Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii

Abstract

The supply of inorganic carbon (Ci; CO2 and HCO3 (-)) is an environmental rate-limiting factor in aquatic photosynthetic organisms. To overcome the difficulty in acquiring Ci in limiting-CO2 conditions, an active Ci uptake system called the CO2-concentrating mechanism (CCM) is induced to increase CO2 concentrations in the chloroplast stroma. An ATP-binding cassette transporter, HLA3, and a formate/nitrite transporter homolog, LCIA, are reported to be associated with HCO3 (-) uptake [Wang and Spalding (2014) Plant Physiol 166(4):2040-2050]. However, direct evidence of the route of HCO3 (-) uptake from the outside of cells to the chloroplast stroma remains elusive owing to a lack of information on HLA3 localization and comparative analyses of the contribution of HLA3 and LCIA to the CCM. In this study, we revealed that HLA3 and LCIA are localized to the plasma membrane and chloroplast envelope, respectively. Insertion mutants of HLA3 and/or LCIA showed decreased Ci affinities/accumulation, especially in alkaline conditions where HCO3 (-) is the predominant form of Ci. HLA3 and LCIA formed protein complexes independently, and the absence of LCIA decreased HLA3 mRNA accumulation, suggesting the presence of unidentified retrograde signals from the chloroplast to the nucleus to maintain HLA3 mRNA expression. Furthermore, although single overexpression of HLA3 or LCIA in high CO2 conditions did not affect Ci affinity, simultaneous overexpression of HLA3 with LCIA significantly increased Ci affinity/accumulation. These results highlight the HLA3/LCIA-driven cooperative uptake of HCO3 (-) and a key role of LCIA in the maintenance of HLA3 stability as well as Ci affinity/accumulation in the CCM.

Keywords: CO2-concentrating mechanism; Chlamydomonas; bicarbonate uptake; chloroplast envelope; photosynthesis.

Fig. 1.

Accumulation and subcellular localization of HLA3 and LCIA. (A) Time-course of accumulation of HLA3, LCIA, and LCI1 proteins in WT cells. For induction of limiting-CO₂ conditions, cells supplied with 5% CO₂ (high CO₂; HC) were centrifuged, suspended in new fresh medium, and cultured with 0.04% CO₂ for 1, 2, 4, 6, and 12 h. Histone H3 was used as a loading control. The total Ci concentrations and calculated CO₂ concentrations after each induction time are also indicated below the figures. Using an HCO₃^–/CO₂ ratio of 4.47 at pH 7.0, CO₂ concentrations were calculated using the equation (pH = pK_a + log₁₀ [HCO₃^–]/[CO₂]), where pK_a was an acid dissociation constant of 6.35. (B) Subcellular localization of HLA3 and LCIA by an indirect immunofluorescence assay. WT cells were grown in very low CO₂ (VLC) for 12 h. DIC, differential interference contrast. (Scale bars, 5 µm.) (C) Immunoblot analysis in isolated plasma membrane (PM) and chloroplast envelope (CE) fractions with antibodies against HLA3, LCI1, H⁺-ATPase, LCIA, and CCP1. Asterisks indicate nonspecific bands.

Fig. 2.

Characterization of an HLA3 insertion mutant. (A) Inorganic carbon (Ci) affinity of WT and HLA3 insertion mutant (Hin-1) grown in very low CO₂ (VLC) for 6 or 12 h. Photosynthetic O₂-evolving activity was measured with different external Ci concentrations at pH 6.2 or 7.8, and the respective K_0.5 (Ci) values, the Ci concentration required for half maximum O₂-evolving activity, were calculated. (B) Ci affinity of WT, Hin-1, and complemented Hin-1 (Hin-1C) grown in high CO₂ (HC) or VLC for 1, 2, 4, 6, and 12 h. O₂-evolving activity was measured at pH 9.0. *P < 0.01 and **P < 0.05 by Student t test. (C) Accumulation and fixation of Ci in WT, Hin-1, and Hin-1C. Cells were grown in VLC for 6 h, and intracellular Ci accumulation (Left) and CO₂ fixation (Right) at pH 9.0 were measured using a silicone oil layer method. SIS, sorbitol impermeable space.

Fig. 3.

Characterization of LCIA insertion mutants and an LCIA/HLA3 double-insertion mutant. (A) Accumulation of LCIA, HLA3, LCI1, and LCIB in WT, LCIA insertion mutants (Ain-1 and Ain-2), and their complemented strains (Ain-1C and Ain-2C). Cells were grown in very low CO₂ (VLC) for 12 h. (B) Inorganic carbon (Ci) affinity of WT, Ain-1, Ain-2, Ain-1C, and Ain-2C grown in VLC for 6 or 12 h. Photosynthetic O₂-evolving activity was measured with different external Ci concentrations at pH 6.2 or 7.8, and the respective K_0.5 (Ci) values, the Ci concentration required for half maximum O₂-evolving activity, were calculated. *P < 0.01. (C) Ci affinity of WT, Ain-1, Ain-2, Ain-1C, and Ain-2C grown in high CO₂ (HC) or VLC for 1, 2, 4, 6, and 12 h. O₂-evolving activity was measured at pH 9.0. *P < 0.01. (D) Accumulation of HLA3 and LCIA in WT and LCIA/HLA3 double-insertion mutants (AHin-1 and AHin-2) grown in VLC for 12 h. (E) Ci affinity of WT and AHin-2 grown in HC or VLC for 6 or 12 h. O₂-evolving activity was measured at pH 6.2, 7.8, or 9.0. *P < 0.01. (F) Accumulation and fixation of Ci in WT and AHin-2. Cells were grown in HC or VLC for 6 h, and intracellular Ci accumulation (Left) and CO₂ fixation (Right) were measured at pH 9.0. SIS, sorbitol impermeable space.

Fig. 4.

Characterization of LCIA- and HLA3-overexpressing strains. Accumulation of inorganic carbon (Ci) (Left) and Ci affinity (Right) in WT and in strains overexpressing LCIA (A), HLA3 (B), LCIA/HLA3 (C), and LCIA/LCI1 (D). Cells were grown in high CO₂-NO₃^– for 12 h, and Ci accumulation was measured at pH 9.0. For Ci affinity, O₂-evolving activity was measured with different external Ci concentrations at pH 6.2, 7.8, or 9.0 and the respective K_0.5 (Ci) values, the Ci concentration required for half maximum O₂-evolving activity, were calculated. *P < 0.01 and **P < 0.05.

Fig. 5.

Molecular masses of LCIA and HLA3 in nondenaturing conditions and effect of the absence of LCIA on HLA3 mRNA accumulation. (A) Molecular masses of LCIA and HLA3 in nondenaturing conditions. Total proteins were solubilized using 1.0% n-dodecyl β-d-maltoside and separated by blue-native PAGE. (B) Quantitative real-time PCR analyses of HLA3 (Upper) and LCIA (Lower) in WT, Ain-1, Ain-2, Ain-1C, Ain-2C, Hin-1, and Hin-1C. These cells were grown in very low CO₂ conditions for 4 h. Expression of each gene was normalized to CBLP. Data in all experiments indicate mean value ± SD from three biological replicates. *P < 0.01.