Linking the Dynamic Response of the Carbon Dioxide-Concentrating Mechanism to Carbon Assimilation Behavior in Fremyella diplosiphon

Environmental regulation of photosynthesis in cyanobacteria enhances organismal fitness, light capture, and associated carbon fixation under dynamic conditions. Concentration of carbon dioxide (CO2) near the carbon-fixing enzyme RubisCO occurs via the CO2-concentrating mechanism (CCM). The CCM is also tuned in response to carbon availability, light quality or levels, or nutrient access—cues that also impact photosynthesis. We adapted dynamic gas exchange methods generally used with plants to investigate environmental regulation of the CCM and carbon fixation capacity using glass fiber-filtered cells of the cyanobacterium Fremyella diplosiphon. We describe a breakthrough in measuring real-time carbon uptake and associated assimilation capacity for cells grown in distinct conditions (i.e., light quality, light quantity, or carbon status). These measurements demonstrate that the CCM modulates carbon uptake and assimilation under low-Ci conditions and that light-dependent regulation of pigmentation, cell shape, and downstream stages of carbon fixation are critical for tuning carbon uptake and assimilation.

ulation of the CCM (30)(31)(32)(33). These efforts are generally limited to single-celled model cyanobacteria and are often inadequate for quickly measuring net C i consumption due to the aqueous nature of these organisms. Several distinct methods for assaying carbon uptake, fixation, and overall photosynthesis have been applied to cyanobacteria. It is perhaps most common to measure O 2 evolution, which probes linear electron flow at photosystem II (PSII) and shows reductions when CCM is compromised (34)(35)(36). Chlorophyll (Chl) fluorescence similarly can be used but requires care in cyanobacteria to avoid interference from phycobilisome absorbance or fluorescence (37). Carbon labeling also has utility for determining rates of carbon assimilation and flux. Due to the equilibration between CO 2 and HCO 3 -, both the media and cytosol can have stores of C i that are separate from what is fixed, so care must be taken to distinguish between stores and the assimilation of CO 2 and HCO 3 - (33,38,39). In general, the aforementioned measurements are limited to endpoint assays and/or are technically challenging.
For terrestrial plants, a robust method derives net gas exchange from a plot of carbon assimilation versus intracellular CO 2 to establish steady-state photosynthetic parameters nondestructively (40). Carbon assimilation versus intracellular CO 2 curves from plants are typically modeled with three distinct regions: at low levels of intercellular C i assimilation, rates are limited by the reaction rate of RubisCO; at higher levels of intercellular C i assimilation, rates are limited by the rate of ribulose-1,5-bisphosphate regeneration (light-limited); and at the highest intercellular C i values, the assimilation curves may show saturation due to maximum utilization of triose phosphate pools (41). Due to the aqueous nature of cyanobacteria and the slow, uncatalyzed equilibration of HCO 3 -with CO 2 , parallel methods have yet to be well established but those that have been examined are promising (32,33). Notably, Douchi et al. recently demonstrated that the response to declining C i can be modeled with a two-phase sigmoidal curve in Synechocystis sp. PCC 6803 (here referred to as Synechocystis) (33), reminiscent of the carbon assimilation versus intracellular CO 2 curves seen in C 4 plants (42,43). Their work supports a biphasic model that indicates rate limitations imposed by the CCM for the lower phase and by the Calvin-Benson cycle (represented by a C i fixation coefficient) for the upper phase. This biphasic model offers a framework for modeling carbon fixation more broadly in cyanobacteria.

FIG 1
Generalized schematic of the carbon-concentrating mechanism (CCM) in a cyanobacterial cell. The CCM is comprised of the flux of C i (as both HCO 3 -and CO 2 ) into a cyanobacterial cell and the carboxysome, a proteinaceous microcompartment which contains RubisCO. This flux of C i and the CCM are regulated and tuned at many points, including by light availability and by the concentration of available CO 2 in the external environment. Light quality and quantity tune multiple aspects associated with CCM and carbon fixation (represented by solid green lines), including tuning phycobilisomes (which are represented by the colored fan-like structures, including the core and rods of hemidiscoidal phycobilisomes typical of Fremyella diplosiphon) that impact the overall efficiency of light harvesting associated with carbon fixation, carboxysome dynamics (size and number per cell), and carbon transporters. Carbon dioxide availability also can impact carbon transporter abundance and carboxysome dynamics (represented by solid black arrows extending from [CO 2 ]).
Dynamic Regulation of Carbon Assimilation in Fremyella ® In this study, we analyzed the carbon fixation characteristics of F. diplosiphon, which exhibits complementary chromatic acclimation (CCA). CCA is a process whereby cells respond to changes in the prevalence of light (primarily red versus green in F. diplosiphon and many other cyanobacteria) by altering the type and abundance of photosynthetic pigments, cell shape, and filament length (44,45). Notably, cyanobacteriochrome RcaE acts as a photoreceptor that controls CCA (46)(47)(48)(49) and contributes to the photoregulation of carboxysome morphology (29). Given the role of RcaE in regulating dynamic organismal responses to light, we hypothesized that this photoreceptor may serve to coordinate critical aspects of cells' dynamic regulation of carbon assimilation and associated organismal fitness. In order to investigate the roles of CCA and CCM regulation in tuning carbon assimilation (e.g., the net rate of CO 2 uptake per unit area), we demonstrate that carbon assimilation can be measured progressively using cyanobacteria in a semiwet state with infrared gas analysis of cyanobacterial discs. We investigated the impact of dynamic environmental factors, including light (quality and quantity), C i availability, and the physiological state of cells during carbon assimilation, on wild-type (WT) F. diplosiphon and a number of mutant strains. We show that dynamic responses of carbon assimilation can be evaluated using carbon response curves (CRCs) in cyanobacteria and, together with measurements such as O 2 evolution, can be used to infer the propensity of cells to exhibit C i uptake and active utilization during oxygenic photosynthesis.

RESULTS
Carbon assimilation measurements of responses of F. diplosiphon to light, inorganic carbon availability, and physiological state. Glass fiber-filtered F. diplosiphon strains (i.e., F. diplosiphon discs) were analyzed in a semiwet state with infrared gas analysis to detect CO 2 uptake and consumption. Carbon assimilation rates in WT and ΔrcaE F. diplosiphon strains were responsive to light intensity, showing light saturation at ϳ100 mol·m Ϫ2 ·s Ϫ1 and ϳ300 mol·m Ϫ2 ·s Ϫ1 in low-light (LL) and HL-acclimated cultures, respectively ( Fig. 2A and B). Thus, 300 mol·m Ϫ2 ·s Ϫ1 was selected for saturating light in further experiments. Under these conditions, strains of F. diplosiphon exhibited changes in carbon assimilation in response to changing carbon levels in a standard CRC (Fig. 2C to F). Blank glass fiber-filtered discs wetted with fresh cell media were used as a control and showed slightly negative assimilation values that became more negative from 600 to 1,000 ppm (see Fig. S1 in the supplemental material). Samples were normalized by optical density at 750 nm (OD 750 ), which had a roughly linear relationship with [Chla] (Fig. S2). As the intercellular C i flux in cyanobacteria is complex and has not been modeled precisely, response curves are presented with the [CO 2 ] levels in the sample chamber (s[CO 2 ]) as the independent variable. As in plants, these CRCs follow a generally sigmoidal curve and are expected to be limited by C i availability at low C i values and by other factors such as light availability when C i levels are saturating. Compensation points (near the point where assimilation becomes negative and which represent equivalent rates of photosynthetic CO 2 flux and respiration) appear to fall between 5 and 25 ppm s[CO 2 ] in cyanobacterial CRCs, which are likely lower than the typical values (25 to 100 ppm intercellular CO 2 ) found in plants (41,50). These observations are consistent with the presence of a CCM in cyanobacteria.
This CRC method was then used to compare cultures acclimated to red light (RL) and green light (GL). The WT strain showed significant differences in carbon assimilation only above 700 ppm CO 2 , i.e., beyond the C i -limited region of the CRCs, with GL-grown cultures reaching higher assimilation levels (Fig. 2C). This result is consistent with previous measurements of O 2 evolution, which revealed similar rates of O 2 evolution for F. diplosiphon grown in low-intensity RL compared to GL at ambient CO 2 , which would correspond to the C i -limited region (37). The ΔrcaE mutant, which has more numerous and smaller carboxysomes than the WT in both RL and GL (29), demonstrated impeded carbon assimilation only under GL conditions. The maximum assimilation rate dropped from ϳ4.0 for the WT to ϳ1.3 mol·m Ϫ2 ·s Ϫ1 for the ΔrcaE mutant in GL. By comparison, the assimilation rate seen with the ΔrcaE mutant was statistically indistinguishable from that seen with the WT under RL conditions ( Fig. 2C and D).
We hypothesized that differences in cellular pigmentation in the WT under RL versus GL conditions contribute to light-dependent differences in the net rate of CO 2 uptake. Thus, we measured the carbon assimilation rate in a ΔrcaC mutant strain with constitutively GL-like pigmentation (51), due to the lack of the DNA-binding regulatory protein RcaC, which acts downstream of RcaE. CRC analysis indicated no differences in the assimilation values for the ΔrcaC strain between RL and GL, with values more similar to the WT values seen under GL conditions (Fig. 2E). This finding suggests that the GL physiological state is partially responsible for the higher assimilation values measured under conditions that employed that light quality in the WT.
In addition to pigmentation differences, WT F. diplosiphon exhibits cell shape differences that are controlled in part by RcaE, with spherical cells in RL and rod-shaped cells in GL (46). We hypothesized that cell shape and its regulation contribute to light-dependent differences in measured carbon assimilation rates, perhaps due to differences in gas diffusion levels in spherical cells compared to rod-shaped cells. Thus, we analyzed carbon assimilation in a ΔbolA mutant strain with an altered, constitutively more spherical cell shape (48). As the strain had WT pigmentation, analysis of the ΔbolA  2 ] for WT (A) and ΔrcaE (B) F. diplosiphon strains grown at low (12 mol·m Ϫ2 ·s Ϫ1 ; white symbols), medium (30 mol·m Ϫ2 ·s Ϫ1 ; gray symbols), and high (100 mol·m Ϫ2 ·s Ϫ1 ; black symbols) WL intensity in air. n ϭ 3 for LL and the ΔrcaE mutant ML, and n ϭ 5 for HL and WT ML. (C to F) Carbon assimilation ("A") response (expressed in mol m Ϫ2 s Ϫ1 ) to CO 2 supplied at 300 mol·m Ϫ2 ·s Ϫ1 for WT (C), the ΔrcaE mutant (D), the ΔrcaC mutant (E), and ΔbolA (F) F. diplosiphon strains grown under ϳ10 to 12 mol·m Ϫ2 ·s Ϫ1 RL (white symbols) or GL (black symbols) conditions. Error bars represent 95% confidence intervals for n Ն 3 from 2 independent biological replicates.
Dynamic Regulation of Carbon Assimilation in Fremyella ® mutant relative to the WT allowed us to separate the potential impacts of pigmentation regulation from the regulation of cell shape. Assimilation values in the ΔbolA mutant showed no differences between RL and GL and were closer to the assimilation values for the WT under RL conditions (Fig. 2F). Since assimilation in the ΔbolA mutant was similar to that measured for spherical WT cells in RL, the regulation of cell shape likely plays a role in CRC behavior whereas pigmentation does not appear to have a significant role.
Effect of nonsaturating light on carbon assimilation. In order to probe for the light-limited regions of the CRC in cyanobacteria, we performed analyses under nonsaturating test light conditions, i.e., using 25 and 50 mol·m Ϫ2 ·s Ϫ1 of light compared to the prior parameters of 300 mol·m Ϫ2 ·s Ϫ1 . WT F. diplosiphon grown under LL conditions had near-saturation carbon assimilation values, even at light measurements as low as 50 mol·m Ϫ2 ·s Ϫ1 (Fig. 3A). However, assimilation was severely impaired 25 mol·m Ϫ2 ·s Ϫ1 above 75 ppm s[CO 2 ] (Fig. 3C). The level of assimilation exhibited by the ΔrcaE mutant was also decreased under nonsaturating light conditions and was indistinguishable from the WT level at 25 mol·m Ϫ2 ·s Ϫ1 (Fig. 3B and D). Carbon assimilation ("A") response (expressed in mol m Ϫ2 s Ϫ1 ) to CO 2 supplied during runs at 300 mol·m Ϫ2 ·s Ϫ1 (black symbols), 50 mol·m Ϫ2 ·s Ϫ1 (gray symbols), or 25 mol·m Ϫ2 ·s Ϫ1 (white symbols) for WT (A and C) and ΔrcaE (B and D) F. diplosiphon strains grown at low (12 mol·m Ϫ2 ·s Ϫ1 ) GL-enriched WL. Panels C and D show data corresponding to 0 to 200 ppm s[CO 2 ] in panels A and B, respectively. Error bars represent 95% confidence intervals for n Ն 3 from 2 independent biological replicates. (E and F) Carbon assimilation ("A") response (expressed in mol m Ϫ2 s Ϫ1 ) to CO 2 supplied at 300 mol·m Ϫ2 ·s Ϫ1 for WT (E) and ΔrcaE (F) F. diplosiphon strains grown at low (12 mol·m Ϫ2 ·s Ϫ1 ; white symbols), medium (30 mol·m Ϫ2 ·s Ϫ1 ; gray symbols), and high (100 mol·m Ϫ2 ·s Ϫ1 ; black symbols) GL-enriched WL intensities in air. Error bars represent 95% confidence intervals for n Ն 4 from 2 independent biological replicates.
Effect of different light intensities during growth on carbon assimilation potential. Since HL is known to induce the components of CCM (4, 5, 27), we hypothesized that growth of F. diplosiphon under conditions of increasing light intensity would support higher assimilation values via induction of C i uptake and increased linear electron flow until the levels of light that were reached were stressful or induced phototoxicity. We used a multicultivator bioreactor system with green-enriched white light (WL) at LL (12 mol·m Ϫ2 ·s Ϫ1 ), medium light (ML; 30 mol·m Ϫ2 ·s Ϫ1 ), or HL (100 mol·m Ϫ2 ·s Ϫ1 ) intensities to measure assimilation rates in the WT and the ΔrcaE mutant. Although the growth rate increased as light intensity increased in both strains ( Fig. S3), cells typically exhibited chlorosis at ϳ7 days after induction of HL, indicating light stress. CRC analysis of the WT indicated that the responses to LL and ML were similar. HL caused a general decreasing trend in CO 2 assimilation levels at high s[CO 2 ] in the WT, with substantial variation, but with assimilation levels significantly lower than those seen under LL or ML conditions at s[CO 2 ] levels of Ն700 ppm (Fig. 3E). In contrast, we observed a general increase in assimilation rates in the ΔrcaE mutant during growth under conditions of increasing light intensity, with assimilation approaching near-WT levels under HL conditions and with significant differences between the levels of CO 2 assimilation for LL compared to HL at higher s[CO2] levels ( Fig. 3E and F). In addition, under conditions of HL acclimation, the two strains exhibited low, indistinguishable assimilation values under nonsaturating light conditions (Fig. S4).
Effect of inorganic carbon availability on carbon assimilation during growth. We next explored the impact of C i availability on CRC behavior. Cells were grown in air or under conditions of C i upshift (3% CO 2 ) or C i downshift (3 days growth in 3% CO 2 followed by a transfer to air for 19 h) in chambers illuminated with 35 to 40 mol·m Ϫ2 ·s Ϫ1 WL (Fig. 4). The WT and ΔrcaE strains exhibited similar carbon assimilation behaviors under conditions of exposure to air ( Fig. 4A and B). The behaviors of these two strains were similar at below 200 ppm s[CO 2 ] under all conditions, and, as expected, the compensation point appeared to decrease as the cultures became more acclimated to lower C i levels and induced high-affinity CCM systems ( Fig. 4C and Carbon assimilation response to C i availability after acclimation to various C i levels. Data represent carbon assimilation ("A") response (expressed in mol m Ϫ2 s Ϫ1 ) to CO 2 supplied at 300 mol·m Ϫ2 ·s Ϫ1 for WT (A and C) and (B and D) ΔrcaE F. diplosiphon strains grown at medium (ϳ35 mol·m Ϫ2 ·s Ϫ1 ) RL-enriched WL intensity in air with 3% CO 2 enrichment (Ci Up; black symbols), without enrichment (Air; gray symbols), or under conditions of C i downshift (Ci Down; white symbols). Panels C and D show data corresponding to 0 to 200 ppm s[CO 2 ] in panels A and B, respectively. Error bars represent 95% confidence intervals for n Ն 4 from 2 independent biological replicates.
Dynamic Regulation of Carbon Assimilation in Fremyella ® D). During acclimation to C i downshift, the two strains also performed similarly to each other in runs under nonsaturating light conditions (Fig. 5). However, the ΔrcaE mutant strain exhibited a deficiency in response to C i levels with reduced assimilation under conditions of C i upshift and a less robust response to C i downshift than the WT at higher s[CO 2 ] levels ( Fig. 4B).
Rates of O 2 evolution in F. diplosiphon strains under RL and GL conditions. To compare our findings to those obtained using established methods and to compare CO 2 uptake with active C i utilization in oxygenic photosynthesis, we analyzed O 2 evolution in WT and ΔrcaE strains that had been acclimated to RL or GL (Fig. 6, white bars). The WT produced O 2 at marginally higher initial rates in GL than were seen with cells grown in RL (P ϭ 0.024). O 2 evolution was significantly decreased in the ΔrcaE mutant relative to the WT under both RL and GL conditions. Whereas CRC analysis uncovered a defect in carbon assimilation only under GL conditions, the ΔrcaE strain showed reduced O 2 evolution rates compared to the WT even after acclimation to RL. We treated cells with 2,6-dichloro-p-benzoquinone (DCBQ; 0.2 mM), which accepts electrons from PSII and enables tests to determine the total number of PSII centers capable of water oxidation (52,53). The WT exhibited similar levels of O 2 evolution in RL with or without DCBQ but exhibited higher O 2 evolution levels in GL after DCBQ was added (Fig. 6). The latter response for the WT was anticipated as the addition of 0.5 mM DCBQ in Synechocystis was previously shown to increase O 2 evolution rates substantially (53). The fact that the rates did not increase in WT F. diplosiphon in RL suggests that this strain utilizes the majority of its PSII complexes that have sufficient excitement to split water (i.e., downstream regulation does not limit the WT in RL) under this light condition. However, in GL, cell activity may be limited by downstream reactions. Furthermore, the decrease of carbon assimilation rates seen under RL compared to GL conditions ( Fig. 2C) may be attributable to the PSII reaction rates, as the WT under RL conditions exhibited lower O 2 evolution rates with and without DCBQ compared to cognate samples in GL. O 2 evolution rates increased in DCBQ-treated ΔrcaE cultures in both RL and GL (Fig. 6). However, the ΔrcaE mutant showed no significant differences FIG 5 Carbon assimilation response to C i availability in nonsaturating light after acclimation to C i downshift. Data represent carbon assimilation ("A") response (expressed in mol m Ϫ2 s Ϫ1 ) to CO 2 supplied at 300 mol·m Ϫ2 ·s Ϫ1 (black symbols) or 25 mol·m Ϫ2 ·s Ϫ1 (white symbols) for WT (A and C) and ΔrcaE (B and D) F. diplosiphon strains grown at medium (ϳ35 mol·m Ϫ2 ·s Ϫ1 ) RL-enriched WL intensity under conditions of C i downshift. Panels C and D show data corresponding to 0 to 200 ppm s[CO 2 ] in panels A and B, respectively. Error bars represent 95% confidence intervals for n Ն 3 from 2 independent biological replicates.

Rohnke et al.
® from the WT under either light condition for DCBQ-treated cultures. This finding suggests that the apparent reduction in the photosynthetic rate of the ΔrcaE mutant under GL conditions (as measured by both carbon assimilation and O 2 evolution rates) is not due to a deficiency in PSII reaction rates but might be associated with aspects of carbon utilization.
Transmission electron microscopy (TEM) analysis of carboxysome morphology in response to light conditions and carbon availability. To contextualize the CRC behaviors and investigate which may be associated with a specific carboxysome morphology, we analyzed carboxysome dynamics under the conditions used for CRC analyses (Fig. 7). In addition to the altered carboxysome size and number in the ΔrcaE mutant compared to the WT in both RL and GL (29), the diameter of carboxysomes decreased in both strains under GL conditions and there were no light qualitydependent changes in carboxysome abundance in either strain. Here, neither the ΔrcaC strain nor the ΔbolA strain showed differences in the size or shape of carboxysomes between RL and GL ( Fig. 7A; see also Fig. 8A and B). Since the WT exhibited a decrease in carboxysome diameter and trended toward higher carboxysome abundance under GL conditions, both the ΔrcaC and ΔbolA strains had significantly larger and fewer carboxysomes than were seen in the WT under GL conditions. Under conditions of increasing light intensity, the WT showed a gradual increase in carboxysome diameter that was significant in comparisons of HL to LL (P ϭ 0.024, Fig. 8C; see also Table 1) and no increase in carboxysome abundance (Fig. 8D). The ΔrcaE mutant showed a similar increasing trend in carboxysome diameter, with HLacclimating cultures showing a significant increase in size (P Ͻ 0.001 [comparing HL to either ML or LL]) (Fig. 8C). Unlike the WT, the ΔrcaE mutant exhibited substantial increases in carboxysome numbers when responding to increased light. The ΔrcaE mutant did not exhibit its characteristic increase in carboxysome abundance compared to the WT until it was acclimated to ML or HL under WL growth conditions (Fig. 8D).
C i availability also impacted carboxysome morphology as expected. While the WT strain showed a characteristic decrease in carboxysome abundance under conditions of C i upshift (Fig. 8F), it also showed an increase in carboxysome diameter (Fig. 8E) (same data as reported in Lechno-Yossef et al. [54]). The C i downshift conditions did not provide sufficient time for complete carboxysome acclimation, which takes 2 to 4 days for Synechococcus elongatus sp. PCC 7942 (here referred to as S. elongatus) (5). While the WT strain under conditions of C i downshift showed carboxysome abundance levels Dynamic Regulation of Carbon Assimilation in Fremyella ® similar to those seen under C i upshift conditions, it exhibited decreased carboxysome size (P ϭ 0.003), which could in part have been due to the transition to the airacclimated state ( Fig. 8E and F). Overall, the ΔrcaE mutant showed a misregulated response to C i availability and a decrease in carboxysome diameter in response to C i upshift (compared to the increase seen in the WT; Fig. 8E) and no significant response with respect to carboxysome abundance (Fig. 8F).
Transcriptional regulation of CCM components measured by quantitative PCR (qPCR) analysis. Given that multiple components of the CCM are expected to be controlled at the transcriptional level in response to light and C i availability (4,27,29,30,55) and the observed changes in carboxysome size for the strains described above, we anticipated changes in regulation of ccm genes under the tested conditions. Thus, we analyzed the CCM components of the F. diplosiphon transcriptome using quantitative PCR (qPCR) analysis (see Table 2 for gene-specific primers). These analyses included carboxysome-related genes in the ccmK1K2LMNO and ccmK3K4 operons; ccmK6, ccmP, rbcL and rbcS (the RubisCO subunits); ccaA1/2 (carboxysomal CA); and alc (the homologue of the RubisCO activase gene [54]). Genes related to C i uptake were also probed, including low-C i -induced cmpA (BCT complex), sbtA, and ndhD3 (NDH-I 3 complex); constitutively expressed ndhD4 (NDH-I 4 complex) and bicA; and a LysR-type transcriptional regulator with homology to cmpR (56) and ccmR (6), the latter two of which are each involved in the transcriptional response to C i availability.
We hypothesized that the photoregulation of CCM components might correspond to the changes in carbon assimilation described above. Thus, we first analyzed strains under RL and GL conditions (Table 3). Whereas the ΔrcaE mutant showed upregulation of ccmM and downregulation of rbcS under RL conditions, more-significant changes were observed under GL conditions, particularly in the downregulation of ccmK3, rbcL, rbcS, and the low-C i induced C i -uptake genes relative to the WT. The regulation of ccmM, rbcL, and rbcS was consistent with prior results (29), as was the downregulation of sbtA and ndhD3 (55). The WT showed few differences between RL and GL conditions; however, alc, bicA, and cmpA were downregulated under GL conditions. For many genes, the ΔrcaE mutant also exhibited downregulation under GL conditions but with more extreme and more frequently statistically significant magnitudes of change. The Under conditions of increasing light intensity (Table 4), WT experienced significant upregulation for selected HCO 3 -transporter genes (likely due to increased linear electron flow), ccmN, and ccmO, alongside downregulation for rbcS (possibly related to HL stress). The ΔrcaE mutant showed the characteristic downregulation of rbcS that was seen under other conditions. Additionally, it exhibited upregulation of ccmK1 and ccmK2 under ML conditions and of ccmK6 under HL conditions, which correlates with the increase in carboxysome abundance ( Fig. 7B; see also Fig. 8D). The ΔrcaE mutant showed a similar upregulation of HCO 3 -transporter genes, ccmN, and ccmO, though not to the same extent as the WT. Finally, in contrast to the nonsignificant increases seen in WT, the ΔrcaE mutant showed significant upregulation of alc. Since the alc gene is important for cellular responses to C i upshift (54), this upregulation might be indicative of altered C i utilization by ΔrcaE cells.
Both the WT and ΔrcaE strains demonstrated significant differential expression of CCM components under conditions of decreasing C i availability ( Table 5). The WT showed a general downregulation in shell protein genes, rbcL, rbcS, and ccmM under conditions of C i downshift, which is consistent with previous findings for Synechocystis (6,57) and S. elongatus (58). It is interesting to consider how these data correlate with the increased carboxysome abundance under conditions of C i downshift reported previously (5,57,59) and in this study ( Fig. 8E and F; C i upshift versus air). As previously noted (54), alc is downregulated under conditions of C i downshift and has been observed to be involved in decreased carboxysome abundance under conditions of C i upshift. Consistent with these expectations, WT also exhibited significant upregulation of the low-C i -induced C i -uptake genes. While the WT upregulated the low-C i -induced C i -uptake genes under both air and C i -downshift conditions, the ΔrcaE mutant did so only under conditions of C i downshift.

Use of the CRC in cyanobacteria.
Our work with F. diplosiphon, a freshwater filamentous cyanobacterium which undergoes CCA in response to light quality, highlights multilayered connections between CCM components, nutrient availability, and the physiological state of the cell (29). Efficiently connecting these factors to overall carbon assimilation is critical to understanding how these organisms (and humans as bio-prospectors) can optimize photosynthesis. We hypothesized that identification of the conditions under which carbon assimilation was disrupted in WT F. diplosiphon or a ΔrcaE mutant strain with compromised CCA would highlight functional roles of CCA in impacting the regulation of CCM and associated carbon fixation and would indicate mechanisms for future analysis.
The use of gas exchange analysis to construct CRCs in cyanobacteria suggests that the acclimation to dominant light quality through CCA has a nuanced impact on overall assimilation behavior. WT F. diplosiphon cells assimilate more CO 2 when acclimated to GL despite having smaller carboxysomes and not being tuned to the red-enriched light of the Li-COR system (Fig. 2C). The disruption of CCA through the loss of the photoreceptor RcaE added layers of complexity; since RcaE influences the stoichiometry of carboxysome components and carboxysome size under both RL and GL conditions (29), we expected a general decrease in net CO 2 uptake and assimilation. Instead, we found GL-specific impairment (Fig. 2D). While the small, more numerous carboxysomes of the ΔrcaE strain may contribute to overall carbon assimilation behavior, this observation cannot explain the higher level of assimilation seen with the WT under GL conditions.  These intriguing initial results prompted further exploration of the assimilation behavior of cyanobacteria. We provide evidence that physiologically relevant CRCs, similarly to the popular carbon assimilation-versus-intracellular CO 2 curves in plants, can be obtained from cyanobacteria in a semiwet state using cyanobacterial discs. Cells showed a dosage response to both light ( Fig. 2A) and CO 2 , two major factors that are relevant to the development of the advanced modeling of photosynthetic parameters in plants (41). CRCs were also sensitive enough to show changes in apparent compensation points based on the physiological state of the cell (Fig. 4C). Traditional O 2 evolution experiments revealed similar trends, with the WT exhibiting higher rates under GL than RL conditions and the ΔrcaE mutant showing higher rates under RL than GL conditions (Fig. 6). Despite this, the two methods differed in comparisons of the WT and ΔrcaE strains under RL conditions; the ΔrcaE mutant exhibited similar C i -uptake rates under RL conditions but a decrease in O 2 evolution, suggesting an impairment in the use of CO 2 for oxygenic photosynthesis in the ΔrcaE mutant. Thus, CRCs of cyanobacterial discs offer novel insight into the CO 2 -uptake behavior of cyanobacteria under a broad range of C i levels. This method also significantly reduces the time required for equilibration between CO 2 and HCO 3 -, which allows dynamic responses to be studied. Thus, it is a promising technique that can be used both as a stand-alone method as a quick measurement of net carbon assimilation and in conjunction with established systems that more deeply probe HCO 3 -/CO 2 flux. In particular, and in contrast to wellestablished procedures that test cyanobacteria's utilization of HCO 3 -, it serves to more directly test the use of CO 2 by cyanobacteria.
The low-C i phase of the CRC (<100 ppm s[CO 2 ]) is driven by C i uptake. The idea of the presence of a C i -limited region at low ppm s[CO 2 ] is supported by data corresponding to the regions of CRCs that do not respond to nonsaturating light at 0 to ϳ100 ppm s[CO 2 ] (Fig. 3A to D) and is consistent with findings reported previously by Douchi et al. (33). Notably, the low-C i region is considerably robust and rarely exhibits differences; e.g., the ΔrcaE mutant is always indistinguishable from the WT in this region. There were only two conditions under which we observed changes to the low-C i region. The slope and compensation point were incredibly responsive to acclimation of the culture to different C i availabilities, with growth under C i downshift conditions prompting a robust assimilation response even at very low C i levels and a reduced apparent compensation point ( Fig. 4C and D). We were tempted to identify this as a light-independent region and so tested a hypothesis predicting that cultures acclimated to C i downshift would not show a change in slope below ϳ100 ppm s[CO 2 ], even analyzed under nonsaturating light conditions. However, nonsaturating light reduced the assimilation slope and increased the compensation point ( Fig. 5C and D). This observation suggests that light availability can affect the low-C i region but only under specific conditions that are related to C i -uptake capacity. Thus, we propose identifying the low-C i region of the cyanobacterial CRC as one that is driven by C i uptake and that is comparable to C i -limited regions of response curves in plants.
The high-C i phase of the CRC (>100 ppm s[CO 2 ]) is responsive to multiple photosynthetic parameters. In line with biphasic models of carbon assimilation in C 4 plants (42,43) and cyanobacteria (33), our work supports the identification of a second region that reaches A max at high C i . However, these data suggest that the high-C i region of cyanobacteria CRCs depends on many variables, including C i availability, carboxysome morphology, linear electron flow, and cell shape.
The components of the CCM that relate to C i uptake appear to have a broad effect on assimilation behavior, consistent with the C i upshift results reported by Douchi et al. (33). Indeed, upregulation of the low-C i -induced genes ( Table 5) was correlated with an increase in assimilation at all s[CO 2 ] levels (Fig. 4A). Since this increase occurred under C i downshift conditions, where WT carboxysomes had not had sufficient time to acclimate to air conditions ( Fig. 8E and F), this is one case where we can neatly attribute a change in assimilation behavior directly to a single major component of CCM (Fig. 9). However, under HL conditions, we saw similar induction of the low-C i -induced genes (Table 4) without the corresponding increase in assimilation (Fig. 3E).
Analysis of the ΔrcaE mutant strain provides some additional lines of inquiry that may offer insight. Unlike the WT results, elevated light intensity increased the maximum assimilation rates of the mutant (Fig. 3F). This may have been because the mutant experienced a greater overall increase in carboxysome volume in response to HL ( Fig. 8C and D), perhaps evidencing the role of the carboxysomes in carbon assimilation behavior as part of a C i fixation parameter. Since the mutant strain maintained a water splitting capacity similar to that seen with WT ( Fig. 6; ϩDCBQ) but showed a decreased net O 2 evolution rate ( Fig. 6; ϪDCBQ) under RL and GL conditions and decreased A max under GL conditions, the ΔrcaE mutant was also less efficient at utilizing light productively. Thus, HL conditions would prove beneficial to the mutant (as evidenced by its increase in assimilation) while being stressful to the more efficient WT. This suggests that carboxysome size or linear electron flow or both contribute to the determination of A max and are the primary contributors to the low A max of the ΔrcaE mutant strain (Fig. 9). Second, the behavior of the ΔrcaE mutant yields insight into the assimilation phenotype of the WT under GL conditions. Though cmpA was downregulated under GL conditions in WL, the ΔrcaE mutant showed much more significant downregulation of low-C i -induced genes (Table 3), which may contribute to the low-assimilation phenotype, and perhaps to the C i -uptake capacity, of the ΔrcaE mutant under GL conditions. If this is the case, then it is probable that the inducible C i -uptake systems contributed but were being masked in the high-carbon-assimilation phenotype of the WT under GL conditions.
Both the ΔrcaC and ΔbolA mutants showed few differences between RL and GL in the experiments performed in this study. Under both RL and GL conditions, the ΔrcaC strain, which was constitutively in a GL-like phenotypic state, showed nearly identical assimilation behaviors that were more similar to those of the WT under GL conditions (Fig. 2E), suggesting that GL acclimation also contributes to the high-assimilation phenotype of the WT. As for the ΔbolA strain, it too showed nearly identical assimilation behavior in both RL and GL but was instead more similar to the WT under RL conditions (Fig. 2F). As the ΔbolA mutant had an enlarged, spherical cell shape under both RL and GL conditions, it is possible that the rod shape of WT F. diplosiphon cells seen under GL conditions enhanced C i uptake and/or cellular CO 2 diffusion.
Impact. This study integrated physiological analyses of the cyanobacterium F. diplosiphon with a novel application of gas exchange analysis to cyanobacteria. Like many cyanobacteria, F. diplosiphon performs CCA, which offers a useful system for studying the impact of light regulation, especially as it relates to photosynthesis. We explored the connection between the loss of RcaE, a cyanobacteriochrome that controls the CCA pathway, and the CCM. Analyses of the CRCs provide a simple method to assay the carbon assimilation phenotype of cyanobacteria, connecting findings on how the stoichiometry of CCM components influences the structure and function of carboxysomes and C i -uptake systems. Preliminary work to identify photosynthetic parameters that are identifiable through CRCs could contribute valuable insight into modeling and understanding the dynamic regulation of photosynthesis in cyanobacteria.

MATERIALS AND METHODS
Growth conditions. General culture inoculation and growth under RL and GL conditions were performed as described previously by Rohnke et al. (29). In brief, we used a short-filament strain of F. diplosiphon with WT pigmentation identified as SF33 (60), a RcaE-deficient mutant strain (the ΔrcaE mutant) characterized previously by Kehoe and Grossman (47), a RcaC-deficient mutant strain (the ΔrcaC mutant) identified in our lab through forward genetics screening, and a BolA-deficient mutant strain (the ΔbolA mutant) described previously by Singh and Montgomery (48). Liquid cultures were inoculated from plated cultures and grown at 28°C under WL in BG-11/HEPES until they were diluted to an initial OD 750 of 0.05 and transferred to experimental conditions. The effect of light intensity was tested in a MultiCultivator MC 1000-OD system (Photon Systems Instruments, Drasov, Czech Republic) equipped with LED WL and autonomous monitoring of OD 680 and OD 720 according to the manufacturer's directions. Since the LED WL was GL dominant, starter cultures grown under GL were used for experiments involving the multicultivator to avoid the WT showing a growth lag as it underwent CCA. Light conditions were set at a constant value of 12 mol·m Ϫ2 ·s Ϫ1 (LL), 30 mol·m Ϫ2 ·s Ϫ1 (ML), or 100 mol·m Ϫ2 ·s Ϫ1 (HL). Since sustained HL conditions ultimately caused chlorosis, when high ODs were needed for harvesting for transmission electron microscopy (TEM) and RNA extraction, the ML and HL cultures were first grown at 12 mol·m Ϫ2 ·s Ϫ1 for 1 to 2 days prior to the onset of ML and HL conditions. Cultures grown this way were allowed to acclimate to the higher light intensity for at least 3 days prior to harvesting. Cultures from all experiments involving HL-grown cultures Dynamic Regulation of Carbon Assimilation in Fremyella ® were harvested prior to the plateauing of OD (within 6 days of HL onset) that preceded substantial cell death.
The effect of carbon availability was tested in Multitron growth chambers (Infors HT, Bottmingen-Basel, Switzerland) at 30°C under WL (ϳ35 to 40 mol·m Ϫ2 ·s Ϫ1 , with RL enrichment) gassed with either unenriched air (air) or air enriched with 3% CO 2 (C i upshift). As described previously by Lechno-Yossef et al. (54) and on the basis of methods described previously by Wang et al. (6), we shifted cultures from C i upshift to air conditions after 3 days of growth and resuspended them in BG11/HEPES that lacked sodium bicarbonate to achieve C i downshift. Cells were harvested for CRC, TEM, or qPCR analysis ϳ19 h after transfer to air (C i downshift).
Carbon response curve analysis using F. diplosiphon discs. OD 750 levels were measured in triplicate for cultures growing under the desired experimental conditions and were harvested between the ODs of 0.6 and 1.2. A total volume equal to 11.8 absorbance units (V ϭ 11.8/OD 750 ) was vacuum filtered through glass fiber filters (Fig. 10) with a pore size that was sufficiently small to capture Ͼ99% of F. diplosiphon cells (Whatman GF/A; Sigma-Aldrich, St. Louis, MO) (47-cm diameter) and a second layer of Whatman grade 1 filter paper to diffuse the filtrate more evenly. The disc diameter was selected to minimize unnecessary surplus surface area for the gas exchange chamber; about 47% of the disc's surface area was exposed to the 6-cm 3 chamber and barely extended past the gaskets. Cyanobacterial discs were handled carefully with forceps, briefly dabbed on filter paper to remove excess wetness, kept on BG11/HEPES agar plates, and analyzed swiftly to minimize environmental perturbation. CO 2 levels were measured with infrared gas analysis by the use of Li-COR Photosynthesis System 6800 (Li-COR, Lincoln, NE), with one end of a strip of damp Whatman grade 1 filter paper placed underneath the disc as a wick. The other end was submerged in double-distilled water (ddH 2 O) to maintain disc dampness for the duration of the experiment, which was found to greatly increase the duration during which the steady state could be maintained to ϳ45 min (data not shown).
The chamber was illuminated by the use of the standard "SunϩSky" (RL-dominant) regime with a leaf temperature of 28°C, a flow rate of 500 mol s Ϫ1 , and a source air with 12 ppm H 2 O. For the standard CRC, the initially supplied CO 2 concentration was 1,000 ppm and the sample was allowed to equilibrate for at least 5 min or until the steady state had been maintained for at least 3 min. The CRC followed a gradient of 1,000, 850, 700, 550, 400, 300, 200, 150, 100, 75, 50, 25, and 5 ppm, followed by a return to 400 ppm with automatic infrared gas analysis-based matching. The sample was allowed to equilibrate for ϳ2 to 3 min at each time point for a total run time of ϳ25 min after initial equilibration. Values for A were calculated as the loss of CO 2 , in mol per m 2 per second, and were corrected for leaks and changes in humidity.
O 2 evolution analysis. O 2 evolution was measured using an Oxytraceϩ O 2 electrode (Hansatech Instruments Ltd., Norfolk, England) illuminated by an acrylic projector bulb. Illumination was maintained at ϳ250 mol·m Ϫ2 ·s Ϫ1 and measured with a LI-250 light meter (Li-COR) equipped with a quantum sensor (model US-SQS/L; Heinz Walz CmbH, Effeltrich, Germany). Cells containing ϳ10 g Chla (determined on the basis of OD 750 extinction coefficients [see Fig. S2 in the supplemental material]) were harvested, washed twice in 3 ml BG11/HEPES that lacked sodium bicarbonate, and resuspended in 1 ml BG11/HEPES that lacked sodium bicarbonate. Cyanobacteria were placed in the chamber and spiked with sodium bicarbonate (Sigma-Aldrich) to reach a final concentration of 2 mM prior to illumination. When applicable, 2,6-DCBQ (Sigma-Aldrich) was then added to reach a final concentration of 0.2 mM and with potassium ferricyanide to reach a final concentration of 1.5 mM to act as the terminal electron acceptor. Cells were allowed to equilibrate at ambient light for ϳ1.5 min and then illuminated. The O 2 evolution V max was recorded as the peak rate that was reached within 10 min of the commencement of illumination.
TEM analysis. For all experimental conditions, TEM analysis was performed according to the methods described previously by Rohnke et al. (29). For the C i -upshift and air conditions, 60 cell sections were randomly selected and analyzed for carboxysome numbers in the WT and the ΔrcaE mutant, with carboxysome diameters measured in 20 of these sections. In all other strains and under all other conditions, 30 cell sections were analyzed, 10 of which were analyzed for carboxysome diameter, as well. Samples were prepared from at least two independent biological replicates. As a modification to the original method, some samples were analyzed using a JEM 1400 Flash TEM (JEOL USA Inc., Peabody, MA) at an operating voltage of 100 V.  abundances of ccmK1, ccmK2, ccmK3, ccmK4, ccmK6, ccmL, ccmM, ccmN, ccmO,  ccmP, ccaA1, ccaA2, alc, rbcL, rbcS, fdiDRAFT81170 (a LysR-type transcriptional regulator gene), cmpA, sbtA, ndhD3, ndhD4, and bicA transcripts were measured relative to the internal control orf10B within total RNA extracts from F. diplosiphon strains grown under various experimental conditions and according to previously described research (29,54) and MIQE guidelines (61). In brief, this involved harvesting ϳ20 ml of exponentially growing cells upon reaching the target OD 750 (ϳ0.5 to 0.6), handling the samples on ice and flash freezing the cell pellet within 1 h of harvesting, and extracting them with a TRIzol reagent incubated at 95°C, followed by wash steps, DNase treatment (TURBO DNA-free kit; Invitrogen, Madison, WI), and RNA quantification using a NanoDrop ND-1000 Spectrophotometer. Reverse transcription was performed using a qScript cDNA SuperMix kit (Quantabio, Beverly, MA), and qPCR was performed using Fast SYBR green master mix (Applied Biosystems, Foster City, CA) in 384-well plates (Applied Biosystems) with a 10-l reaction volume, with each procedure performed according to the instructions of the manufacturer. Probe sequences are provided in Table 2. RNA quality was assayed using gel electrophoresis, and genomic contamination was controlled for by verifying that no templatecontrol samples had quantification cycle (C q ) values greater than 5 cycles higher than the respective unknowns. The data reflect three technical replicates for each of at least three independent biological replicates and are presented using the delta C q method (ΔC q ) in order to foster analyses of comparisons between several strains and conditions. Chlorophyll extraction. Chla was measured spectrophotometrically according to the methods described previously by de Marsac and Houmard (62) for use with F. diplosiphon (63). Samples were harvested in parallel with CRC analysis as a secondary validation of normalization by OD 750, and at least three independent biological replicates were analyzed.
Statistical analysis. Experiments were performed with n Ն 3 from at least 2 biological replicates for all experiments. Statistical significance was evaluated using Student's t tests performed in R.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.