1. ABSTRACT: Phytoplankton production in the Southern Ocean is controlled by complicated interactions of light, nutrients, and iron availability. In early 2002, the Southern Ocean Iron Experiment (SOFeX), was completed in the Southern Pacific (along ~ 170º W). Two iron enriched patches were created North and South of the Polar Front with initially distinct silicic acid concentrations, ~ 1 µM and ~ 60 µM, respectively. Pulse amplitude modulated (PAM) fluorometry was employed for measuring phytoplankton electron transport chain between Photosystem II and Photosystem I for whole water (bulk) and single-celled photochemical efficiency of Photosystem II (F v /F m ). Bulk measurements of photochemical efficiency for both PAM show increases as phytoplankton from iron- enriched patches are relieved from iron stress. For single-celled analysis with PAM, a time course from the South Patch will be presented along with late North Patch data. For the Southern Patch, a distinct increase in F v /F m was detected for all diatom genera analyzed. For late Northern Patch quantum yield, the dominant species did not exhibit a high photochemical efficiency. A Study of Phytoplankton Photochemical Response During the Southern Ocean Iron Experiment Using Pulse Amplitude Modulated (PAM) Fluorometry Jill A. Peloquin* and Walker O. Smith, Jr. College of William and Mary, Virginia Institute of Marine Science, Gloucester Point, VA, *Contact: jillp@vims.edu INTRODUCTION: Phytoplankton production in the Southern Ocean is controlled by complicated interactions of light, nutrients, and iron availability. In early 2002, the Southern Ocean Iron Experiment (SOFeX), was completed in the Southern Pacific (along ~ 170º W). Along approximately 170  W, there is a strong silicic acid gradient (2 to 60 µM) from 55° S to 65° S (Mengelt et al. 2001). Thus, north of the Polar Front (PF) flagellates and prymnesiophytes dominate and south of the PF, a mixed assemblage of diatoms is historically present (Mengelt et al. 2001). Two iron enriched patches were created north and south of the PF (Figure 1) with initially distinct silicic acid concentrations, ~ 1 µM and ~ 60 µM, respectively. To maintain high (ca. 1 nM) concentrations of iron in the surface waters, iron was deployed 3-4 times, several days apart. Variable fluorescence is a powerful tool to assess the photochemical state of phytoplankton cells. A photon entering a cell has one of three fates: it can drive photochemistry, be remitted as fluorescence, or emitted as heat. Thus, by understanding fluorescence characteristics, one can gain insight to photochemistry. Pulse amplitude modulated (PAM) fluorometry was employed for measuring phytoplankton electron transport chain between Photosystem II and Photosystem I for whole water (bulk) and single-celled photochemical efficiency of Photosystem II (F v /F m ). Our primary objective was to, for the bulk phytoplankton assemblage: 1. Measure changes in photochemical yield with iron enrichment. 2. Use fluorescence-based rapid light curves (1 min) to estimate fluorescence signature versus irradiance (light harvesting potential) for phytoplankton cells over the course of SOFeX. We then modeled the curves and compared them to determine whether or not these experiments are a sensitive indicator of iron stress. Our primary objective was to, for phytoplankton single cells: 1. Measure genera specific photochemical yield with iron enrichment. 2. Model the iron enrichment response in order to intercompare compare genus-specific responses. Figure 2. A. Time course of cooling block for microscopy-based PAM on iron deplete cells. Green bar represents the time at which water bath was turned off. Samples were never kept on block more than 1 hour for experimental sampling. B. Surface sample taken at approximately 9:00 am LT, from 30 m on December 7 th, 2001 during SOFeX. Bulk measurement of phytoplankton was allowed to low light adapt (10 µmol m -2 sec -1 ) on ice and re- measured at set time intervals. This figure clearly shows a marked increase with time for the quantum yield of photochemistry. Since SOFeX samples were taken at all times during the course of the day, samples were allowed to low light adapt for 40 minutes before measurement. This removed most effects of photoinhibition on the photosystem for comparable cells. Figure 3. Whole water measurements of the quantum yield vs. the time elapsed since the onset of iron enrichment for SOFeX for the south patch. Bars indicate the approximate time where iron additions began. Circled data points are out-stations. The bulk measurements of quantum yield of photochemistry clearly increase with time and iron enrichment. Measurements approached maximum physiological quantum yield (0.70), but never reached it for 20 m water over the course of the experiment for the Southern Patch. METHODS: Samples were taken from the CTD rosette (20 m) and directly put on ice. Two instruments were utilized, a submersible PAM used bench-top (for analysis of the bulk assemblage) and a microscopy-based PAM (for single celled analysis). For the microscopy-PAM, the stage was adapted to keep samples cool (Figure 2A).All samples were low-light adapted (Figure 2B) before measurement. To estimate electron transport rate, yields were determined according to an internally programmed light curve routine. Quantum yields are determined at each light level and then normalized to chlorophyll specific light absorption, light level, and 0.5 (for light partitioning between PSI and PSII). These responses are then modeled according to the Platt et al. (1980) equation as follows adapted for fluorescence: Figure 1. Location of iron enriched patches RESULTS/DISCUSSION: The majority of results presented are specific to the south patch (south of the PF; Si replete). Analysis of this patch was more effective due to time spent on it and the fact that the species present were larger and non-motile. Thus, the microscopy-based PAM was effectively used for this patch. Since the north patch was dominated by small flagellates in iron deplete conditions, a true OUT station or time 0 was impossible to measure to compare IN stations with. 1. For the south (Figure 3) and north patch (data not shown), the bulk phytoplankton photochemical yield increased with time since the first iron enrichment as determined with PAM. OUT stations increased slightly with time. Cells never reached physiological maximum (0.70 for PAM). It is unclear why they never reached the physiological maximum; it is possible that there were confounding complications between light and iron (Sunda and Huntsman 1997) or that this depth was not absolutely released from iron depletion. The north patch shows similar trends (data not shown) although there are fewer stations. RESULTS/DISCUSSION (cont.) 2.All rapid light curves were fitted and the parameters for IN stations were plotted with time in the iron enrichment experiment (Figure 4). At this time, the estimates of I k and F max show no clear trend (data not shown). However, the slope of the curve (alpha) increases with time. It is unclear at this time whether these type of measurements may absolutely be appropriate as an indicator of iron depletion. One complication is that the phytoplankton assemblage is shifting to larger species with inherently different light harvesting and fluorescence characteristics. Another parameter to consider is the 0.5 coefficient as static partitioning of light between PSI and PSII. It is well established that in iron limiting conditions, this ratio is dynamic. The utilized model is not able to take this fluctuation accurately into consideration. More detialed work will have to be completed for these data. Clearly, the next step will be to compare these results with carbon-based production versus irradiance experiments. 3.Quantum yields from single cells (south patch) were binned into genus-specific responses. An example of a typical response is shown in Figure 5. All quantum yields for cells followed throughout the course of the experiment increased with time. An interesting feature of these measurements is the extremely low measurements (  0.2 - 0) for iron deplete cells. Low measurements were consistently found for all diatom genera, but these may be slightly lower than for true maximum/potential F v /F m due to microscopy techniques (finding cells with low light). 4.The single-celled responses were fit with a modified Platt et al. (1980) model to ascertain the fluorescence maximum and a time (specific to the SOFeX experiment) saturation parameter (Table 1). Cells with lower time values or high F values may be inherently more efficiently at responding to rapid increases in in situ iron concentrations (Figure 6). It is important to note that since these time values have not yet been converted to iron concentrations, these results only describe the response measured with time (and not iron) for this specific experiment. An additional time offset will have to be included in the model, at present model parameters for time are relative. An interesting comparison will be with genera-specific biomass to see if these photochemical signatures translate into phytoplankton biomass accumulation. 5.We were able to measure single cells from the IN stations for the return to the North patch when pennate diatoms dominated the patch (Figure 7). The dominate species of Pseudonitzia sp. exhibited a low F v /F m compared to the bulk assemblage, whereas Phaeocystis sp. tended to show higher values of photochemical efficiency. This suggests that the Pseudonitzia sp. was entering a senescent phase of growth (driven by Si depletion?). ACKWOLEDGEMENTS: Funding provided by NSF to the Southern Ocean Iron Experiment. Thanks to Chief Scientist Ken Johnson and the Captain and crew of the R/V Revelle. I am indebted to Scott Polk who kindly offered to present this poster so that I could return to the Antarctic in a timely fashion. Thanks also to fellow SOFeX scientists for engaging conversations and project feedback. Table 1. Complete table of genus-specific model values. Figure 7. Quantum yields of photochemistry upon return to Northern patch. Error bars are standard errors. WORKS CITED: Mengelt, C. M Abott, J. Barth, R. Letelier, C. Measures, S. Vink. 2001. Phytoplankton pigment distribution in relation to silicic acid, iron and the physical structure across the Antarctic Polar Front, 170  W, during austral summer. Deep-Sea Research II. 48: 4081-4100. Platt, T, CL Gallegos, WG Harrison 1980. Photoinhibition of photosynthesis in natural assemblages of marine phytoplankton. Journal of Marine Research. 38:687-701. Sunda, WG and SA Huntsman. 1997. Interrelated influence of iron, light and cell size on marine phytoplankton growth. Nature. 390: 389-392. Figure 4. The response of alpha (the slope of the increase in fluorescence versus irradiance experiment) for the Southern (above) and Northern patch. Alpha tends to increase with time, likely due to the physiological change in the photosystem or in the phytoplankton assemblage. Figure 5 (left). Response of single cells. Error bars represent standard errors for each time point. Circled points are OUT stations (control). Figure 6 (right). Above, the maximum quantum yield over the course of measurement. Below, the saturating time parameter for fluorescence increase. Centric diatoms 90 0 10 20 30 40 50 60 70 80 Pseudonitchizia sp. Fragilariopsis sp. Asteromphalus sp. Corethron sp. Chaetoceros sp. Bulk assemblage 0 0.1 0.2 0.3 0.4 0.5 0.6 Asteromphalus sp. Centric diatoms Pseudonitchizia sp. Fragilariopsis sp. Rhizoslenia sp. Chaetoceros sp. Corethron sp. Bulk assemblage Time (hours) F v /F m B A Time (hrs) Time (min) F = F s [1 – exp(  E/F s )] exp (-  F s ) F m = F s [   E k = F m /  Parameters important to this study are F m, E k and . F m is the maximum fluorescence level,  (dimensionless) describes the initial slope of the curve, and E k (  mol m -2 s -1 ) is the light saturation parameter. Single cells were measured, quantum yields were determined and binned into genus-specific responses. This response was then fit with an adapted Platt et al. (1980) to solve for the maximum F m, E k and . B