Photochemistry and Photobiology, 2014, 90: 522–532

Kinetics of Photosynthetic Response to Ultraviolet and Photosynthetically Active Radiation in Synechococcus WH8102 (CYANOBACTERIA) Glaucia M. Fragoso*§1, Patrick J. Neale1, Todd M. Kana2 and Alicia L. Pritchard1 1 2

Smithsonian Environmental Research Center, Edgewater, MD University of Maryland Center for Environmental Science, Cambridge, MD

Received 22 August 2013, accepted 25 October 2013, DOI: 10.1111/php.12202

ABSTRACT

several orders of magnitude (3,4). With the sequencing of the Synechococcus genome and emergence of new molecular tools, it is possible to document in unprecedented detail the mechanisms used to respond to high PAR, UV and other interacting environmental stresses, such as the production of reactive oxygen species (ROS) (5–7). Planktonic cyanobacteria, and Synechococcus specifically, have several responses related to the particular structure of their photosynthetic apparatus, which have induction/relaxation time scales of minutes to hours. Protective mechanisms in Synechococcus that cause nonphotochemical quenching (NPQ) to dissipate the excess excitation energy not used for photochemistry are primarily mediated by a carotenoid-binding protein, known as Orange Carotenoid Protein (OCP) (5,8,9). State transitions, a mechanism that regulates the distribution of absorbed energy between PSI and PSII, are also assumed to be a photoprotective mechanism in cyanobacteria, including Synechococcus (10). In spite of all these mechanisms of photoprotection, damage to the PSII reaction center still occurs in Synechococcus (and other algae and plants as well) as a consequence of exposure to high PAR and UV, targeting, among other sites, the reaction center D1 protein (encoded by psbA). A unique feature in Synechococcus is that psbA exists as a multigene family, and extended high PAR and/or UV exposure can lead to a shift in gene expression toward synthesis of a more damage-resistant form—D1:2 (11) More recently, another photoprotective mechanism that relieves the excess photosynthetic electron flow from PSII has been recently proposed for Synechococcus as well as other picophytoplankton. An alternate electron transfer pathway, involving a plastoquinol terminal oxidase (ptox) or a ptox-like enzyme has been reported to be a sink for a significant fraction of photosynthetic electron transport under high irradiance (5,12). This mechanism may compensate for the constitutively low PSI to PSII ratio in Synechococcus, which would be favored in an iron-poor environment. However, in contrast to our understanding of the more damage-resistant form—D1:2, little is known about the dynamics of ptox activity and whether it is important under UV exposure. There is a critical gap in understanding the spectral and temporal response to full spectral irradiance of phytoplankton in the mid and low latitude open ocean, in particular with respect to the productively important Synechococcus. To date, most studies on the spectral-temporal response of phytoplankton to combined PAR + UV exposure have focused on assemblages or species in areas where the stratospheric ozone depletion has resulted in

The picoplanktonic cyanobacteria, Synechococcus spp., (N€ageli) are important contributors to global ocean primary production that can be stressed by solar radiation, both in the photosynthetically active (PAR) and ultraviolet (UV) range. We studied the responses of PSII quantum yield (active fluorescence), carbon fixation (14C assimilation) and oxygen evolution (membrane inlet mass spectrometry) in Synechococcus WH8102 under moderate UV and PAR. PSII quantum yield decreased during exposure to moderate UV and UV+PAR, with response to the latter being faster (6.4 versus 2.8 min, respectively). Repair processes were also faster when UV+PAR exposure was followed by moderate PAR (1.68 min response time) than when UV was followed by very low PAR (10.5 min response time). For the UV+PAR treatment, the initial decrease in quantum yield was followed by a 50% increase (“rebound”) after 7 min exposure, showing an apparent photoprotection induction. While oxygen uptake increased with PAR, it did not change under UV, suggesting that this oxygen-dependent mechanism of photoprotection, which may be acting as an electron sink, is not an important strategy against UV. We used propyl gallate, an antioxidant, to test for plastid terminal oxidase (ptox) or ptox-like enzymes activity, but it caused nonspecific and toxic effects on Synechococcus WH8102.

INTRODUCTION The picoplanktonic cyanobacteria, Synechococcus spp., are ubiquitous in marine and freshwater environments and are important contributors to global ocean primary production. In the open ocean, Synechococcus population densities are highest in the surface layer, where their dominance has been associated with the ability to tolerate a wide range of environmental stressors, such as chronically low nutrient concentrations (including N, P and Fe) and variable and high ultraviolet (290–400 nm for incident irradiance) and photosynthetically active radiation (PAR) (400–700 nm) (1,2). The light experienced by Synechococcus in the surface layer can fluctuate over broad timescales and over *Corresponding author email: [email protected] (Glaucia Fragoso) §Current Address: Ocean and Earth Science, National Oceanography Centre Southampton, University of Southampton Waterfront Campus, European Way, Southampton, SO14 3ZH, UK © 2013 The American Society of Photobiology

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Photochemistry and Photobiology, 2014, 90 episodic increases in UV-B, e.g. Southern Ocean (13–15), or coastal and freshwater environments (16). Previous studies have shown the time dependence of Synechococcus photosynthetic characteristics under PAR and UV radiation (7,11,17). However, it is difficult to relate the results of these studies to in situ responses because they have used a low time resolution (hours– days) and the irradiance treatments differ strongly in their spectral composition from solar radiation. Laboratory studies usually use fluorescent lamp sources, which emit a much higher proportion of short wavelength UV-B (280–315 nm) and UV-A (315–400 nm) relative to long wavelength UV-A and PAR compared to solar radiation and potentially distorts the magnitude of the response to the irradiance treatment (18). Moreover, most of UV radiation that penetrates into the upper water column is in the UV-A range because it is attenuated less than UV-B and may penetrate down to 50 m in the water column, particularly in clear waters of the oligotrophic oceans (19,20). Although UV-B is more damaging on a per photon basis through DNA damage (21), most inhibition of primary production and degradation of photosynthetic pigments is caused by the much larger flux of UV-A irradiance (22,23). Here, we report on the short-term responses of Synechococcus WH8102, an isolate from the Sargasso Sea, a low latitude oligotrophic gyre, to levels of UV radiation (UVR) and PAR that best simulate solar radiation of a typical mixed layer. Our approach was to use variable fluorescence measurements of effective PSII quantum yield to obtain the highly resolved temporal response (kinetics) of inhibition and recovery from UV and/or PAR exposure on the time scale of minutes to hours. These types of kinetics are consistent with underlying physiological dynamics of an equilibrium between “damage”—all those processes which cause loss of photosynthetic activity and “repair”—processes that counteract such loss with this basic concept applying to both excess PAR (24) and UV (23) exposure. To obtain results that are relevant to the mixed layer, we used spectral treatments with similar proportions of UV-B and UV-A to PAR as in the underwater environment. In addition, to gain insight into suspected underlying mechanisms that may influence the photoinhibition response, we conducted measurements of photosynthetic carbon assimilation and photosynthetic oxygen cycling using 18O2 to trace lightdependent oxygen uptake and possible ptox(-like) activity.

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conditions and fluorescence was measured before and after the addition of 10% HCl using a Turner 10-AU fluorometer calibrated with chl a from spinach (Sigma). Kinetics of inhibition and recovery. The quantum yield of PSII photochemistry was measured using a portable underwater Pulse-AmplitudeModulated (PAM) fluorometer, the Diving-PAM/B (Walz, Germany) with a blue Light Emitting Diodes (LED) (470 nm) excitation. In most cases, the modulation frequency of the measuring beam (MB) was 20 kHz, resulting in a background PAR exposure of 14 lmol photons m2 s
1. An aliquot of culture (2 mL) was placed in a quartz cuvette, which was optically coupled to the PAM using a custom fabricated acrylic rod. Actinic irradiance was provided by a 150 W Xenon Lamp (Osram) filtered through a (1) GG400 filter for PAR-only illumination (control), (2) WG335 filter for UV+PAR and (3) WG280 and UG-11 filters for the UV treatment. The first two filters were selected to manipulate the spectral UV and PAR irradiance derived from the xenon lamp in an attempt to mimic the ongoing radiation from the surface mixed layers of oligotrophic gyres, where Synechococcus are abundant (Fig. 1). On the other hand, the UV treatment, in which PAR was filtered out (Fig. 1), enabled us to separately test the effect of the UV component of the actinic irradiance. Spectral irradiance in each treatment was measured with a custom built spectroradiometer system, details of the system are given by (26). Sample aliquots were air cooled during exposure and temperature was checked periodically to ensure that no heating occurred. Metal screens were used to further adjust overall irradiance for the PAR only and UV+PAR treatment. For example, samples were initially monitored without actinic irradiance (measuring beam only = MB), and then acclimated to PAR-only exposure of 70 and 350 lmol photons m2 s
1 remaining at each irradiance level until PSII quantum yield approached a steady state (5–10 min). The actinic beam was blocked (MB only) for 30 s while filters were changed, during which time one yield measurement was made. Then, cells were exposed to moderate UV and PAR levels (or just PAR for the control) for 30 min. This longer exposure was at an irradiance of 480 lmol photons m2 s
1 for PAR, to which was added 27 W m
2 of UV for the UV+PAR treatments. To keep the UV exposure moderate, very little UV-B was transmitted to the sample (0.1 W m
2). The recovery was monitored for 30 min after removal of UV and subsequent exposure to PAR only at 480 lmol photons m2 s
1 followed a period of no actinic irradiance, i.e. exposure to PAR from only the MB (14 lmol

MATERIALS AND METHODS Culture and growth conditions. A Synechococcus sp. culture, strain WH8102, was provided by the Provasoli–Guillard National Center for Marine Algae and Microbiota (NCMA). The growth media was filtered seawater from the Gulf Stream with salinity at 36 psu and enriched with SN media (25). Axenic Synechococcus sp. culture was grown semicontinuously at 26°C with constant aeration and illuminated with 77 lmol photons m2 s
1 PAR irradiance provided by cool-white fluorescence lamps under 16:8 light:dark photoperiod. Cellular density and chlorophyll concentration. Cell density was counted using Neubauer hematocytometer (Hausser Scientific, Horsham, PA, USA) under a epifluorescence microscope using dim light. Growth rate (l, d
1) was calculated as the average slope of ln N(t) versus time from a triplicate growth curve during exponential growth phase, where N (t) is cell abundance on day t. The culture was inoculated at an initial abundance of 2.5 9 105 cells mL
1 and used for experiments with abundance in the range 106–107 cells mL
1. Triplicate chlorophyll samples were filtered under low vacuum ( 10 min) (Fig. 6). The carbon assimilation rate is average for the same exposure conditions as the PAM measurements (see methods). A smaller decrease was observed in net O2 evolution (average of 28.2  5.4%) than in CO2 fixation (average of 51.4  12.3%). PGAL as an oxidase inhibitor and oxygen scavenger To assess whether increased electron transport from PSII to an oxidase (potentially ptox or a ptox-like enzyme) contributed to the time-dependent enhancement of quantum yield of Synechococcus WH8102, the antioxidant PGAL was added 30 min after cells were exposed to different light treatments (moderate PAR, UV and UV+PAR). In all treatments, the addition of PGAL resulted in a progressively lower quantum yield of PSII, with a steady reduction in yield over time toward zero (data not shown). In all cases, PGAL appeared not to be a specific inhibitor of ptox (or ptox-like enzyme), as it imposed a variety of responses in quantum yield, ultimately lowering it to zero, an indication of possible multiple toxic effects.

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Figure 3. Kinetic analysis of inhibition and recovery of quantum yield under moderate UV+PAR (part of time course shown in Fig. 2 c). a) General form of the function used to fit inhibition kinetics, with arrows indicating the definition of the three yield levels and delay time in Eq. (1), b) Measurements and fitted inhibition curve for a 30 min moderate high UV+PAR exposure and c) Measurements and fit for subsequent recovery of quantum yield in moderate PAR alone.

DISCUSSION The PSII activity of Synechococcus WH8102 cells, either measured through effective quantum yield of photosynthesis (ΦPSII) or Eo, declined exponentially as soon as they were exposed to moderate UVR irradiance. Most studies focus on the photosynthetic damage by UV-B in Synechococcus sp. (7,35–37). Notably, our moderate irradiance treatment included mainly UV-A radiation in combination with PAR and exposure to UV-B was very limited, which is representative of average spectral exposure in the mixed layer (Fig. 1) (19). This irradiance treatment is consistent with the major role that UV-A has been shown to have in the inhibition of aquatic photosynthesis in general (reviewed by 38). Moreover, there are few studies of the photosynthetic response of Synechococcus to UV-A (39), or UV-A dominated solar radiation (22), which indicate that UV-A at natural levels is also a potent inhibitor of photosynthesis. More specifically for Synechococcus, Biological Weighting Functions (BWFs) for the spectral dependence of UV inhibition have been defined in a related study (40). When these are applied to a typical irradiance spectra for the near-surface oligotrophic ocean (cf. Fig. 1), the predictions confirm that UV-A makes the greatest overall

contribution to inhibition of the photosynthesis in Synechococcus (both WH8102 and WH7803 strains). The time scale of UV inhibition of photosynthesis is also important information considering that the light experienced by Synechococcus in the surface layer can fluctuate over broad timescales—from minutes to hours (4). While there are a few previous studies of the time dependent decreases in PSII activity of Synechococcus under light stress, these have not concerned the short-term response to UV. Six et al. (7) studied the short-term response to a 10-fold increase in PAR for several picoplanktonic cyanobacteria, with a 15 min sampling interval. Unlike our results, PSII quantum yield of WH8102 decreased continuously during a 90-min PAR exposure with no indication of an approach to steady state. Their experimental protocol employed a full step change from low to high PAR without intermediate steps to allow induction of NPQ. Garczarek et al. (11) also showed progressive declines in PSII function of Synechococcus WH7803 during a 5-h UV+PAR exposure (0.5–1 h sample interval), even for high PAR (350 lmol m
2 s
1) acclimated cultures. Their exposures included relatively more UV-B than other studies. PSII quantum yield sampled at 3 h intervals was also inversely related to diel light exposure in Synechococcus

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Glaucia M. Fragoso et al. the residence times of the water may be only few minutes, Synechococcus WH8102 responds to the full range of irradiance in the mixed layer, even with activated repair processes and photoprotective mechanisms. Kinetics of inhibition and recovery

Figure 4. Rates of gross oxygen evolution (black circle) and uptake (empty circle) and net oxygen evolution (gray circle) as a function of irradiance in Synechococcus sp. WH8102. Arrow and gray line indicates the decline of the rate of oxygen uptake (Uo = 495 mol O2 mol chl
1 h
1) when irradiance was abruptly lowered to 82 lmol photons m2 s
1 right after the measurement of oxygen exchange at the maximum irradiance (1705 lmol photons m2 s
1).

WH7803 (17). They observed no additional negative effects of UV+PAR compared to PAR alone when cultures were grown under continuous (but diel varying) PAR+UV (mainly UV-A). The methodology of all these previous studies differed from this study in that they measured the maximum quantum yield after a short (5–10 min) period of postexposure dark adaptation. This measurement mainly reflects the degree of NPQ and PSII reaction center damage (cf. “spikes” in Fig. 2). In contrast, we have measured the effective PSII quantum yield which is more representative of electron transport under actinic exposure, and we preacclimated samples to PAR to ensure full induction of NPQ before commencing UV+PAR exposure. This enabled the first demonstration of an immediate effect of the simulated solar UV radiation representative of an oligotrophic mixed layer in inducing photosynthetic inhibition in Synechococcus WH8102. Moreover, the high time resolution of our measurements documented that a steady state was attained under each exposure condition (discussed more below). The rapid UV-induced inhibition response suggests that, in the context of vertical mixing where

The establishment of a lower asymptote of the ΦPSII in Synechococcus WH8102 indicates that, although loss of photosynthetic activity was significant in both treatments, ongoing repair processes were able to offset damage caused by moderate UV and UV+PAR (27,28,41,42). Loss of photosynthetic activity or rate of inhibition (rinh) was significantly faster when cells were exposed to moderate UV+PAR treatment (0.41 min
1) than UV (0.18 min
1) or PAR exposure (0.16 min
1). One possible explanation of different inhibition kinetics is that UV+PAR affects more targets in cyanobacteria than UV or PAR alone, including both D1 and D2 protein subunits of the PSII reaction center (35) and other independent targets of the PSII complex (37). More importantly, repair and recovery time were also faster under UV+PAR when they were returned to moderate PAR exposure as compared to recovery in the UV treatment. Although low to moderate PAR levels have been suggested to stimulate PSII activity repair in Synechocystis PCC 6803 cells exposed simultaneously to UV (35,37,43), this is the first time that a rebound effect of the ΦPSII has been documented in Synechococcus WH8102 under concomitant exposure of UVR and PAR. A similar response (though less pronounced and slower) was observed by Sobrino and Neale (44), where the ΦPSII of the diatom T. pseudonana slightly increased after 25 min of photoinhibitory UV exposure. The rebound of the ΦPSII has been suggested to be an initial phase of acclimation to UV; nonetheless, the underlying mechanisms involved in photoprotection remains elusive. Induction of increased photoprotection under moderate UV+PAR exposure was also evident in our companion study to model the spectral dependence of UV effects on photosynthesis (14C incorporation) in Synechococcus WH8102 (40). A combined BWF/Photosynthesis–Irradiance model for Synechococcus was defined in which one parameter is a measure of repair capacity (Emax). It was observed that a 1 h pre-exposure of the culture to moderate UV+PAR (versus remaining at growth PAR) more than doubled the Emax estimated from photosynthesis subsequently measured under a range of UV+PAR treatments. The increase in Emax is in the same range as rebound associated increase in quantum yield reported here.

Table 2. Summary of parameter estimates for nonlinear regression fits of the photosynthesis measurements (gross and net Eo and carbon fixation; n = 2) to a photosynthesis–irradiance curve and the photosynthetic quotient ratio (ratio of the maximum rate of net Eo to carbon assimilation) in Synechococcus sp. WH8102. See methods for definition of curve-fit parameters. The realized maximum is the curve-fit-based estimate of the actual rate of oxygen exchange at light saturation. NA = Not applicable. Gross O2 evolution

Dark rate of gas exchange (molO2/CO2 chl
1 h
1) Maximum rate of gas exchange (molO2/CO2 chl
1 h
1) Saturating Irradiance (lmol m
2 s
1) Realized Maximum (PsB-R) (molO2/CO2 chl
1 h
1) R2

Net O2 evolution

Carbon fixation

Value

Error

Value

Error

Value

Error

14 2757 205.4 2771 0.99

84 124


359 1899 147.4 1540 0.99

68 93


23 844 122.7 821 0.97

30 31 10.8

NA

NA

NA

Photosynthetic quotient ratio 2.25

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Figure 5. A comparison of photosynthetic rates as a function of PAR irradiance using fitted photosynthesis–irradiance curves for gross (Gross Eo, black circle) and net oxygen evolution (Net Eo, gray circle) and carbon assimilation (triangle). Rates are averages (n = 2).

Some possible explanations for the rebound of the quantum yield are that (1) rate of ΦPSII damage decreased, (2) the rate of ΦPSII repair increased and (3) excitation pressure on PSII decreased. These possible mechanisms are not mutually exclusive. The excitation pressure on PSII could arise from the activity of a ptox-like enzyme that provided an additional sink for reductant from PSII. Regardless of whether a ptox or ptox-like oxidase was active in Synechococcus, Uo was the same as dark respiration under UV+PAR exposure, which is inconsistent with an enhanced oxidase activity as a response to UV stress (see further discussion). Therefore, it becomes highly unlikely that the rebound effect of the ΦPSII in Synechococcus WH8102 is related to any oxygen-dependent mechanism of photoprotection, including ptox. Instead, the rebound effect may be related to the increased repair resulting from the shifts in the expression of the multiple PSII-D1 genes (psbA) found in cyanobacteria under moderate UVR (45). Synechococcus strains possess three to six copies of these genes. The products of which (D1:1 and D1:2 isoforms) appear to protect the PSII reaction center complex under UV-B and low PAR through rapid turnover of the D1:1 isoform prevalent under low PAR conditions and replacement with the more damage-resistant, D1:2 protein isoform (11,45). Accumulations of the gene transcripts for resistant D1 isoforms (psbAII and psbAIII) were found within 15 min of moderate UVB (0.4 W/m2) and low PAR exposure (50 lmol photons m2 s
1) (45). Therefore, it is possible that this more resistant D1 protein form became active within the first 10 min of UV + PAR exposure, which might have resulted in the rebound effect observed for ΦPSII. More recently, Berg et al. (46) reported an increased expression of D1:2 in Synechococcus WH8102 under high PAR, although cells were not exposed to UV. Mella-Flores et al. (17) observed an UV-induced increase in the daily PSII repair rate in Synechococcus WH7803 on simulated midday light levels, as a result of a potential replacement of the D1:1 by D1:2 isoform. While the integration of a more resistant D1 into the Synechococcus PSII pool would explain the increase in the operating yield, we cannot exclude that other mechanisms that decrease

Figure 6. Time course of variation in the rates of oxygen a) gross, b) net evolution (square) and c) uptake (triangle) of Synechococcus sp. WH8102 under moderate PAR (black, 500 lmol photons m2 s
1) or UV+PAR exposure (gray, 27 W m
2 and 500 lmol photons m2 s
1, respectively). The repeated symbols and shading represent duplicate runs. The dashed line in c) indicates average dark respiration rates (n = 4).

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Glaucia M. Fragoso et al.

damage or increase repair might have also been involved in the occurrence of this rebound effect. In contrast, no rebound effect occurred under UV exposure which only received PAR from the measuring beam. So a combination of UV and at least low PAR (50 lmol m
2 s
1 in the study of Campbell et al. (45)) may be needed to trigger the shift in psbA gene expression, if these are related. The slower inhibition rate suggests that the inherent rate of damage to PSII was slow under these conditions; nevertheless, a significant reduction in yield still occurred mainly because PSII repair was also slow under the 14 lmol m
2 s
1 of PAR from the measuring beam. Oxygen uptake and photoprotection mechanisms under high PAR It has been suggested that Synechococcus WH8102 may have evolved to express an oxidase-dependent enzyme that acts as an electron valve upstream of PSI, which keeps PSII photochemically active at high PAR despite the lower levels of PSI compared to PSII (3,5). Indirect evidence of this process was observed in an open ocean picophytoplankton assemblage from the Atlantic, where a disparity between the irradiance saturation values of PSII electron flow (1985 lmol photons m
2s
1) and CO2 assimilation (131 lmol photons m
2s
1) was observed (12). In our study, CO2 assimilation started to saturate at lower irradiances (123 lmol photons m2 s
1) than PSII electron transport (147 and 205 lmol photons m2 s
1 for net and gross Eo) in Synechococcus WH8102. The disparity between these saturation values is consistent with the results of Mackey et al. (12) and their hypothesis of an electron sink between PSII and PSI. In our study, however, this disparity was smaller than reported by Mackey et al. (12), suggesting that such an electron sink is less active in Synechococcus WH8102 in culture than in open ocean picophytoplankton. Our results also showed that as PAR increases above saturation for carbon assimilation, further increases in linear electron transport occurred coupled to increased O2 uptake. This process was reversible, suggesting that oxygen may be involved in photoprotection mechanisms in Synechococcus WH8102 under high PAR. However, it is uncertain if this is due to an oxidase, such as ptox or ptox-like enzyme that might be involved in those processes. PGAL has been considered an inhibitor specifically for ptox and been widely used in experiments to test the activity of ptox or ptox-like enzymes in Synechococcus (5) and other algae (12,47). Nonetheless, our results show that, under same concentrations, PGAL has a broad toxic effect and is unsuitable for a definitive test for ptox activity in phytoplankton. Moreover, the results of Berg et al. (46) showed low rates of ptox expression, implying that the activity of ptox should be limited in Synechococcus WH8102. Oxygen-dependent photoprotective mechanisms that act as an alternate sink of electrons in the photosynthetic electron transport chain, such as the Mehler reaction (also known as the water–water cycle) have already been reported to occur in another strain of Synechococcus WH7803 (32) and may also be present on WH8102. Although we cannot definitively discriminate between Mehler reaction versus oxidase activity as accounting for the PAR-stimulated Uo in Synechococcus WH8102, our result support evidence that an oxygen-dependent mechanism of photoprotection is active and may act as an alternative sink of electrons under high PAR.

Oxygen uptake, evolution and carbon fixation as a function of UV exposure Although it is clear that Uo may provide additional photoprotection in Synechococcus WH8102 when cells are exposed to high PAR, Uo levels were not greater than dark respiration when cells were exposed to moderate levels of UVR and PAR. Similar results were observed by Heraud and Beardall (48) where they found that UVR had no effect on oxygen consumption as a demand of repair in Phaeodactylum tricornutum, Dunaliella tertiolecta and Wolozynskia sp. The gross Eo time course did not show a ~7 min “rebound” effect as observed for PSII quantum yield, possibly due to coarser time resolution in the MIMSderived rate data when Synechococcus WH8102 were exposed to UV+PAR. Nonetheless, both parameters showed a similar decline, showing that UVR combined with PAR caused a considerable impairment to the PSII activity. Berg et al. (46) do not relate inhibition of maximum PSII quantum yield (Fv/Fm) in WH8102 with PSII damage under high PAR, instead, they relate it to an increase in phycobilisome fluorescence, which boosts dark adapted fluorescence yield (F0) and consequently reduces Fv/Fm. However, in our case, it is evident that high UV+PAR caused inhibition of PSII activity, given that light-saturated gross and net Eo rates were also reduced. The absence of Uo (greater than dark respiration) in Synechococcus WH8102 when exposed to UV+PAR suggests that either (1) enzymes involved in Uo, i.e. ptox or ptox-like, or any other oxygen-dependent mechanism of photoprotection are insensitive to moderate UV or (2) part of the UV-induced effect is on PSII function, thus a “safety valve” is not needed because less reductant goes through PSII. In any case, it is clear that Uo is not an important strategy of photoprotection for Synechococcus WH8102 under moderate UV+PAR as opposed to PAR. Inhibition of CO2 fixation was greater under UV+PAR (relative to the PAR only control) than PSII noncyclic electron transport, represented as net Eo under the same exposure. Thus, UV appears to have a greater effect on carbon assimilation than PSII. It remains unclear what mechanism(s) account for this difference, as many components of the photosynthetic apparatus are sensitive to UV damage (49). The larger response of CO2 fixation could be an indication of primary effects of UV exposure on the activity of ribulose 1,5-biphosphate carboxylase (Rubisco), the main CO2 -fixing enzyme in C3 phototrophs. UV-B has been demonstrated to directly damage Rubisco from higher plants through a cross-linking mechanism (50), but it remains unknown if a similar response occurs in Synechococcus. Clearly, more work is needed to understand how UV exposure affects the interaction between the “light” and “dark” reactions. Our results do advance the application of models of photosynthetic response to UV to assess the impact of solar UV on primary productivity in the ocean. As Synechococcus rapidly attains a balance between damage and repair processes under UV+PAR exposures, photosynthetic response can be modeled as a function of weighted irradiance (c.f. 28). To support such a model, BWFs for UV inhibition of photosynthesis in Synechococcus have been developed and will be presented in a separate report (40). The present work helps to validate the predictions of a coupled BWF and photosynthesis–irradiance curve model (BWF/P-E). Based on the measured irradiance spectra for the UV+PAR treatment used in this study and independently estimated BWFs (data not

Photochemistry and Photobiology, 2014, 90 shown), the model predicts a decrease in carbon fixation of 49  5% for WH8102 grown at medium light and 26°C compared to the observed 53% decrease. Future work will apply these models to predict the impact of UV on primary productivity of Synechococcus in the ocean given present day and altered atmospheric conditions. Acknowledgements—This research was supported by NASA grant NNX09AM85G to Brian Thomas, Patrick J. Neale and Adrian Melott and NSF grant OCE- 0727488 to Todd Kana. The authors thank Erica Kiss (UMCES, Horn Point Laboratory), who assisted with the MIMS experiments and Emily Roberts for constructive comments and criticisms throughout the development of this manuscript.

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