Volume 7, Issue 10, Pages 1545-1559 (October 2014) Integration of Carbon Assimilation Modes with Photosynthetic Light Capture in the Green Alga Chlamydomonas reinhardtii  Hanna Berger, Olga Blifernez-Klassen, Matteo Ballottari, Roberto Bassi, Lutz Wobbe, Olaf Kruse  Molecular Plant  Volume 7, Issue 10, Pages 1545-1559 (October 2014) DOI: 10.1093/mp/ssu083 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Availability and Type of Carbon Source Triggers NAB1 Accumulation. (A) Immunodetection of NAB1 in whole cell protein extracts derived from cultures grown in the presence (+) or absence (–) of acetate (Ac). Cultures were either bubbled with air (–CO2) or CO2-enriched (3% (v/v)) air (+CO2). (B) Immunodetection of NAB1 after growth in the presence (+) or absence (–) of carboxymethyl cellulose (CMC) and air levels of CO2 (–CO2). Lower panels (A, B): Coomassie-Brilliant Blue-stain (CBB) serving as a loading control. Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Limited CO2 Supply Activates the NAB1 Promoter Resulting in Protein Accumulation. Photoautotrophically cultured cells (3% (v/v) CO2 in air) expressing the NAB1:gLuc reporter construct were re-suspended in media with (+Ac) or without acetate (–Ac) and bubbled with air (–CO2) or CO2-enriched air (3% (v/v) CO2) for 4h. (A) NAB1 and LHCBM6 mRNA level assessed by qRT–PCR. (B) Reporter activity in culture supernatants determined by luminescence quantification. (C) Densitometric scanning of NAB1 (αNAB1) and LHCBM6/8 (αL6/8) immunoblot signals (D). Phototrophic cultivation (–Ac/+CO2) served as the reference condition (set to 1). Error bars represent SD (n = 3 for (A, B); n = 6 for (C)). Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Antenna Size Adjustment during the Transition from Mixotrophy to Photoheterotrophy Requires NAB1-Mediated Translation Control. (A, B) Immunoblot detection of NAB1 (αNAB1), LHCBM isoforms 6 and 8 (αL6/8), all LHCBMs (αLHCII), and D1 protein (αD1) in whole cell samples derived from cultures of the parental strain (wt; (A)) and the NAB1 k.o. mutant (B). Cultures acclimated to high CO2 (3% (v/v)) concentrations (+, t0) were further exposed to high CO2 supply (black bar), air levels of CO2 (–, white bar), or fluctuating CO2 levels in 6-h intervals (–/+). (C) qRT–PCR quantification of LHCBM6 transcript levels in parental strain (wt) and NAB1 k.o. mutant at t0 and after 12h of mixotrophic (+CO2) or photoheterotrophic (–CO2) growth with level at 0h set to 1. Indicated are mean values with standard deviations (n = 3). (D) Mean chlorophyll a/b ratios at t12h (n = 3). (E–G) Chlorophyll a fluorescence analyses performed 12h after changing the CO2 supply to either low (–CO2) or high (+CO2) concentrations. (E) Fluorescence induction kinetics in the presence of DCMU (left panel) and the calculated functional PSII antenna size (1/τ; right panel). (F) Effective PSII quantum yield ΦPSII and (G) excitation pressure. Mean values are shown together with standard deviations (n = 9 for (E, F); n = 3 for (G)). Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 The Absence of NAB1 Reduces Growth Performance under Conditions of Fluctuating and Low CO2 Supply. Parental strain (wt; (A, B, D)) and NAB1 k.o. mutant (A, C, and D) were cultivated as was done for the immunoblot studies shown in Figure 3 to analyze the effects of continuous CO2 supply (black; +CO2), air bubbling (light gray; –CO2), or fluctuating CO2 provision on the growth performance of both cell lines. Growth was determined as dry biomass (A) and cell density (B–D). (D) Detailed view on the initial growth phase in wt (solid lines) and k.o. mutant (dashed lines) cultures. All data represent mean values ± SD of three biological replicates with triplicate measurements (n = 9). Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 A Functional NAB1-Mediated Adjustment of the PSII Antenna Depends on Tight Nuclear Promoter Control. Cultures of the NAB1 oex strain were grown for immunodetection (A) and the determination of Chl a/b ratios at t12h (B) as described in Figure 3. Growth was determined as dry biomass (C) and cell density (D) with data representing means and SD (n = 6). Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 NAB1 Accumulates under Conditions that Induce a State II Transition and Accumulation Requires Photosynthetic Electron Transport and the LHCII Kinase STT7. Wild-type cells (upper panels), the state transition kinase-deficient mutant stt7 (lower panels), and the STT7-complemented strain (STT7 comp., second row) were cultivated phototrophically in minimal medium and 3% (v/v) CO2 prior to acetate addition and CO2-limitation (air bubbling). (A) Representative 77 K fluorescence spectra of the time course experiment performed in the presence (right panels, +DCMU) or absence (left panels, no inh.) of the PET inhibitor DCMU (20 μM). (B) PSI/PSII fluorescence ratios at 77 K determined as F712/F685 in the absence (dark bars, no inh.) or presence of DCMU (light bars, +DCMU). Data represent means and SD of at least two biological replicates. (C) Immunodetection of NAB1 in cell lysates of wild-type cells (wt), stt7, and complemented strain (STT7 comp.) before (0h, +CO2) and 2–6h after CO2-limitation and acetate supply (+Ac), in the presence (+DCMU) or absence of inhibitor (no inh.). Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 NAB1-Mediated PSII Antenna Size Control Is a Long-Term Response to CO2-Limitation. (A) A Chlamydomonas cell exposed to organic carbon and saturating CO2 concentrations. The Calvin cycle (C3-cycle, C3C) consumes high amounts of NADPH provided by linear electron transport that requires a large absorption at PSII (yellow flash). Activity of the NAB1 promoter in the nucleus (gray) is low (red cross) resulting in a reduced availability of the LHCII translation repressor. (B) Limiting CO2 supply reduces NADPH consumption in the C3-cycle causing increased PSII excitation pressure that has to be relieved by lowering PSII light capture. A retrograde signal (dashed arrow) activates the nuclear NAB1 promoter to increase the extent of LHCII translation repression in the cytosol. (C) NAB1-mediated translation repression as a long-term response replaces state transitions as a fast mechanism of excitation pressure control. Molecular Plant 2014 7, 1545-1559DOI: (10.1093/mp/ssu083) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions