Trophic status of Chlamydomonas reinhardtii influences the impact of iron deficiency on photosynthesis
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To investigate the impact of iron deficiency on bioenergetic pathways in Chlamydomonas, we compared growth rates, iron content, and photosynthetic parameters systematically in acetate versus CO2-grown cells. Acetate-grown cells have, predictably (2-fold) greater abundance of respiration components but also, counter-intuitively, more chlorophyll on a per cell basis. We found that phototrophic cells are less impacted by iron deficiency and this correlates with their higher iron content on a per cell basis, suggesting a greater capacity/ability for iron assimilation in this metabolic state. Phototrophic cells maintain both photosynthetic and respiratory function and their associated Fe-containing proteins in conditions where heterotrophic cells lose photosynthetic capacity and have reduced oxygen evolution activity. Maintenance of NPQ capacity might contribute to protection of the photosynthetic apparatus in iron-limited phototrophic cells. Acetate-grown iron-limited cells maintain high growth rates by suppressing photosynthesis but increasing instead respiration. These cells are also able to maintain a reduced plastoquinone pool.
KeywordsAcetate Algae LhcSR Non-photochemical quenching Respiration
- PSI, PSII
Photosystem I, photosystem II
Light harvesting complex I
High performance liquid chromatography
- SDS PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Although iron (Fe) is the fourth most abundant element in the Earth’s crust, its low bioavailability makes it a limiting nutrient for life. In nature, iron is mostly found as stable Fe3+-oxides, which are insoluble in aerobic environments at biological pH (Guerinot and Yi 1994). Iron’s control on photosynthetic systems has been notably demonstrated by the stimulation of algal blooms following the addition of nanomolar concentrations of iron to several open ocean locations that receive very low natural iron inputs (e.g., Martin et al. 1994; Boyd et al. 2000). Besides oceanic plankton communities, iron-deficiency has been well documented in plants and in heterotrophs. Organisms have developed complex systems for iron acquisition and for adjusting their biochemistries to survive in low iron environments (reviewed in Guerinot 1994; Marschner and Römheld 1994; Philpott 2006; Walker and Connolly 2008).
The majority of iron in algae and plants is believed to be associated with the chloroplast (Raven 1988; Briat et al. 2007). In oxygenic photosynthesis, iron is a cofactor in PSII, PSI, the cytochrome b6/f complex, and in algae, cytochrome c6 as well. The abundance of these proteins is reduced during iron-deficient growth (Singh et al. 2003). PSI seems to be a focus during iron limitation, probably due to its high iron content (12 Fe per PSI) (Sandmann and Malkin 1983). The ratio of PSI/PSII changes from 4:1 to 1:1 under iron deficiency in cyanobacteria (Straus 1995), and a diatom evolved to low ambient iron has a constitutive PSII/PSI ratio of about 10:1 (Strzepek and Harrison 2004).
A reduction in the number of reaction centers decreases the ability of the photosynthetic apparatus to use light energy, and iron-limited algae and cyanobacteria show decreased PSII function, inter-photosystem electron transport, carbon fixation rates, and ultimately decreased growth (Greene et al. 1992; Vassiliev et al. 1995; Ivanov et al. 2000). To compensate for the change in the abundance of photosystems, cyanobacteria modify their remaining photosystem I to maximize light harvesting while minimizing photooxidative damage (reviewed in Michel and Pistorius 2004; Kouril et al. 2005). In addition to these changes, some iron-containing electron carriers are replaced completely by iron-independent substitutes such as the well-characterized switch from ferredoxin to flavodoxin (Laudenbach et al. 1988; Sandmann et al. 1990; La Roche et al. 1995, 1996; Erdner et al. 1999). This phenomenon is known as metal sparing.
After the photosynthetic apparatus, the respiratory electron transport chain represents the major use of iron within a photosynthetic cell. Iron limitation should also impact its activity, and indeed, studies in land plants indicate that iron limitation causes a decrease in iron-containing respiratory complexes, oxygen consumption, and growth rate (Pascal and Douce 1993; López-Millán et al. 2000; Andaluz et al. 2006; Vigani et al. 2009). Iron limitation in heterotrophic bacteria also significantly impacts electron flow, oxygen consumption, and growth rates (Rainnie and Bragg 1973; Hubbard et al. 1986; Tortell et al. 1996).
Chlamydomonas reinhardtii, in the green plant lineage, is a reference organism for the study of chloroplast metabolism and photosynthesis. This unicellular alga can grow phototrophically in the light, heterotrophically with acetate in the dark, or mixotrophically on acetate in the light. In an experimental situation, four stages of iron nutrition can be distinguished (La Fontaine et al. 2002; Moseley et al. 2002; Long et al. 2008). Iron-replete, with 20-μM Fe in the medium, corresponds to the iron content of standard laboratory growth medium (Harris 2009). In the iron-deficiency state (1–3-μM Fe, depending on the strain) the cells are preparing for the onset of iron limitation by up-regulating iron transporters and adjusting their biochemistry to minimize iron utilization, but their chlorophyll content is not or only marginally affected. Fluorescence kinetics and low temperature fluorescence studies indicate an impact on PSI light harvesting as well as electron transfer (Moseley et al. 2002). Iron-limited cultures (0.2-μM Fe) are visibly chlorotic owing to the programmed destruction of reaction centers and LHCIs (Moseley et al. 2002; Naumann et al. 2005). The involvement of a di-iron aerobic cyclase encoded by CHL27 in chlorophyll biosynthesis may also contribute to chlorosis (Tottey et al. 2003). Finally, in the iron-excess situation (200-μM Fe), the cells are phenotypically indistinguishable from iron-replete cells at normal light intensities but are sensitive to excess excitation energy (>500 μmol photons m−2 s−1) (Long and Merchant 2008).
We investigated the iron nutrition response of Chlamydomonas in acetate versus minimal medium to distinguish the impact of deficiency on bioenergetic pathways. There were striking differences in the response of the photosynthetic apparatus depending on the trophic status of the cultures. Iron-limited, photoheterotrophically grown cells maintained high growth rates by apparently suppressing photosynthesis while maintaining relatively high rates of respiration. This contrasts with autotrophic cells, which had efficient photosynthetic systems throughout the spectrum of iron nutritional status, but lost overall photosynthetic capacity at the onset of iron limitation.
Materials and methods
Strains and growth
Chlamydomonas reinhardtii strain 4A+ (137c background, courtesy of J.-D. Rochaix, University of Geneva) was used in this study. Starter cultures were maintained either photo-heterotrophically in standard Tris–acetate–phosphate (TAP) medium or in autotrophic medium lacking acetate (TP) at 24°C at a light intensity of 95 μmol photons m−2 s−1 and constant shaking (Harris 2009). For TP medium, acetic acid was omitted from the medium and the pH was adjusted to 7.4 with HCl. Autotrophic cells were also bubbled with sterile air. Media containing various amounts of iron were prepared and inoculated as in (Terauchi et al. 2009). No significant differences in chemical speciation at equilibrium in TP vs. TAP or in TP versus HSM (which is commonly used in other studies) were predicted using Visual Minteq software (http://www.lwr.kth.se/English/OurSoftware/vminteq). Cells were collected in mid-exponential phase (1–2 × 106 cells per ml) for all analyses.
Measurement of iron content
Samples were prepared as described by Petroutsos et al. (2009) and iron content was determined by inductively coupled plasma-mass spectroscopy (Agilent 7500 ICP-MS, detection limit 0.01 ppb) using the standard addition method in Helium mode.
Protein isolation for immunoblot analysis
2 × 108 cells were collected by centrifugation at 2500×g for 3 min, washed in 10-mM sodium phosphate, pH 7.0, resuspended in 300 μl of the same buffer, and stored at −80°C. For denaturing gel electrophoresis, cells were lysed by freeze/thaw cycling (Howe and Merchant 1992), and protein concentration was determined by the Lowry method against a Bovine Serum Albumin standard.
Proteins were separated by SDS-PAGE and immunodetection was carried out essentially as by Terauchi et al. (2009) except that membrane protein samples were incubated at 65°C for 20 min prior to separation by SDS-PAGE and transferred to a polyvinylidene difluoride membrane in transfer buffer containing 0.04% SDS. Primary antibody dilutions were: Fd, 1:10 000; Cyt f, 1:1000; D1, 1:500; PsaD, 1:1000; LhcSR, 1:1000; Fox1, 1:300; Nuo6, 1:2000; Nuo7, 1:2000; Nuo8, 1:3000, Cox2b, 1:5000, CF1, 1:10 000. Antisera against Fd, Cyt f, Fox1, Cox2b, and CF1 were from Agrisera. Antisera against Nuo6–Nuo8 were kindly provided by Patrice Hamel, and antisera against D1, PsaD, and LhcSR were kindly provided by Susan Preiss, Jean-David Rochaix, and Michel Guertin, respectively.
Oxygen evolution rates were measured using a standard Clark-type electrode (Hansatech Oxygraph with a DW-1 chamber). Photosynthetic rate in situ was calculated as: oxygen evolution at 217 μmol photons m−2 s−1 minus oxygen consumption in the dark. For all other oxygen evolution measurements, cells were collected by centrifugation as described above, resuspended in medium and dark acclimated at 25°C for 10 min. Chlorophyll a per sample ranged from 10 to 20 nmol/ml. Cells were placed in the cuvette and nitrogen gas was used to purge dissolved oxygen to about 50% saturation. The respiration rate was measured as oxygen consumption for 5 min in the dark. Changes in oxygen concentration were measured for 30 s at: 3, 8, 21, 46, 71, 84, 88, 218, 358, 544, 650, 927, 1350, and 1735 μmol photons m−2 s−1 sequentially. 500 μl of cells was removed from the cuvette at the end of the light sequence, centrifuged at 14,000×g for 5 min, and the pellets were resuspended and extracted in 80% acetone for several hours. Chlorophyll a concentrations were estimated as described previously (Porra et al. 1989; Porra 2002). These data were used to assemble photosynthesis–irradiance curves. Net oxygen evolution rates were normalized to chlorophyll a, and photosynthetic parameters were derived by fitting light saturation curves to the equation: P = Pmax tanh (αI/Pmax) using Matlab, where P is the oxygen evolution rate at a given light intensity (I) (Neale and Melis 1986).
Cells (1 ml) were collected by centrifugation at 14,000×g in a table-top centrifuge. The medium was removed by aspiration and the pellet was immediately frozen in liquid nitrogen and held at −80°C. The abundance of chlorophyll a and xanthophyll cycle pigments was determined by HPLC after extraction in 100% acetone according to Müller-Moulé et al. (2002).
Chlorophyll a fluorescence
Chlorophyll a fluorescence was measured using a Hansatech FMS2 pulse-modulated fluorometer. Cells were dark acclimated for 15 min and gently filtered onto 13-mm diameter Millipore AP20 glass fiber filters. These filters were placed into the manufacturer’s leaf clip and an actinic light intensity of 217 μmol photons m−2 s−1 was used to probe the photo-physiology of the algal cells. Chlorophyll a fluorescence parameters were assayed and calculated according to the definitions of Baker (2008).
Growth of photoheterotrophic versus phototrophic Chlamydomonas
Growth rate of photoheterotrophic versus phototrophic cells in response to iron nutrition
Acetate μ (day−1)
CO2 μ (day−1)
0.96 ± 0.12
0.56 ± 0.04
0.90 ± 0.04
0.59 ± 0.07
1.44 ± 0.15
0.68 ± 0.11
1.68 ± 0.08
0.74 ± 0.06
Phototrophic cells accumulate more Fe than photoheterotrophic cells
Photosynthetic and respiratory capacity of photoheterotrophic versus phototrophic cells
Photosynthetic and respiratory rates of acetate versus CO2-grown cells in various iron concentrations
3.1 ± 0.8
−2.1 ± 0.4
5.2 ± 1.4
−0.8 ± 0.1
3.4 ± 0.7
−1.9 ± 0.2
5.9 ± 0.8
−0.8 ± 0.2
4.9 ± 1.2
−1.9 ± 0.6
6.0 ± 0.6
−0.6 ± 0.0
6.7 ± 0.8
−2.5 ± 0.4
6.1 ± 0.6
−0.7 ± 0.1
Photosynthetic efficiency of photoheterotrophic versus phototrophic cells
1.7 ± 0.1
0.014 ± 0.001
130 ± 6
3.6 ± 0.2
0.013 ± 0.004
300 ± 140
1.9 ± 0.0
0.014 ± 0.002
140 ± 21
2.8 ± 0.2
0.021 ± 0.001
140 ± 16
2.3 ± 0.2
0.024 ± 0.002
95 ± 1
2.5 ± 0.2
0.022 ± 0.003
120 ± 11
2.7 ± 0.4
0.023 ± 0.002
120 ± 25
2.7 ± 0.2
0.022 ± 0.002
120 ± 7
Maximum quantum efficiency of PSII in photoheterotrophic versus phototrophic cells
Maximum quantum efficiency of PSII in phototrophic versus photoheterotrophic cells in response to iron nutrition
0.54 ± 0.07*
0.72 ± 0.01
0.67 ± 0.01
0.70 ± 0.02
0.73 ± 0.02
0.72 ± 0.01
0.73 ± 0.01
0.72 ± 0.01
0.74 ± 0.01
0.72 ± 0.01
0.74 ± 0.01
0.72 ± 0.00
Abundance of Fe-containing components in energy transducing membranes
Although photosynthesis requires more iron due to the high abundance of photosynthetic complexes in the thylakoid membrane, the demand for iron per monomer is greater for respiration. Complex I requires the most iron, containing a total of 8 iron–sulfur clusters (6 [Fe4S4] and 2 [Fe2S2]) for a total of 28 Fe atoms per complex I (Cardol et al. 2004; Sazanov 2007; Remacle et al. 2008). Complex II binds a total of 9 Fe atoms in the form of 3 iron–sulfur clusters (1 [Fe2S2], 1 [Fe3S4], and 1 [Fe4S4]) and 1 heme. Complex III contains 5 Fe atoms bound to 1 [Fe2S2] and 3 heme molecules, and complex IV utilizes 2 heme molecules to reduce oxygen to water. Since complex I contains the most iron, the abundance of iron-binding subunits of complex I was investigated. Surprisingly, similar to photosynthetic proteins, complex I subunits Nuo6 (Fe/S-binding) and Nuo7 (non-Fe/S-binding) were maintained in iron-limited (0.1-μM Fe) phototrophic cells, but decreased approximately 2-fold in heterophototrophic iron-limited cells, even though iron-limited heterophototrophic cells had a higher rate of oxygen consumption (Fig. 7; Table 2). Fe/S-binding Nuo8 was also more abundant in phototrophic when compared to photoheterotrophic cells (Fig. 7).
Since oxygen consumption rates measured in Table 2 are a direct result of complex IV activity, complex IV (Cox2b) abundance was investigated. Like complex I proteins, Cox2b was also maintained in phototrophic cells, and was slightly increased in iron-limited photoheterotrophic cells (Fig. 7), in agreement with the insensitivity of respiratory rate to iron limitation in the presence of acetate (Table 2). Collectively, these results indicate that phototrophic cells accumulate more iron, and are therefore able to maintain both photosynthetic and respiratory electron transport chain proteins, and this correlates with their increased capacity for iron accumulation, resulting probably from increased expression of iron uptake components.
Respiration is preferred over photosynthesis in iron-limited Chlamydomonas
In this study, we investigated the impact of iron limitation on photosynthesis and respiration of Chlamydomonas in the presence and in the absence of acetate. Overall, the results indicated that respiration is the preferred bioenergetic pathway in Chlamydomonas cells when a substrate is available. Photoheterotrophic cells, given the option to grow phototrophically or heterotrophically, suppressed photosynthetic iron-containing proteins before iron-containing respiratory proteins in response to decreasing iron nutrition (Fig. 7). In the presence of acetate, iron-limited cells could respire at a rate approximately three times that of iron-replete phototrophic cells (Table 2). In addition, the growth rate of severely iron-limited photoheterotrophic cells was still faster than the growth rate of iron-replete photoautotrophic cells (Table 1; Fig. 1). These results are consistent with theoretical predictions of iron use efficiencies (carbon fixed into cellular biomass per unit Fe per unit time), which suggest that cells growing via respiration alone are more efficient than those employing photosynthesis (Raven 1988). Collectively, these data indicate that when given a choice, it is more effective for the organism to use respiration instead of photosynthesis.
In a study of the response of photoheterotrophic Chlamydomonas to iron-starvation using a proteomics approach, photosynthetic proteins were decreased while respiratory proteins were increased, suggesting the prioritization of respiration over photosynthesis in iron deficiency (Naumann et al. 2007). In that study, a 20% decrease in the abundance of respiratory complex I subunits was observed in iron-starved cells, while all other respiratory components were increased in abundance. This may be due to the fact that the Fe in Fe/S is more labile than Fe bound to heme (Fridovich 1997; Imlay 2006; Jang and Imlay 2007). In agreement with these results, the decrease of complex I subunits in iron-limited photoheterotrophic cells and an increase in Cox2b were also observed in this study (Fig. 7).
Phototrophic cells are less sensitive to iron deficiency
In general, phototrophic cells are less sensitive to iron-deficiency than are photoheterotropic cells. This could be observed at the level of growth rate, where the difference in growth rate of iron-replete versus iron-limited cells was much more drastic in photoheterotrophic (57%) than in phototrophic (75%) conditions (Table 1; Fig. 1). Iron-limited phototrophic cells were also visually less impacted with respect to chlorosis than photoheterotrophic cells (data not shown), and this was confirmed by HPLC analysis of chlorophyll a levels (Fig. 3). A similar trend was observed for oxygen evolution rates. While oxygen evolution rates were decreased at least 50% in response to iron limitation in acetate-grown cells, they were only decreased 10% in phototrophic iron-limited cells relative to iron-replete conditions (Table 2). The lack of sensitivity is also noted with respect to respiration and the maintenance of respiratory and photosynthetic complexes (Fig. 7). We attribute this to the higher iron content (and hence reservoir) in phototrophic versus photoheterotrophic cells (Fig. 2). It is possible that the excess iron is stored in ferritin or the vacuole of phototrophic cells and provided as needed as cells divide and deplete iron from the medium (Long et al. 2008; Roschzttardtz et al. 2009). Although the lower abundance of ferritin as measured by immunoblot analysis in phototrophic cells (Supplemental Fig. 1; Busch et al. 2008) might argue against this possibility, we note that in neither study was the iron content of ferritin assessed. Since the mechanisms for regulating iron loading and unloading of ferritin are not known, storage in ferritin remains a formal possibility. Another possibility is that more iron may be stored in the vacuole of phototrophic cells relative to photoheterotrophic cells and mobilized in a situation of iron-deficiency by up-regulation of vacuolar efflux transporters. Both the vacuole and the ferritin have been implicated as possible sites of iron storage in Chlamydomonas as well as in other plants (Semin et al. 2003; Lanquar et al. 2005; Kim et al. 2006; Long et al. 2008; Briat et al. 2009). According to ferroxidase expression, which we use as a sentinel of iron nutritional status, phototrophic cells are not iron-deficient until the iron in the medium is lowered to 0.1 μM (Fig. 7), which supports the model of iron storage in phototrophic cells. The delayed degradation of PSI and expression of ferroxidase in phototrophic cells was also observed in an iron starvation time course experiment of cells grown in TAP versus HSM medium (Busch et al. 2008).
It is interesting to note that the abundance of de-epoxidized xanthophyll cycle pigments was increased in photoheterotrophic iron-limited cells when compared to phototrophic iron-limited cells (Fig. 5), and LhcSR proteins were expressed at similar levels (Fig. 7), yet iron-limited photoheterotrophic cells were clearly impaired in NPQ (Fig. 4). This result suggests that the presence of de-epoxidized xanthophyll pigments and LhcSR proteins is not sufficient for NPQ, and that another factor is necessary to induce NPQ. Although photoheterotrophic iron-limited cells can generate a thylakoid lumen pH low enough to induce the xanthophyll cycle, it is possible that the decreased capacity for photosynthetic electron transport in these cells is unable to maintain a lumen pH that is low enough to induce NPQ to the same extent as in phototrophic cells. This result could also indicate that LhcSR proteins are required for functions other than NPQ.
We noted that the plastoquinone pool of iron-limited photoheterotrophic cells was more reduced, even in the dark (Fig. 6). The reduction of plastoquinone is known to occur in Chlamydomonas by chlororespiration via a nucleus-encoded type-II NAD(P)H dehydrogenase (Mus et al. 2005; Jans et al. 2008; Desplats et al. 2009). In the light, one possibility is that the observed reduction of the plastoquinone pool in iron-limited photoheterotrophic cells is due in part to a reduced number of PSI centers in iron-limited cells (Moseley et al. 2002).
In conclusion, in the presence of acetate, iron-limited Chlamydomonas cells maintain high growth rates by suppressing photosynthesis and prioritizing respiration, while phototrophic cells maintain efficient photosynthetic systems throughout the spectrum of iron status, but still lose overall photosynthetic capacity at the onset of iron deficiency, which is delayed in phototrophic cells (0.1-μM Fe vs. 1-μM Fe in photoheterotrophic cells) due to their increased iron content.
We thank Patrice Hamel for antibodies against Nuo6-8, Susanne Preiss for antibodies against D1, Michel Guertin for antibodies against LhcSR, and Jean-David Rochaix for antibodies against PsaD. We are grateful to Janette Kropat for the measurement of iron shown in Fig. 2 and to Marina Sharifi for assistance with HPLC analysis, to Davin Malasarn for his assistance with Visual Minteq and to Naomi Ginsberg for extrapolating the data shown in Table 3 using Matlab. This research was supported by grants from the Department of Energy (DE-FD02-04ER15529) to S.S.M. and from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (FWP number 449A449B) to K.K.N. Aimee Terauchi was supported by an Institutional Ruth L. Kirschstein National Research Service Award (GM070104) and a Dissertation Year Fellowship from the UCLA graduate division.
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