The Physiological Responses Of Marine Synechococcus Strains To Iron Limitation

Iron supply limits primary production in the ocean, especially HNLC (high-nutrient low-chlorophyll) regions whose area is one-third of the world’s ocean. The important role of iron in oceanic productivity, biogeochemical cycle, and global climate can be seen from many iron enrichment experiments in HNLC regions since1988. Synechococcus species are ubiquitous and abundant in major oceanic regimes, underlying their ecological importance as significant contributors to the total photosynthetic biomass in the ocean. They also play a key role in pelagic food-web structure via energy transfer within the microbial loop, It has been estimated that35%-100%of the Synechococcus standing stock can be grazed per day. Picophytoplankton such as Synechococcus had previously been assumed not to be strongly limited by iron in HNLC regions because only the diatoms bloomed by escaping grazing pressure upon iron enrichment. But there were also some evidences indicated that Synechococcus also iron limited in the oceans. The physiolgical and biochemical response to iron limitation has been well studied in diatoms and other marine eukaryotic algae, but there are much less physiological information about ecological important Synechococcus.Iron plays a catalytic role in many biochemical reactions as a cofactor of enzymes and proteins involved in photosynthetic and respiratory electron transports, nitrogen assimilation and many other metabolic processes. But the majority of intracellular iron is required in the photosynthetic apparatus. Iron concentrations in coastal waters are higher than those in open ocean waters. Selection pressure imposed by iron limitation has resulted in oceanic eukaryotic diatoms less susceptible to iron limitation than coastal species. More iron was predicted to be required by using nitrate as the nitrogen source compared to ammonium. We did not know whether the oceanic Synechococcus strains were more tolerant to iron limitation than the coastal strains. Furthermore, there are conflicting data over whether ammonium, rather than nitrate, supports a higher growth rate under iron limited conditions. Iron concentrations are depleted in the surface ocean but light intensity decreased with depth in the ocean. According to the prediction as well as some data on diatoms, cellular iron demand enhanced under low irradiation but the physiological consequences of Fe limitation would increase susceptibility to PSII photoinhibition at high irradiance. Thus further studies need to be done about the interactive influences of iron and light on the growth and photosynthesis of green and red coastal synechococcus strains. Synechococcus sp. PCC7002has been proved to release extracellular Fe3+chelating agents (siderophore) to cope with iron limitation. It is worth us to explore whether PCC7002possessed a competitive advantage when they mixed-cultured with the strains that do not produce these ligands. In these studies, Synechococcus strains were grown in Aquil medium that was developed specifically for studying trace-metal physiology in algae, and the Metal Clean technique will be used to rigorously control the trace metal contaminations all through our experiments. The F0value measured by Water-PAM fluorometer was used to monitor the growth of Synechococcus. Water-PAM and FIRe fluorescence techniques as well as Flow cytometry technique were used to study the physiolgical response of different Synechococcus strains to iron limitation. The species-specific differences in physiology responses to iron limitation and the effects of iron in untilization of nitrogen and light in marine Synechococcus were explored. The results could help us to well evaluate the effects of iron limitation on primary productivity of the picophytoplankton, and to open out the effects of iron on the phytoplankton community composition of the ocean ecosystem. These results also provide us some bases to further research on biochemical and molecular mechanisms to cope with iron limitation in different strains. The mail results are as follows:1. The minimal fluorescence yield is a reliable and easy biomass measurement of picophytoplankton Synechococcus under semicontinuous batch culture. Two coastal Synechococcus strains (PCC7002and CC9311) and one oceanic strain (WH8102) were cultured with4-1000nM Fe in Aquil medium. The cell concentration and minimal fluorescence yield (F0) were measured daily by Flow cytometry and Water-PAM fluorometer in the exponential growth stage, and the growth rates obtained from these two methods showed little difference. Compared with those under iron-replete condition, their growth rates were significantly decreased by59%for WH8102at15nM Fe, by37%for CC9311at15nM Fe and by57%for PCC7002at4nM Fe. Among these three strains, PCC7002was the most tolerant to iron limitation while WH8102was the most sensitive to iron limitation. The linear correlation was established between F0value and cell concentration although Fo value per cell varied depending of the strains and iron levels. Under iron-replete condition, the minimal fluorescence yield per cell was100-fold higher for phycoerythrin-lacking strain PCC7002than two phycoerythrin-containing strains WH8102and CC9311. Under iron-deplete condition, it was increased respectively by128%and7%for WH8102and CC9311but was decreased by30%for PCC7002. This is mainly related to differences in the pigment composition of phycobilisomes of the various strains and to different effect of iron limitation on photosynthetic pigment contents. Furthermore, F0value per cell concentration for PCC7002and CC9311showed little difference throughout the light and dark diel cycle. However, it was significantly higher for WH8102in the daytime than in the dark. In a word, it is a reliable and easy method to assay the specific growth rate by measuring F0value at the steady state of cultures. 2. Different responses of photosynthesis and flow cytometric signals to iron limitation and nitrogen source in coastal and oceanic Synechococcus strains (Cyanophyceae). This study have been compared the photosynthesis and flow cytometric signals of four Synechococcus strains grown under different iron concentrations with either nitrate or ammonium as the sole nitrogen source. Two oceanic strains were much more sensitive to iron limitation than two coastal strains. The inhibition of iron limitation on the growth, maximal PSII photochemical yield, maximal rate of relative electron transport and photochemical quenching of the two oceanic strains was higher than for their coastal counterparts. Under iron limitation condition, the connectivity factor between individual photosynthetic units (p) increased for the two coastal strains while decreased for the two oceanic strains. Furthermore, iron limitation accelerated the QA re-oxidation of the two oceanic strains and the PQ pool re-oxidation of the two coastal strains. Under iron limitation condition, the cell size of the two coastal strains and intracellular pigment concentrations of the two oceanic strains decreased while the side light scatter/front light scatter (SS/FS) ratio of the two coastal strains increased. In contrast to iron limitation, nitrogen source only marginally affected the photosynthesis of the four Synechococcus strains. Ammonium enhanced the growth of the two coastal strains under iron-replete condition. For the two oceanic strains, ammonium increased their cell size and decreased their SS/FS ratio and intracellular pigment concentrations under iron-deplete and iron-replete conditions. Previously studies indicated that oceanic eukaryotic diatoms were less susceptible to iron limitation than coastal species. These were opposite to our results in Synechococcus. Further works need to be done to understand the biochemical mechanisms responsible for these physiological responses to iron limitation between oceanic and coastal Synechococcus strains.3. Different responses of growth, photosynthesis and flow cytometric pigments fluoresence to iron and light in one green and one red coastal synechococcus strains (Cyanophyceae). One green (PCC7002) and one red (CC9311) coastal synechococcus strains were cultured under different iron(10and1000nM) and light conditions (10and60μmol photons·m-1·s-1). The results indicated that PCC7002was more sensitive to iron limitation under low light while CC9311was more easily to suffer from photoinhibition under iron limitation. Anymore, CC9311was more sensitive to iron limitation than PCC7002both under high light and low light. The flow cytometric chl a, PE and PC fluorescence of the two strains decreased under iron limitation and high light conditions. Decrease in aPSII for PCC7002cultured under iron and light colimitation and for CC9311under iron limitation and photoinhibition conditions might resulted from decrease in the amount of pigments serving one RCII. Under low iron and low light conditions, the linear electron transport rate (rETRmax) decreased but PQ pool re-oxidation(1/τPQ) accelerated for PCC7002which might result from the stimulation of cyclic electron transport around PSI. Under low iron and high light conditions, the rETRmax decreased but Qa re-oxidation and PQ pool re-oxidation (1/τPQ) accelerated for CC93311which might result from the stimulation of cyclic electron transport around PSI and PSII respectively. Samples of PCC7002and CC9311cultured under different light and iron conditions were illuminated at300μmol photons-m·2-s·1for30min and then transferred to10μmol photons·m-2·s-1for60min for recovery. The Fv/Fm values were measured after high light treatment and low light recovery. The results indicated that CC9311was more susceptible to high light exposure and had lower capacity to recover from this photoinhibitory stress compared to PCC7002. Anymore, cells cultured at low light and low iron conditions were more easily to suffer from photoinhibition and had lower capacity to recover from photoinhibition. These might result from their lower NPQ (nonphotochemical quenching). Our results suggested that iron and light interaction might be important for vertical separation of red and green strains of synechococcus.4. Physiology response to iron limitation and effects of iron on the competition of PCC7002between WH8102or CC9311. PCC7002which could produce siderophore involved in iron sequestration under iron limitation conditions was predicted to own competitive advantage. In monoculture, the cell yieds and relative growth rate of WH8102and CC9311was inhibited more than PCC7002under iron limitation. Anymore, celluar ROS content and percentages of dead cells of WH8102and CC9311were also effected more by iron limitation. This results indicated that PCC7002was much more tolerant to iron limitation than WH8102and CC9311. Front light scatter (FS, related to cell size) of CC9311and WH8102increased significantly whereas the FS of PCC7002decreased significantly under iron limitation. Iron limitation effected cell cycle behavior and decreased DNA content of the three strains. The cell-cycle behavior of CC9311and WH8102were different from PCC7002, which corresponds to the slow and fast growth case of cell-cycle model respectively. Percentages of cells in G1phase decreased under low iron for WH8102and CC9311while PCC7002presented a broad and narrow unimodal under iron replete and deplete conditions respectively. Compared to monoculture, cell yieds, relative growth rate, Chl a fluorescence, FS and DNA content of PCC7002were decreased while celluar ROS content and percentages of dead cells increased in mixed-culture under iron limitation. However, there were no significant differences between the physiological characteristics for PCC7002in monoculture and mixed-cultured under iron replete conditions, and so is the physiological characteristics of WH8102or CC9311both under iron deplete and replete conditions. We concluded that cells of PCC7002in mixed-culture suffered from more heavily iron stress than it in monoculture under iron limitation conditions.