66 and the knockdown mutants. A, RT-qPCR shows expression of CAH4/5 genes in knockdown lines cah4/5-1 and cah4/5-2 and in D66. Cells were grown in MIN media for 48 h in high CO2 conditions before transferring them to ambient CO2 for 12 h before harvesting for RNA. Error bars represent standard deviation from three biological replicates. Transcript levels were calculated using 2−ΔΔCT relative to the reference gene CBLP and reported relative to the corresponding WT D66 cells. The asterisk indicates the value is significantly different from the control (*P < 0.05 by Student’s t test). B, Western blot showing protein levels of CAH4/5 in knockdown lines cah4/5-1 and cah4/5-2 and D66 grown in ambient CO2 (0.04% CO2 (v/v) in air. Cells were initially grown in MIN media in the light for 48 h in high CO2 conditions before incubating them for 12 h at ambient CO2. The top panel is an immunoblot using an antibody raised against CAH4; the bottom panel is SDS–PAGE of the samples stained with Coomassie Blue.
Figure 4
Figure 4
Growth of mutants cah4/5-1 and cah4/5-2 at different CO2 levels. Growth analysis showing D66, cah4/5-1, cah4/5-2, cia3, and cia5. Cells were diluted to 6.6 × 106 cells mL−1, followed by 1:10 serial dilution three times at very low CO2, ambient CO2, and high CO2 at pH 7.2, pH 7.8, and pH 8.4, respectively. Cells were grown for 6 d. The cia3 and cia5 mutants were included as a CCM-deficient control. Cells were initially grown in TAP media at ambient CO2 in the light before spotting them onto plates.
Figure 5
Figure 5
Photosynthetic oxygen evolution of the cah4/5 knockdown RNAi lines and D66. Ci affinity and K0.5(Ci) were estimated for cah4/5-1, cah4/5-2, and D66 acclimated to ambient CO2 for 12 h at (A) pH 7.2, (B) pH 7.8, and (C) pH 8.4. K0.5 (Ci) values (Ci concentration needed for half maximum oxygen evolution) were calculated from the O2 evolution versus Ci curves. Asterisk indicates the value is significantly different from the control (*P < 0.05 by Student’s t test). Cells were grown in MIN media for 48 h in high CO2 conditions before incubating them for 12 h at ambient CO2 at the indicated pH. Each point in O2 evolution versus Ci curves represents the mean and sd of three technical replicates from a representative experiment. Error bars in K0.5 (Ci) values indicate sd.
Figure 6
Figure 6
Ci uptake of D66 and cah4/5 RNAi knockdown lines at pH 7.8. The silicone oil method was used to estimate Ci fixation and Ci accumulation (see “Materials and methods”). Cells were grown in elevated CO2 and then acclimated to ambient CO2 for 12 h prior to the assays. Cells were depleted of endogenous Ci in a 6-mL chamber with a Clark electrode before performing the assays. Time courses of CO2 fixation (A) and Ci accumulation (B) are shown at pH 7.8. Each point represents the mean and sd of three technical replicates from a representative experiment.
Figure 7
Figure 7
Ci uptake of D66 and cah4/5 RNAi knockdown lines at pH 8.4. The silicone oil method was used to estimate Ci fixation and Ci accumulation (see “Materials and methods” section). Cells were grown in elevated CO2 and then acclimated to ambient CO2 for 12 h prior to the assays. Cells were depleted of endogenous Ci in a 6-mL chamber with a Clark electrode before running the assays. Time courses of CO2 fixation (A) and Ci accumulation (B) are shown at pH 8.4. Each point represents the mean and SD of three technical replicates from a representative experiment.
Figure 8
Figure 8
Mitochondrial localization in WT D66 cells with change in CO2 levels. TEM of sectioned Chlamydomonas WT cells at (A) high CO2 and (B) ambient CO2 levels. WT cells were grown in MIN media for 48 h in high CO2 before incubating them for 12 h at their respective conditions. Areas shown by the rectangles are enlarged (right) to reveal mitochondrial structures. Scale bar, 2 µm (A), 2 µm (B), and 500 nm (enlargements).
Figure 9
Figure 9
CAH5 protein localization in WT cells with changes depending on the CO2 levels during growth. Confocal images were taken of CSI_FC1G05 cells expressing pLM005-CAH5-Venus-3xFLAG at (A) high CO2 and (B) ambient CO2 levels. WT cells were grown in MIN media for 48 h in high CO2 before incubating them for 12 h at their respective conditions. Scale bar, 4.2 µm (A), 2.5 µm (B).
Figure 10
Figure 10
Model showing the proposed physiological role of mitochondrial CAH4/5 in the CCM of Chlamydomonas. In (A) known CAs (CAH1, CAH2, CAH3, and LCIB) are indicated in the periplasmic space, the chloroplast stroma, and the thylakoid lumen, respectively. Dotted lines indicate the leakage of CO2 from the pyrenoid and how it is recaptured by different layers of CAs. PGA, phosphoglyceric acid. B, The proposed recapturing of CO2 in mitochondria. The CO2 leakage arises from the chloroplast, mitochondrial respiration, and photorespiration.
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