| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online October 1, 2004; 10.1104/pp.104.047936 Plant Physiology 136:3301-3312 (2004) © 2004 American Society of Plant Biologists Carbon Status Constrains Light Acclimation in the Cyanobacterium Synechococcus elongatus1Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada E3B 6E1 (T.D.B.M., R.A.B., D.A.C.); and Department of Biology and Coastal Wetlands Institute, Mount Allison University, Sackville, New Brunswick, Canada E4L 1G7 (D.A.C.)
Acclimation to one environmental factor may constrain acclimation to another. Synechococcus elongatus (sp. PCC7942), growing under continuous light in high inorganic carbon (Ci; approximately 4 mM) and low-Ci (approximately 0.02 mM) media, achieve similar photosynthetic and growth rates under continuous low or high light. During acclimation from low to high light, however, high-Ci cells exploit the light increase by accelerating their growth rate, while low-Ci cells maintain the prelight shift growth rate for many hours, despite increased photosynthesis under the higher light. Under increased light, high-Ci cells reorganize their photosynthetic apparatus by shrinking the PSII pool and increasing Rubisco pool size, thus decreasing the photosynthetic source-to-sink ratio. Low-Ci cells also decrease their reductant source-to-sink ratio to a similar level as the high-Ci cells, but do so only by increasing their Rubisco pool. Low-Ci cells thus invest more photosynthetic reductant into maintaining their larger photosystem pool and increasing their Rubisco pool at the expense of population growth than do high-Ci cells. In nature, light varies widely over minutes to hours and is ultimately limited by daylength. Photosynthetic acclimation in S. elongatus occurs in both high and low Ci, but low-Ci cells require more time to achieve acclimation. Cells that can tolerate low Ci do so at the expense of slower photosynthetic acclimation. Such differences in rates of acclimation relative to rates of change in environmental parameters are important for predicting community productivity under variable environments.
Photosynthetic organisms, including cyanobacteria, must balance the absorption of light energy and its use, primarily in the fixation of inorganic carbon (Ci; Fujita, 1997  ; Huner et al., 1998). In the light, photosynthetic organisms produce reductant from the photooxidation of water that must be rapidly consumed through reduction of molecules such as Ci, nitrogen, or O2 to avoid overreduction of intermediate electron carrier pools and buildup of reactive byproducts (Sonoike et al., 2001; Kana et al., 2002). In the freshwater environments of Synechococcus elongatus (sp. PCC7942; Rippka and Herdman, 1992), however, the availability of the principal Ci reductant sink is highly variable (Cole et al., 1994). Cyanobacteria have evolved to cope with variations in light and Ci and have mechanisms to acclimate from one light regime or Ci condition to another.
In aquatic environments, light can vary widely within the lifetime of individual cells, on timescales of seconds to hours, from wave focusing, vertical mixing, cloud cover, and the daily cycle of the sun (Schubert et al., 1995
S. elongatus has at least four Ci uptake systems (Badger and Price, 2003
We hypothesized that the induced CCMs in low-Ci cells would, however, constrain the rate and amplitude of light acclimation. Light and Ci are major environmental factors that interactively constrain the organization of the photosynthetic apparatus. In cyanobacteria, abundant complexes, including phycobilisome antennae, two photosystems, intersystem electron transport components, and terminal electron sinks, absorb and convert light energy into reductant (electrons) and chemical energy (ATP) for the biosynthesis of cell components and the maintenance of cell functions, including the self-same photosynthetic apparatus. The organization of the photosynthetic apparatus is in a dynamic balance (Kana et al., 1997
In this study, we quantified the organization and performance of the photosynthetic apparatus in high- and low-Ci cells, and how that organization and performance changed during and after acclimation to a steep increase in light. We measured overall photosynthetic function before and after the light increase by light response curves of O2 evolution (net reductant production), PSII function using fluorescence signals, and PSI function using specific absorbance change signals (Fig. 1). These complementary methods are noninvasive and detect the activities of the photosystems in situ (Krause and Weiss, 1991
High Carbon Status Allowed Rapid Exploitation of Increased Light Under steady illumination of 50 µmol photons m–2 s–1, S. elongatus cultures grew at nearly identical rates of about 2 doublings per day, under markedly different high- and low-Ci levels (Fig. 2). Similarly, high- and low-Ci cultures under long-term, continuous 500-µmol photons m–2 s–1 both grew at about 2.8 doublings per day. To minimize complications from self-shading as cell numbers increased in the shifted cultures, separate cultures grown under continuous high-light (HL) illumination to about 1.5 µg chlorophyll (Chl) a mL–1 were used to determine the growth rates of cultures fully acclimated to HL.
Under continuous illumination, the induced CCM activity could produce a high enough intracellular Ci to allow the low-Ci cells to maintain a growth rate similar to the high-Ci cells, as found previously (Rodríguez-Buey and Orús, 2001 ), over a large part of the tolerable light range for this organism. During acclimation to HL, however, the high-Ci cells quickly exploited the higher light and increased their growth rate to nearly double their preshift values within 4 h of the shift to HL, after which their growth rate began to decline again toward the continuous HL value (Fig. 2). By contrast, low-Ci cells could not quickly exploit the light increase, and instead initially suffered a decline in growth rate after the shift to HL, which only recovered to values similar to preshift growth rates after 4 h of growth under HL, with subsequent slow increase to the continuous HL value.
Both the high- and low-Ci cells had a similar large capacity for net photosynthetic reductant production, measured as net O2 evolution, before and after the shift to HL, which increased only somewhat after growth under HL for 6 h (Fig. 3). The greatest change in achieved O2 evolution occurred because of the shift from the light-limited range of the light response curve to the light-saturated range immediately upon exposure to HL, causing an approximate doubling of O2 evolution in both the high- and low-Ci cells. The light-saturated O2 evolution rose further by 25% in both high- and low-Ci cells after 6 h of acclimation to HL. The light response curves in both high- and low-Ci cells showed slight drops in their initial slope (
Changes in Photosystem Excitation Balance after HL Shift The balance of the redox states of PSII and PSI were remarkably different between high- and low-Ci cells in response to the HL shift (Fig. 4). In both high- and low-Ci cells, by 6 h after the HL shift the proportion of open PSII centers at 500 µmol m–2 s–1 nearly doubled (Fig. 4A). There was also near doubling of the proportion of open PSI centers in the high-Ci cells but not in the low-Ci cells (Fig. 4B). The pressure was generally balanced between PSII and PSI in both high- and low-Ci cells under low light (LL) before the light shift (Fig. 5). Excitation pressure under HL in the high-Ci cells transiently fell disproportionately on PSI immediately after the light shift (low ratio of open PSI to open PSII) but returned to balance between PSII and PSI by 6 h after the light shift. In low-Ci cells, however, this excitation imbalance toward PSI after the light shift was larger and sustained. Even those low-Ci cells grown in continuous HL maintained an excitation pressure imbalance toward PSI.
Stoichiometric Changes in the Photosynthetic Apparatus Even though high- and low-Ci cells had similar growth rates under the same illumination before the light shift, the stoichiometric organization of the photosynthetic apparatus in the two cell types was remarkably different (Table I). The high-Ci cells before the HL shift had a lower PSI-to-PSII ratio and a higher PSII functional absorption cross-section compared to low-Ci cells. The product of photosystem pool size and functional absorption cross-section is a measure of the functional absorption capacity allocated to the photosystem pool. The ratio of functional absorption capacity of PSII was approximately equal between PSII and PSI in the high-Ci cells, while the functional absorption capacity of PSI was nearly twice that of PSII in the low-Ci cells, suggesting a higher priority on PSI activity likely for cyclic electron flow to power the CCMs. After the shift to HL, both PSII and PSI functional absorption capacities dropped by about 60% in the high-Ci cells. In low-Ci cells, however, PSI functional absorption capacity dropped disproportionately to also bring the capacities of PSII and PSI to approximate equality after the light shift. Despite the different photosystem organization of the high- and low-Ci cells, the excitation pressure on turnover rate of PSII were approximately the same in high- and low-Ci cells under LL before the light shift, and allowed a similar increase in PSII openness after the shift to HL. Rubisco rose substantially in both high- and low-Ci cells (Fig. 6) after the light shift. The ratio of PSII absorption capacity to Rubisco concentration is an index of the allocations to photosynthetic reductant source versus sink in the cell because the source of reductant is PSII, and the primary sink for reductant is carbon fixation in the Rubisco-mediated Calvin cycle. The source-to-sink allocation ratio was larger in high-Ci cells than in low-Ci cells before the light shift, and they both dropped considerably after the light shift to converge at approximately the same value after 6 h in HL (Table I).
The drop in source-to-sink allocation in high-Ci cells occurred primarily via a sharp decline in the functional PSII content of the cells, and to a lesser extent the absorption cross-section (  II) of PSII. Photosynthetic rate was maintained in the smaller functional PSII pool by increasing PSII turnover rate ( –1) and the speed of electron transfer from PSII to PSI, leading to the observed increase in Ek. By contrast, low-Ci cells maintained their functional PSII pool and decreased their source-to-sink ratio only by increasing their Rubisco pool. These cells did not substantially increase the PSII turnover rate, nor did they decrease the time required for reductant to travel from PSII to PSI (tred), as compared to high-Ci cells. Our estimation of cell numbers based upon A750 may be underestimated after 6 h of growth in HL, as after extended growth at HL, cells per A750 eventually increase slightly. This may have led to a slight overestimate in the per-cell pool sizes of pigments, photosystems, and Rubisco in the postlight shift samples reported in Table I; however, this potential overestimation would have no effect on photosystem stoichiometries, source-to-sink relationships, or other parameters not standardized to cell number.
High- and low-Ci cells showed similar increases in O2 evolution but different growth rates after the HL shift, indicating a disparity in the photosynthetic reductant required for growth of the two cell types. High-Ci cells maintained a more balanced growth yield of photosynthetic reductant than did the low-Ci cells (Fig. 7). Before the light shift, all of the reductant could be accounted for by cellular growth in the high-Ci cells. Our growth yield of photosynthetic reductant was potentially overestimated, as literature values of the elemental composition of different strains of Synechococcus vary (Bertilsson et al., 2003
Regardless of the factors contributing to error in the reductant requirements of cell growth, it is clear that the low-Ci cells produced more reductant relative to their requirement for balanced growth than did the high-Ci cells. The growth yield of reductant in LL, low-Ci cells was 25% less than the high-Ci cells, with about 87% of the reductant required for growth. After the shift to HL, the growth yield of reductant dropped in both high- and low-Ci cells, as photosynthesis shifted up the light response curve and increased more than did growth. In the high-Ci cells, the drop was relatively small and returned to preshift balance after 6 h under HL, while in the low-Ci cells the drop was more pronounced and sustained. These depressions of the growth yield of photosynthetic reductant may indicate an imbalanced investment of electrons between growing the cell population and the macromolecular pools contained in those cells, or a dumping of excess electrons into the reduction of carbon for extracellularly excreted organic compounds (Otero and Vincenzini, 2004 ).
The induction of a high-affinity CCM allows S. elongatus, under continuous illumination and mineral nutrient repletion, to maintain photosynthesis and growth at remarkably similar rates across a wide range of external Ci concentrations. These similar rates were maintained in part through compensatory changes of photosystem stoichiometry in cells grown in high- and low-Ci, a phenomenon originally described in Anacystis nidulans (currently named S. elongatus) as CO2 control over their pigmentation (Eley, 1980 ). In the prelight shift cultures grown for many generations at 50 µmol m–2 s–1, the high- and low-Ci cells also achieved balanced excitation of their photosystems, despite their differences in photosystem stoichiometry and Ci status. The photosystem stoichiometry difference between high- and low-Ci cells and restriction of the carbon sink in the low-Ci cells, however, caused different rates of acclimation to a sudden increase in light. High-Ci cells quickly exploited the higher light and were able to substantially increase their growth rate. Low-Ci cells, by contrast, which had a strong ability to concentrate the otherwise limiting low external Ci concentration, did so at the expense of rapid acclimation and did not increase their growth rate after the HL shift. After the shift to HL, the growth rate of the low-Ci cells remained at or below the preshift levels for many hours, despite a rapid and large increase in reductant produced; thus, the growth yield of photosynthetic reductant dropped after the shift to HL. The remaining reductant production in the initial hours following the light shift was likely invested in the rapid accumulation of an increased Rubisco pool while simultaneously increasing PSII synthesis to maintain a steady PSII pool in the face of more rapid PSII photoinactivation under HL, as well as in presumed increases of CCM capacity to better coordinate Ci accumulation with the increased photosynthetic rate.
The light response curves of net photosynthetic electron production, measured as O2 evolution, remained remarkably similar before and after the shift to HL, with the increase in electron flux primarily a result of the change in light with only limited changes in the light response of O2 evolution. The stability of the light response curves was despite great changes in the photosynthetic apparatus, particularly in the PSII pool, and may help maintain biosynthesis and growth rate in the face of a sudden and potentially photoinhibitory 10-fold increase in light. In both the high- and low-Ci cells, there was a substantial initial decline in PSII pool size that did not affect the saturated rate of photosynthesis under the new HL condition. Several authors have demonstrated a similar large capacity of PSII in excess of the rate of net reductant produced, as O2 evolved or CO2 fixed (Sukenik et al., 1987
Such a stability of O2 evolution light response curves can be seen in several light acclimation studies in cyanobacteria (e.g. Vierling and Alberte, 1980
Several studies, including ours, have shown that changes in photosystem stoichiometry, and less so in the pigmentation of the photosystems, are an important component of light acclimation in cyanobacteria (Manodori and Melis, 1984
In this study, we observed a transient but large increase in the functional PSI-to-PSII ratio in high-Ci cells shifted to HL, largely through a drop in the functional PSII pool size. This drop in functional PSII allowed cells to maintain efficient reductant flow into carbon fixation while it relieved excess pressure on the electron transport system and allowed a rapid, but ultimately transient, growth burst. This transient compensatory pattern in the PSII pool has been observed in natural phytoplankton assemblages periodically mixed into HL surface waters, and in cultured diatoms and green algae transiently exposed to HL (Vasilikiotis and Melis, 1994
The large change in photosystem stoichiometry in the high-Ci cells in response to the light shift allowed a rapid and balanced relief of excitation pressure on both PSII and PSI. This relief of excitation, and progressive balancing of the redox state of the two photosystems after the shift to HL, shows that the changing photosystem stoichiometry and source-to-sink ratios worked to increase electron transport while minimizing dangerous overenergization of the photosynthetic system. Presumably, the overpressured and imbalanced excitation of the two photosystems upon HL exposure signaled regulatory systems to alter photosystem content (Durnford and Falkowski, 1997
In the light-limited preshift conditions, reductant produced from net O2 evolution by photosynthesis was efficiently invested into balanced cell growth in both high- and low-Ci cells, but after the light increase, half or more of the measured reductant was not accounted for by balanced cell growth. The low growth rate in low-Ci cells may be explained by the reduction of material for building intracellular pools during acclimation at the expense of population growth, including increased Rubisco, maintained PSII and possibly a greater pool of CCM components to better serve the increased photosynthetic rate with more Ci. Electrons generated by photosynthesis can also flow to fates other than reduction of carbon, nitrogen, and sulfur for generation of new organic material. These electrons are also diverted into saturation reactions in existing fatty acids, pyruvate decarboxylation, Glu synthesis, enzyme regulation through reduced thioredoxin (Chitnis, 1996
The observed acclimatory changes in pigments, redox balance of the photosystems, and investment of reductant in growth in high- and low-Ci cells occurred at similar rates when expressed on a scale of generational (i.e. relative to growth rate) rather than chronological time. Thus, if arguing relative to the growth of the population, the cells acclimated to the change in light at a similar rate. Indeed, high- and low-Ci cells grown continuously under HL and fully acclimated to it achieved remarkably similar growth rates, photosystem excitation balance, and reductant investment into balanced population growth. Thus, given enough chronological time, high- and low-Ci cells can acclimate comparably to wide shifts in light intensity, albeit through distinct mechanisms. Nevertheless, chronological time constrains achieved acclimation regardless of the generational time spent acclimating to new conditions. HL conditions necessarily last less time than the daylength, and only those cells able to chronologically rapidly exploit new light levels will gain growth benefits of transiently increased light.
During acclimation to HL in the high-Ci cells, growth rate was transiently substantially higher than in cells finally acclimated to the continuous HL condition. This growth burst led to a much larger population of cells in the limited natural time available, which could allow resource sequestration or shading of competing phytoplankton species. Conversely, the growth burst in high-Ci cells was sustained long enough to cover any naturally likely period of increased light. Concurrent with the rise in growth rate in the high-Ci cells was a larger drop in photosystem content and pigmentation than in the low-Ci cells and a smaller rise in Rubisco than in the low-Ci cells. These changes occur in the context of a higher electron transport per photosystem in the high-Ci cells compared to the low-Ci cells, suggesting less investment of photosynthetic reductant needed to be made into the photosynthetic apparatus of the high-Ci cells. High-Ci cells shifted to HL could thus initially invest more photosynthetic reductant in cell growth, supporting the observed growth burst. Once macromolecular complexes of the photosynthetic system declined to levels appropriate to the new light regime, investment may have been withdrawn from accelerated cell growth and returned to balanced pigment and photosystem production, which may explain the slower long-term growth rate in high-Ci cells grown continuously in HL. S. elongatus is a model for researching circadian phenomena in cyanobacteria, and investment into the photosynthetic apparatus is modulated with a circadian rhythm when exposed to a light-dark cycle (Suzuki and Johnson, 2001
Light acclimation strategies in S. elongatus are strongly dependent upon the external Ci concentration. In nature, Ci concentration in freshwater habitats varies from less than our low-Ci level to greater than our high-Ci level, and fluctuates greatly through the year (Cole et al., 1994
Culture Conditions and Light Shift Experiments
Cultures of Synechococcus elongatus were separated into distinct metabolic populations of high-Ci status growing in media [Ci] of about 4 mM, achieved through bubbling with 5% CO2-enriched air at about 40 mL min–1, or low-Ci status growing in media [Ci] of about 0.02 mM, achieved through bubbling with unadulterated air at about 370 µmol CO2 mol–1 at about 200 mL min–1. The growing medium was BG-11 (Rippka et al., 1979 The high- and low-Ci cultures were grown at 35°C, under 50 µmol photons m–2 s–1 fluorescent light. These populations were maintained axenically for days to weeks (many tens of generations), continually reinoculated from late log-phase growth into fresh BG-11 medium to keep them in rapid growth at the above conditions. Culture lines were restarted approximately monthly, from axenic cultures maintained on agar plates. Experimental cultures from each high- and low-Ci population were separately inoculated into polycarbonate tissue culture flasks at approximately 0.2 µg Chl a mL–1 and grown under continuous light at 50 µmol photons m–2 s–1 in a controlled environment chamber (Conviron, Winnipeg, Canada) on an orbital shaking table. Experimental cultures were approximately 120 mL volume, 1 cm deep, and continuously and rapidly mixed in the flasks to minimize light gradients and differential shading of the cells. Gas, either 5% CO2 or ambient air, was supplied via syringe needles extending through a rubber septum sealing the flasks so that the gas was bubbled directly into the medium. Gas was vented from the overlying airspace through a second syringe needle in the rubber septum.
Experimental cultures were grown through approximately three generations to about 1.5 µg Chl a mL–1 at 50 µmol photons m–2 s–1 (LL), and then were exposed to 500 µmol photons m–2 s–1 (HL). This HL level is near the upper end of their tolerable range and has been previously used for studying HL acclimation in this cyanobacterium (Schaefer and Golden, 1989
Several times before and then intensively for the 6 h after the light shift, samples of approximately 1 mL volume were removed via sterile syringes pierced through the rubber septum sealing the flask. Whole-cell spectra were measured with a Spectronic Unicam spectrophotometer (VWR Canlab, Mississauga, Canada) to calculate population growth rate and per-cell concentrations of phycocyanin and Chl a. We modified the Myers et al. (1980)
Oxygen evolution, Chl fluorescence, and P700 absorbance changes were measured simultaneously in subsamples taken from the experimental cultures. The 1.65-mL subsamples were suspended in a temperature-controlled quartz cuvette, with fiber-optic light guides to conduct actinic light, saturation light pulses, and probe light for Chl fluorescence and differential absorbance measurements of P700 oxidation. The subsamples were capped with a Clarke-type oxygen electrode modified to seal the sample from the air (YSI, Yellow Springs, OH) and stirred from below with a micromagnetic stirrer. The signals from the oxygen electrode were amplified by a Hansatech control box (King's Lynn, UK). A Walz XE-PAM modulated fluorometer was used to monitor Chl fluorescence and a XE-ST single turnover xenon discharge lamp (Walz, Effeltrich, Germany) was used for measurement of PSII functional absorption cross-sections. A PAM-101 Chl fluorometer modified with the P700DW dual-wavelength differential absorbance probe (Walz) was used to measure the oxidation state of P700, the reaction center of PSI (Klughammer and Schreiber, 1994 Signals generated from the oxygen content of the sample, Chl fluorescence, absorbance changes from P700, and the light level in the cuvette were simultaneously digitized at 20 Hz by a LabPro data logger (Vernier Software, Portland, OR) and displayed real time through a computer interface. These data were subsequently summarized into relevant Chl fluorescence and P700 oxidation parameters by custom-designed software (written by T.D.B. MacKenzie). The performance of the automated data summarizing was verified by manually summarizing a subset of the collected data.
Light response curve plots of photosynthetic performance as a function of light intensity (Henley, 1993
, where P is the Chl-specific rate of O2 evolution (µmol O2 µmol Chl–1 s–1), Pmax is the maximum rate of light-saturated O2 evolution (µmol O2 µmol Chl–1 s–1),  is the initial slope of the light response curve under limiting light (µmol O2 µmol photons m2 µmol Chl–1), E is the light intensity (µmol photons m–2 s–1), and R is the respiration rate (µmol O2 µmol Chl–1 s–1).
Light response curves can be divided into two sections, a light-limited region in which the photosynthetic rate rises steeply and nearly proportionately with increasing light,
Ek is considered the optimum light intensity for photosynthesis along the light response curve (Falkowski and Raven, 1997
We determined
We measured the time required for electrons from PSII to reach PSI and begin reducing the P700 pool, tred, after the application of a fast-rising and intense saturation light pulse (Fig. 1). Changes in the speed of electron transfer from PSII to PSI indicate changes in the effective size of the intersystem electron transport chain relative to photosystem content.
We also used light response curves to study the functions of PSII and PSI individually. We measured the proportion of open photosystems, those available for photochemistry, in the PSII and PSI pools across a range of light intensities simultaneously with the O2 evolution measurements. We used the established qP parameter calculated from Chl fluorescence (van Kooten and Snel, 1990
).
We used a parameter analogous to the PSII fluorescence parameter qP for analyzing the redox state of PSI, based upon P700 oxidation states during steady-state illumination and in response to a saturating light. On exposure to a saturation pulse of intense and fast-rising white light (9,000 µmol m–2 s–1; half-rise about 2 ms;
have recently demonstrated that the proportion of reduced (open) PSI centers, like the proportion of open PSII centers, is directly related to the photosystem's photochemical potential, and, thus, we interpret changes in PSIopen similarly to PSIIopen.
In vivo PSII functional, or photochemically active, absorption cross-sections were estimated using pump and probe fluorometry. Chl fluorescence yield of PSII was measured before, and 100 µs after, exposure to microsecond-length light pulses with a dose of approximately 0.1 to 10 photons nm–2. Fluorescence yields were fit to the one-hit Poisson model of Mauzerall and Greenbaum (1989)
 is fluorescence yield induced over the background level by a light dose of E photons nm–2,  is the maximum inducible fluorescence yield over the background level, and II is the functional absorption cross-section of PSII in nm2.
In vivo PSI functional absorption cross-sections were estimated by measuring the rapid (<10 ms) P700 oxidation kinetics during exposure to the fast-rising saturating light (Fig. 1). A second LabPro data logger and computer were used for digitizing P700 oxidation changes at 10,000 Hz, and the level of P700 oxidation at each point during the profile was compared to the accumulated photon dose applied from the 9,000 µmol photons m–2 s–1 white-light saturation pulse. Total light exposure during the rising profile of P700 oxidation ranged up to approximately 25 photons nm–2, comparable to the range of quantum dose for the PSII cross-section measurements. Like the fluorescence response of PSII, the P700 oxidation response was fit to quantum dose by the Poisson model, and the resultant functional absorption cross-section was denoted
We estimated the numbers of photosystems in the cells using
Photosystem Chl molecules were taken as 36 per PSII monomer (Ferreira et al., 2004
Rubisco Quantitation We centrifuged 1-mL aliquots of medium (containing between 1.5 and 3 µg Chl) at 12,000g for 5 min immediately after withdrawal from the culture and stored the cell pellets at about –20°C until extraction. The protein extraction occurred in 200 µL of solubilization buffer (140 mM Tris base, 105 mM Tris-HCl, 0.5 mM EDTA, 2% LDS, 10% glycerol), which was frozen in liquid nitrogen, then thawed while sonicating with a Branson Ultrasonics (Danbury, CT) model 450 sonicator for 50 s in two cycles. Dithiothreitol was added to the solubilization buffer (final concentration 50 mM), and the samples were incubated at 65°C for 5 min to denature and solubilize the proteins. The protein samples were centrifuged at 12,000g for 2 min to pellet insoluble cell debris, then frozen and stored at approximately –20°C until use. The protein samples were separated on precast 10% linear BisTris polyacrylamide NuPage gels (Invitrogen, Carlsbad, CA) in an XCell electrophoresis system (Invitrogen). The samples were loaded on an equal-cell-number basis (2.3 x 106 cells per lane; approximately 50–80 pmol Chl a per lane), then run at 200 V for 1 h in 1x MES buffer (Invitrogen). At least two lanes per gel were loaded with spinach Rubisco (AgriSera, Vännäs, Sweden) as a quantitative standard. The separated proteins were transferred onto PVDF membrane (Bio-Rad Laboratories, Hercules, CA) with 1x NuPage transfer buffer in the XCell (Invitrogen) at 30 V for 1 h. The membrane was immediately placed in a 2% ECL Advance blocking buffer (Amersham-Pharmacia Biotech, Uppsala) in Tris-buffered saline (TBS-T: 20 mM Tris base, 500 mM NaCl, 0.1% Tween 20, pH 7.6) for 1 h at room temperature. The membrane was then incubated with a Global anti-RbcL (large subunit of Rubisco) antibody raised in chicken (AgriSera) at a dilution of 1:50,000 in 10 mL of 2% blocking buffer for 1 h at room temperature. This antibody is directed against a region of the RbcL protein identical in sequence in S. elongatus and from spinach. The membrane was exhaustively rinsed in TBS-T and then incubated with a HRP-conjugated antichicken secondary antibody raised in goat (Abcam Scientific, Cambridge, UK) at a dilution of 1:50,000 in 10 mL of blocking buffer for 1 h at room temperature. Again, the membrane was exhaustively rinsed in TBS-T and then detected with ECL Advance chemiluminescent development reagents (Amersham-Pharmacia Biotech) according to the manufacturer's protocol. The chemiluminescent blots were imaged with a Fluor-S Max camera system (Bio-Rad) and signals were quantified using Quantity One software (Bio-Rad).
Growth Investment of Biosynthetic Electron Transport
The exponential function in Equation 9 calculates the growth rate (µE) at a specific light level (E) as the change in cell number, cell–1 s–1, multiplied by the empirically estimated carbon content per cell, to compare to oxygen produced by photosynthesis at that light (PE), converted to equivalent electrons s–1. The number of carbon atoms per cell, 3.26 x 1010 for high-Ci cells and 2.27 x 1010 for low-Ci cells, was estimated by quantifying CO2 produced after combustion of cell samples. The cell samples were rinsed with distilled water and dried at about 70°C under dry N2 gas in glass ampules. The ampules were sealed, containing excess O2, then combusted at 500°C overnight. CO2 resulting from the sample combustion was quantified with an infrared CO2 gas analyzer (Qubit Systems) using standards of 100 mmol Gly solution (Sigma, St. Louis), dried and combusted in the same manner as the cell samples. The 5.919 electrons per carbon reduced were estimated as the sum of the electrons required to reduce carbon, nitrogen, and sulfur in the ratio of their estimated abundance in the cells. Electrons required to reduce carbon alone were estimated based on the reactions
We thank AgriSera for supplying the Global anti-RbcL antibody and Dr. Amanda Cockshutt and Mr. Chris Brown for optimizing the protein immunodetection protocols. Received June 15, 2004; returned for revision July 29, 2004; accepted August 2, 2004.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) operating and equipment grants (to D.A.C.), NSERC postgraduate and University of New Brunswick Board of Governors scholarships (to T.D.B.M.), and the Mount Allison Coastal Wetlands Institute funded by the Canada Foundation for Innovation and Atlantic Canada Opportunities Agency.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.047936. * Corresponding author; e-mail dcampbell{at}mta.ca; fax 506–364–2505.
Allen J (2003) Cyclic, pseudocyclic and noncyclic photophosphorylation: new links in the chain. Trends Plant Sci 8: 15–19[CrossRef][Web of Science][Medline] Anderson J, Park Y-I, Chow WS (1998) Unifying model for the photoinactivation of Photosystem II in vivo under steady-state photosynthesis. Photosynth Res 56: 1–13
Badger MR, Price GD (2003) CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. J Exp Bot 54: 609–622 Behrenfeld M-J, Prasil O, Kolber ZS, Babin M, Falkowski PG (1998) Compensatory changes in Photosystem II electron turnover rates protect photosynthesis from photoinhibition. Photosynth Res 58: 259–268[CrossRef] Bertilsson S, Berglund O, Karl DM, Chisholm SW (2003) Elemental composition of marine Prochlorococcus and Synechococcus: implications for the ecological stoichiometry of the sea. Limnol Oceanogr 48: 1721–1731 Bukhov NG, Carpentier R (2003) Measurement of photochemical quenching of absorbed quanta in photosystem I of intact leaves using simultaneous measurements of absorbance changes at 830 nm and thermal dissipation. Planta 216: 630–638[Medline]
Campbell D, Hurry VM, Clarke AK, Gustafsson P, Öquist G (1998) Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation. Microbiol Mol Biol Rev 62: 667–683 Clarke AK, Soitamo A, Gustafsson P, Öquist G (1992) Rapid interchange between two distinct forms of cyanobacterial photosystem II reaction-center protein D1 in response to photoinhibition. Proc Natl Acad Sci USA 90: 9973–9977 Chitnis PR (1996) Photosystem I. Plant Physiol 111: 661–669[CrossRef][Web of Science][Medline] Chitnis PR (2001) Photosystem I: function and physiology. Annu Rev Plant Physiol Plant Mol Biol 52: 593–626[CrossRef][Web of Science][Medline]
Cole JJ, Caraco NF, Kling GW, Kratz TK (1994) Carbon dioxide supersaturation in the surface waters of lakes. Science 265: 1568–1570 Durnford DG, Falkowski PG (1997) Chloroplast redox regulation of nuclear gene transcription during photoacclimation. Photosynth Res 53: 229–241[CrossRef] Eley J (1980) Effect of carbon dioxide concentration on pigmentation in the blue-green alga Anacystis nidulans. Plant Cell Physiol 12: 311–316
Escoubas JM, Lomas M, Laroche J, Falkowski PG (1995) Light intensity regulation of cab gene transcription is signaled by the redox state of the plastoquinone pool. Proc Natl Acad Sci USA 92: 10237–10241 Falkowski P, Raven JA (1997) Aquatic Photosynthesis. Blackwell, Oxford
Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Architecture of the photosynthetic oxygen-evolving center. Science 303: 1831–1838 Fujita Y (1997) A study on the dynamic features of photosystem stoichiometry: accomplishments and problems for future studies. Photosynth Res 53: 83–93[CrossRef]
Fujita Y, Murakami A, Ohki K (1987) Regulation of photosystem composition in the cyanobacterial photosynthetic system: the regulation occurs in the response to the redox state of the electron pool located between the two photosystems. Plant Cell Physiol 28: 283–292 Heldal M, Scanlan DJ, Norland S, Thingstad F, Mann NH (2003) Elemental composition of single cells of various strains of marine Prochlorococcus and Synechococcus using X-ray microanalysis. Limnol Oceanogr 48: 1732–1743 Henley WJ (1993) Measurement and interpretation of photosynthetic light-response curves in algae in the context of photoinhibition and diel changes. J Phycol 29: 729–739[CrossRef][Web of Science] Herbert SK, Martin RE, Fork DC (1995) Light adaptation of cyclic electron transport through photosystem I in the cyanobacterium Synechococcus sp. PCC 7942. Photosynth Res 46: 277–285[CrossRef] Ho TY, Quigg A, Finkel ZV, Milligan AJ, Wyman K, Falkowski PG, Morel FMM (2003) The elemental composition of some marine phytoplankton. J Phycol 39: 1145–1159[CrossRef][Web of Science] Huner NPA, Maxwell DP, Gray GR, Savitch LV, Laudenbach DE, Falk S (1995) Photosynthetic response to light and temperature: PSII excitation pressure and redox signalling. Acta Physiol Plant 17: 167–176 Huner NPA, Öquist G, Sarhan F (1998) Energy balance and acclimation to light and cold. Trends Plant Sci 3: 224–230[CrossRef][Web of Science] Kana TM, Geider RJ, Critchley C (1997) Regulation of photosynthetic pigments in micro-algae by multiple environmental factors: a dynamic balance hypothesis photoacclimation. New Phytol 137: 629–638[CrossRef] Kana R, Lazár D, Prásil O, Naus J (2002) Experimental and theoretical studies on the excess capacity of Photosystem II. Photosynth Res 72: 271–284[CrossRef][Medline]
Kaplan A, Helman Y, Tchernov D, Reinhold L (2001) Acclimation of photosynthetic microorganisms to changing ambient CO2 concentrations. Proc Natl Acad Sci USA 98: 4817–4818 Klughammer C, Schreiber U (1994) An improved method, using saturating light pulses, for the determination of photosystem I quantum yield via P700+-absorbance changes at 830 nm. Planta 192: 261–268[CrossRef][Web of Science] Krause GH, Weiss E (1991) Chlorophyll fluorescence and photosynthesis: the basics. Annu Rev Plant Physiol Plant Mol Biol 42: 313–349[CrossRef][Web of Science]
Kulkarni RD, Schaefer MR, Golden SS (1992) Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942. J Bacteriol 174: 3775–3781 Li Q, Canvin DT (1997) Oxygen photoreduction and its effect on CO2 accumulation and assimilation in air-grown cells of Synechococcus UTEX 625. Can J Bot 75: 274–283
Li L-A, Tabita F (1994) Transcription control of ribulose bisphosphate carboxylase/oxygenase activase and adjacent genes in Anabaena species. J Bacteriol 176: 6697–6706 Lourenco SO, Barbarino E, Mancini-Filho J, Schinke KP, Aidar E (2002) Effects of different nitrogen sources on the growth and biochemical profile of 10 marine microalgae in batch culture: an evaluation for aquaculture. Phycologia 41: 158–168 MacIntyre HL, Kana TM, Anning T, Geider RJ (2002) Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria. J Phycol 38: 17–38[Web of Science]
Manodori A, Melis A (1984) Photochemical apparatus organisation in Anacystis nidulans (Cyanophyceae). Plant Physiol 74: 67–71
Markager S, Vincent W (2001) Light absorption by phytoplankton: development of a matching parameter for algal photosynthesis under different spectral regimes. J Plankton Res 23: 1373–1384 Mauzerall D, Greenbaum NL (1989) The absolute size of a photosynthetic unit. Biochim Biophys Acta 974: 119–140 McGinn PJ, Price GD, Badger MR (2004) High light enhances the expression of low-CO2-inducible transcripts involved in the CO2-concentrating mechanism in Synechocystis sp. PCC6803. Plant Cell Environ 27: 615–626[CrossRef]
Melis A, Brown JS (1980) Stoichiometry of system I and system II reaction centers and of plastoquinone in different photosynthetic membranes. Proc Natl Acad Sci USA 77: 4712–4716
Miskiewicz E, Ivanov AG, Huner NPA (2002) Stoichiometry of the photosynthetic apparatus and phycobilisome structure of the cyanobacterium Plectonema boryanum UTEX 485 are regulated by both light and temperature. Plant Physiol 130: 1414–1425 Müller C, Reuter W, Wehrmeyer W, Dau H, Senger H (1993) Adaptation of the photosynthetic apparatus of Anacystis nidulans to irradiance and CO2-concentration. Bot Acta 106: 480–487
Murakami A, Fujita Y (1991a) Regulation of photosystem stoichiometry in the photosynthetic system of the cyanophyte Synechocystis PCC 6714 in response to light intensity. Plant Cell Physiol 32: 223–230
Murakami A, Fujita Y (1991b) Steady state of photosynthetic electron transport in cells of the cyanophyte Synechocystis PCC 6714 having different stoichiometry between PS I and PS II: analysis of flash-induced oxidation-reduction of cytochrome f and P700 under steady state of photosynthesis. Plant Cell Physiol 32: 213–222
Murakami A, Kim S-J, Fujita Y (1997) Changes in photosystem stoichiometry in response to environmental conditions for cell growth observed with the cyanophyte Synechocystis PCC 6714. Plant Cell Physiol 38: 392–397 Muramatsu M, Hihara Y (2003) Transcriptional regulation of genes encoding subunits of photosystem I during acclimation to high-light conditions in Synechocystis sp. PCC 6803. Planta 216: 446–453[Web of Science][Medline]
Myers J, Graham JR, Wang RT (1980) Light harvesting in Anacystis nidulans studied in pigment mutants. Plant Physiol 66: 1144–1149 Ogawa T, Kaplan A (2003) Inorganic carbon acquisition systems in cyanobacteria. Photosynth Res 77: 105–115 Öquist G, Chow WS, Anderson JM (1992) Photoinhibition of photosynthesis represents a mechanism for long-term regulation of photosystem II. Planta 186: 450–460 Ort DR, Baker NR (2002) A photoprotective role for O2 as an alternative electron sink in photosynthesis? Curr Opin Plant Biol 5: 193–198[CrossRef][Web of Science][Medline] Otero A, Vincenzini M (2004) Nostoc (Cyanophyceae) goes nude: extracellular polysaccharides serve as a sink for reducing power under unbalanced C/N metabolism. J Phycol 40: 74–81[Web of Science] Pfannschmidt T (2003) Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci 8: 33–41[CrossRef][Web of Science][Medline] Prasil O, Kolber Z, Berry J, Falkowski P (1996) Cyclic electron flow around photosystem II in vivo. Photosynth Res 48: 395–410[CrossRef] Raven JA (2003) Inorganic carbon concentrating mechanisms in relation to the biology of algae. Photosynth Res 77: 155–171[CrossRef][Medline] Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyano-bacteria. J Gen Microbiol 11: 1–61 Rippka R, Herdman M (1992) Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture, Catalogue and Taxonomic Handbook. Institut Pasteur, Paris Rodríguez-Buey ML, Orús MI (2001) The response of Synechococcus PCC7942 (Cyanophyta) to changes in CO2 supply in relation to the acclimation of the CO2-concentrating mechanism. I: physiological study. J Plant Physiol 158: 325–334 Rosenqvist E (2001) Light acclimation maintains the redox state of the PS II electron acceptor QA within a narrow range over a broad range of light intensities. Photosynth Res 70: 299–310
Schaefer MR, Golden SS (1989) Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids. J Biol Chem 264: 7412–7417 Schubert H, Matthijs H, Mur LR, Schiewer U (1995) Blooming of cyanobacteria in turbulent water with steep light gradients: the effect of intermittent light and dark periods on the oxygen evolution capacity of Synechocystis sp. PCC 6803. FEMS Microbiol Ecol 18: 237–245 Shibata M, Ohkawa H, Katoh H, Shimoyama M, Ogawa T (2002) Two CO2-uptake systems: four systems for inorganic carbon acquisition in Synechocystis sp. strain PCC6803. Funct Plant Biol 29: 123–129[CrossRef]
Smith B, Melis A (1988) Photochemical apparatus organization in the diatom Cylindrotheca fusiformis: photosystem stoichiometry and excitation distribution in cells grown under high and low irradiance. Plant Cell Physiol 29: 761–769
Sonoike K, Hihara Y, Ikeuchi M (2001) Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC6803. Plant Cell Physiol 42: 379–384 Sukenik A, Bennett J, Falkowski P (1987) Light-saturated photosynthesis—limitation by electron transport or carbon fixation. Biochim Biophys Acta 891: 205–215[CrossRef] Suzuki L, Johnson CH (2001) Algae know the time of day: circadian and photoperiodic programs. J Phycol 37: 933–942[CrossRef][Web of Science] van Kooten O, Snel JFH (1990) The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res 25: 147–150[CrossRef]
Vasilikiotis C, Melis A (1994) Photosystem II reaction center damage and repair cycle: chloroplast acclimation strategy to irradiance stress. Proc Natl Acad Sci USA 91: 7222–7226 Vierling E, Alberte R (1980) Functional organization and plasticity of the photosynthetic unit of the cyanobacterium Anacystis nidulans. Physiol Plant 50: 93–98 Walters RG, Horton P (1993) Theoretical assessment of alternative mechanisms for non-photochemical quenching of PS II fluorescence in barley leaves. Photosynth Res 36: 119–139[CrossRef] Webb M, Melis A (1995) Chloroplast response in Dunaliella salina to irradiance stress effect on thylakoid membrane protein assembly and function. Plant Physiol 107: 885–893[Abstract]
Woodger FJ, Badger MR, Price GD (2003) Inorganic carbon limitation induces transcripts encoding components of the CO2-concentrating mechanism in Synechococcus sp. PCC7942 through a redox-independent pathway. Plant Physiol 133: 2069–2080 This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
< 695 nm) saturation pulse of about 9,000 µmol photons m–2 s–1 for 0.5 s. The Chl fluorescence parameters Fm' (maximum fluorescence), Fs (steady-state fluorescence), and Fo' (minimum fluorescence) and the P700 oxidation parameters P700ox (fully oxidized), P700s (steady-state oxidation), P700red (fully reduced P700), and tred (the time for reductant to travel through the intersystem electron transport chain to PSI) are indicated on the respective traces and are described in the text. Note the time axis is logarithmic to better show the details of the rapid induction kinetics in the context of the other fluorescence and oxidation parameters.
, and connected to the light-shifted cells by dotted lines (values are means, n = 6 ± SEM).
-cyanobacteria, such as S. elongatus, which have evolved in an environment with fluctuating Ci (Raven, 2003
to produce C0 for carbohydrate-C and most protein-C, and 
to produce C2– for lipid-C. We assumed an 84:16 C0:C2– ratio from literature values of lipid content (Lourenco et al., 2002
as well as two additional electrons required for the reductive amination reaction to assimilate the ammonium into amino acids. The only nitrogen source in the medium was nitrate; S. elongatus does not fix nitrogen, the amount of nitrogen required was based upon the average N content of four Synechococcus strains, 0.146 N per C (Bertilsson et al., 2003
and an estimated S content of the cells of 0.0066 S per C (Heldal et al., 2003
