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Copper (Cu) concentration is greatly reduced in the open sea so that phytoplankton must adjust their uptake systems and acclimate to sustain growth. Acclimation to low Cu involves changes to the photosynthetic apparatus and specific biochemical reactions that use Cu, but little is known how Cu affects cellular metabolic networks. Here we report results of whole transcriptome analysis of a plastocyanin-containing diatom, Thalassiosira oceanica 1005, during its initial stages of acclimation and after long-term adaptation in Cu-deficient seawater. Gene expression profiles, used to identify Cu-regulated metabolic pathways, show down-regulation of anabolic and energy-yielding reactions in Cu-limited cells. These include the light reactions of photosynthesis, carbon fixation, nitrogen assimilation and glycolysis. Reduction of these pathways is consistent with reduced growth requirements for C and N caused by slower rates of photosynthetic electron transport. Up-regulation of oxidative stress defense systems persists in adapted cells, suggesting cellular damage by increased reactive oxygen species (ROS) occurs even after acclimation. Copper deficiency also alters fatty acid metabolism, possibly in response to an increase in lipid peroxidation and membrane damage driven by ROS. During the initial stages of Cu-limitation the majority of differentially regulated genes are associated with photosynthetic metabolism, highlighting the chloroplast as the primary target of low Cu availability. The results provide insights to the mechanisms of acclimation and adaptation of T. oceanica to Cu deficiency. This article is protected by copyright. All rights reserved.

Uptake of Cu by

Copper (Cu) is an essential cofactor of photosynthetic and respiratory redox proteins in phytoplankton and a scarce resource in parts of the open sea. Although its importance for growth is well recognized, the molecular mechanisms by which phytoplankton respond and acclimate to Cu deficiency are not well known. In this study, we identified the dominant Cu-regulated proteins and measured key physiological traits of Thalassiosira oceanica (CCMP 1005) under Cu-limiting and sufficient conditions. Growth limitation of T. oceanica occurred at environmentally relevant Cu concentrations (1 nM) as a result of decreased photosynthetic efficiency (ΦPSII). In Cu-limited cells, levels of plastocyanin decreased by 3-fold compared to Cu-replete cells and rates of maximum photosynthetic electron transport were reduced. Proteins associated with light harvesting complexes also declined in response to Cu limitation, presumably to adjust to reduced photosynthetic electron flow and to avoid photodamage to the photosystems. Key enzymes involved in carbon and nitrogen assimilation were down-regulated in low-Cu cells, as were steady state rates of C and N uptake. Relatively fewer proteins were up-regulated by Cu limitation, but among them were two enzymes involved in fatty acid oxidation (FAO). The increase in FAO may be a sign of increased turnover of cellular lipids caused by damage from oxidative stress. A putative transcription factor containing three, repetitive methionine motifs (MpgMgggM; MpgMggM) increased significantly in Cu-limited cells. The collective results provide a general description of how plastocyanin-dependent diatoms adjust metabolism to cope with chronic Cu deficiency.

Copper concentration is so low in some remote parts of the sea it limits phytoplankton growth, but may be high enough in coastal and estuarine regions to be toxic. Acclimation to variations in Cu concentration thus requires a tightly regulated Cu transport system to help maintain Cu homeostasis. In marine species, the molecular mechanisms of Cu transport are not known. We studied Cu-responsive genes and uptake in Thalassiosira oceanica at environmentally relevant Cu concentrations varying between 0.012 and 12 900 pmol Cu' l-1 . Copper uptake rate assessed at high Cu concentration was three-fold faster in Cu-limited than in Cu-replete cells, confirming the existence of an inducible uptake pathway in this diatom. Four putative CTR-type Cu transporters (ToCTR1, ToCTR2, ToCTR3a and ToCTR3b) identified in the transcriptome shared conserved features with known high-affinity Cu(I) transporters. Expression of the CTR genes was upregulated as Cu concentration declined and cells maintained maximum rates of growth. Further decreases in Cu led to decreased growth rate and increased abundance of ToCTR3a/b. Both ToCTR3a and 3b restored growth of a Cu transport mutant, Saccharomyces cerevisiae ctr1Δctr3Δ, in Cu-deficient medium and increased the uptake rates of Cu(I) and Cu(II). Thus, ToCTR3a/3b is a high-affinity Cu(I) transporter that, in conjunction with the other ToCTRs, may enable T. oceanica to survive in Cu-deplete ocean environments and respond to natural variation in Cu availability.

Thalassiosira oceanica (CCMP 1005) was grown over a range of copper concentrations at saturating and sub-saturating irradiance to test the hypothesis that Cu and light were interacting essential resources. Growth was a hyperbolic function of irradiance in Cu-replete medium (263 fmol Cu′ · L−1) with maximum rates achieved at 200 μmol photons · m−2 · s−1. Lowering the Cu concentration at this irradiance to 30.8 fmol Cu′ · L−1 decreased cellular Cu quota by 7-fold and reduced growth rate by 50%. Copper-deficient cells had significantly slower (P < 0.0001) rates of maximum, relative photosynthetic electron transport (rETRmax) than Cu-sufficient cells, consistent with the role of Cu in photosynthesis in this diatom. In low Cu medium (30.8 fmol Cu′ · L−1), growth rate was best described as a positive, linear function of irradiance and reached the maximum value measured in Cu-replete cells when irradiance increased to 400 μmol photons · m−2 · s−1. Thus, at high light, low Cu concentration was no longer limiting to growth: Cu concentration and light interacted strongly to affect growth rate of T. oceanica (P < 0.0001). Relative ETRmax and Cu quota of cells grown at low Cu also increased at 400 μmol photons · m−2 · s−1 to levels measured in Cu-replete cells. Steady-state uptake rates of Cu-deficient and sufficient cells were light-dependent, suggesting that faster growth of T. oceanica under high light and low Cu was a result of light-stimulated Cu uptake. This article is protected by copyright. All rights reserved.

Cyanobacteria are globally important primary producers and abundant in some iron-limited ocean environments. The ways in which they take up iron are largely unknown, but reduction of Fe3+ is an important step in the process. Here we report a special iron permease in Synechocystis, cFTR1, that is required for Fe3+ uptake following Fe2+ re-oxidation. The expression of cFTR1 is induced by iron starvation, and a mutant lacking the gene is abnormally sensitive to iron starvation. The cFTR1 protein localizes to the plasma-membrane and contains the iron-binding motif “REXXE”. Point-directed mutagenesis of the REXXE motif results in a Fe-transport deficient phenotype. Measurements of iron (55Fe) uptake rate show that cFTR1 takes up Fe3+ but Fe2+. The function of cFTR1 in Synechocystis could be genetically complemented by the iron permease, Ftr1p, of Saccharomyces cerevisiae, that is known to transport Fe3+ produced by the oxidation of Fe2+ by a multi-copper oxidase. Unlike yeast Ftr1p, cyanobacterial cFTR1 probably obtains Fe3+ primarily from the oxidation of Fe2+ by oxygen. Growth assays show that the cFTR1 is required during oxygenic, photoautotrophic growth but not when oxygen production is inhibited during photoheterotrophic growth. In cyanobacteria, iron reduction/re-oxidation uptake pathway represents important adaptations of cyanobacteria in oxygenated environments. This article is protected by copyright. All rights reserved.

Copper concentration is so low in some remote parts of the sea it limits phytoplankton growth, but may be high enough in coastal and estuarine regions to be toxic. Acclimation to variations in Cu concentration thus requires a tightly regulated Cu transport system to help maintain Cu homeostasis. In marine species, the molecular mechanisms of Cu transport are not known. We studied Cu-responsive genes and uptake in Thalassiosira oceanica at environmentally relevant Cu concentrations varying between 0.012 and 12 900 pmol Cu' l

The response of heterotrophic bacteria in a mesoscale iron (Fe) enrichment was measured in the northeast subarctic Pacific Ocean in July 2002. Addition of FeSO4 increased the dissolved Fe concentration in the fertilized patch to 2–3 nmol L−1 and triggered an increase in concentration of Fe(III)-binding ligands that complexed all of the dissolved Fe. Two to three days later, leucine incorporation rate and specific growth rate of bacteria doubled. Physiological markers of bacterial Fe nutritional state varied during the experiment as the microbial community assimilated the added Fe. Cellular uptake rate of an iron-siderophore complex, 55Fe-ferrioxamine B (FB), increased twofold to fourfold over background values and then declined by day 4.5. The fastest rate of Fe-FB uptake on day 2.5 coincided roughly with a transient increase in outer-membrane, 55Fe-FB-binding protein(s) in bacteria and with the peak in ligand concentration. Maximum potential uptake rate of inorganic Fe (Vmax) was 8 zmol Fe bacterium−1 h−1 prior to Fe enrichment and then decreased by a factor of four within 2.5 d of fertilizing the patch as bacteria became Fe sufficient. Vmax gradually increased by day 6.5 as the bacterial community re-entered iron deficiency. Similar changes in growth and Fe uptake kinetics were observed after a second Fe addition. Heterotrophic bacteria in the subarctic Pacific were Fe-deficient and responded directly to Fe addition by up-regulating pathways for Fe-siderophore acquisition and assimilating complexed Fe. The observation that increases in Fe uptake pathways and production were synchronous is consistent with the hypothesis that bacterial growth was directly limited by Fe.

Ice-retreat from the Cape Bathurst Polynya occurred in late May and early June 2004, and was quickly followed by a decrease in dissolved nutrient concentration at the sea sur- face. Concentration plots of NO3 � versus PO4 3� , and Si(OH)4 versus NO3 � showed the ratios of nutrient uptake in the surface layer were in proportions expected for diatom growth (N:P 13.1:1; Si:N 1.8:1) and that water-column NO3 � was depleted before PO4 3� and Si(OH)4. The temporal changes in integrated nitrate, phosphate and silicate concentrations were well described by logis- tic models that showed maximum consumption rates of 11.8, 0.82, and 17.8 mmol m �2 d �1 , respec- tively, and a total seasonal draw-down of 210 ± 19 mmol NO3 � m �2 . If we include estimates of NO3 � supply by advection, nitrification and freshwater dilution, then the amount of NO3 � consumed could be 25�33% higher. Uptake of NO3 � above the 1% isolume was balanced by an equivalent (94%) increase in particulate N over a 15 d period beginning at ice break-up. Thus, the amount of particulate spring new production associated with NO3 � disappearance was estimated to be 16.1 ± 1.5 g C m �2 .

Uptake of 15 N-labelled nitrate, ammonium, and urea was measured over a quasi-annual cycle in the Cape Bathurst Polynya in the Amundsen Gulf and on the Mackenzie Shelf, during the Canadian Arctic Shelf Exchange Study (CASES) in 2003 and 2004. Before the phytoplankton bloom and in autumn, nitrogen uptake was slow, representing less than 5% of annual consumption. Uptake rates increased exponentially after ice retreat and within 3 wk reached a maximum of 38.6 mmol N m ―2 d ―1 . During spring, NO 3 ― uptake supported new production of 166 mmol N m ―2 and f-ratios rose from 0.1―0.2 to 0.6―0.9. Filter fractionation showed that GF/F filters retained 93.1 ± 1.3% of the 15 N incorporated into particulate matter, suggesting that phytoplankton were responsible for the majority of the N uptake. Although free-living bacteria took up relatively more 15 N in autumn and in the lower part of the euphotic zone than phytoplankton, their assimilation of inorganic N had little effect on water column integrated f-ratios or new production. Urea supplied almost half the N assimilated by phytoplankton annually and about 80 % of the regenerated production during the spring bloom. Total new production, estimated from water column integrated 15 N-nitrogen uptake rates and linear models that interpolated rates over unsampled periods, was 342―415 mmol N m ―2 yr ―1 . Total annual N production for the region was 1.24-1.48 mol N m ―2 yr ―1 .

Assessments of carbon and nitrogen (N) assimilation in Canadian Arctic waters confirmed the large contribution of subsurface chlorophyll maxima (SCM) to total water-column production from spring to late fall. Although SCM communities showed acclimation to low irradiance and greater nitrate (NO3−) availability, their productivity was generally constrained by light and temperature. During spring–early summer, most of the primary production at the SCM was sustained by NO3−, with an average f-ratio (i.e., relative contribution of NO3− uptake to total N uptake) of 0.74 ± 0.26. The seasonal decrease in NO3− availability and irradiance, coupled to the build up of ammonium (NH4+), favoured a transition toward a predominantly regenerative system (f-ratio = 0.37 ± 0.20) during late summer and fall. Results emphasize the need to adequately consider SCM when estimating primary production and to revisit ecosystem model parameters in highly stratified Arctic waters.

Cyanobacteria are globally important primary producers and abundant in many iron-limited aquatic environments. The ways in which they take up iron are largely unknown, but reduction of Fe

Synechococcus species are important primary producers in coastal and open-ocean ecosystems. When nitrate was provided as the sole nitrogen source, nickel starvation inhibited the growth of strains WH8102 and WH7803, while it had little effect on two euryhaline strains, WH5701 and PCC 7002. Nickel was required for the acclimation of Synechococcus WH7803 to low iron and high light. In WH8102 and WH7803, nickel starvation decreased the linear electron transport activity, slowed down QA reoxidation, but increased the connectivity factor between individual photosynthetic units. Under such conditions, the reduction of their intersystem electron transport chains was expected to increase, and their cyclic electron transport around PSI would be favored. Nickel starvation decreased the total superoxide dismutase (SOD) activity of WH8102 and WH7803 by 30% and 15% of the control, respectively. The protein-bound (63) Ni of the oceanic strain WH8102 comigrated with SOD activity on nondenaturing gels and thus provided additional evidence for the existence of active NiSOD in Synechococcus WH8102. In WH7803, it seems likely that nickel starvation affected other metabolic pathways and thus indirectly affected the total SOD activity.

Cyanobacteria are known to survive in iron-deficient environments, but the ways in which they acquire Fe and acclimate are not completely understood. Here we report a novel gene sll1263 that is required for Synechocystis sp. strain PCC 6803 to grow under iron-deficient conditions. sll1263 encodes a putative cation diffusion facilitator protein (CDF) that shows 50% amino acid similarity with ferrous iron efflux protein (FieF) of heterotrophic bacteria. In bacteria, the gene product is involved in metal export from the cell, but in Synechocystis sll1263 plays a role in iron uptake. The results show that expression of sll1263 was induced by iron-deficient conditions and its inactivation significantly decreased the growth rate of an sll1263(-) mutant. Other genes known to be required for Fe acquisition were also strongly up-regulated in the mutant even in the presence of high Fe. Overexpression of sll1263 increased growth under iron deficiency but reduced growth under high-iron stress, suggesting that the gene product was involved in iron uptake rather than detoxification. Expression of FieF in the sll1263(-) mutant was unable to rescue the Fe-deficient phenotype, but Sll1263 completely restored it. Measurements of cellular iron content and the iron uptake rate showed that they were significantly less in the sll1263(-) mutant than in the wild type, consistent with a role for sll1263 in iron uptake. We hypothesize that the low-iron habitats and high-iron requirements of cyanobacteria may be the reason why cyanobacterial CDF protein functions in Fe uptake and not efflux as in non-photosynthetic bacteria.

Synechococcus species are important primary producers in coastal and open-ocean ecosystems. When nitrate was provided as the sole nitrogen source, nickel starvation inhibited the growth of strains WH8102 and WH7803, while it had little effect on two euryhaline strains, WH5701 and PCC 7002. Nickel was required for the acclimation of Synechococcus WH7803 to low iron and high light. In WH8102 and WH7803, nickel starvation decreased the linear electron transport activity, slowed down QA reoxidation, but increased the connectivity factor between individual photosynthetic units. Under such conditions, the reduction of their intersystem electron transport chains was expected to increase, and their cyclic electron transport around PSI would be favored. Nickel starvation decreased the total superoxide dismutase (SOD) activity of WH8102 and WH7803 by 30% and 15% of the control, respectively. The protein-bound (63) Ni of the oceanic strain WH8102 comigrated with SOD activity on nondenaturing gels and thus provided additional evidence for the existence of active NiSOD in Synechococcus WH8102. In WH7803, it seems likely that nickel starvation affected other metabolic pathways and thus indirectly affected the total SOD activity.

The effects of selenium deficiency on the siliceous and nonsiliceous components of the planktonic marine diatom Thalassiosira pseudonana (Hust.) Hasle and Heimdal (clone 3H) are examined using light and electron microscopy. Selenium deficiency induces elongation along the pervalvar axis initially as a result of chain formation caused by the failure of sibling cells to separate and subsequently by cell elongation via the production of hyaline girdle bands. In Se-deficient cultures cell elongation involves the blockage of both mitotic and cytokinetic components of cell division. Selenium deficiency results in ultrastructural alterations in the reticular membrane system and in mitochondrial and chloroplast membranes. Various types of inclusions are seen in vacuolar areas and the accumulation of lipid reserves is evident in Se-deficient cells. These results provide indirect evidence for a metabolic Se requirement in this algal species.

An obligate requirement for selenium is demonstrated in axenic culture of the coastal marine diatom Thalassiosira pseudonana (clone 3H) (Hust.) Hasle and Heimdal grown in artificial seawater medium. Selenium deficiency was characterized by a reduction in growth rate and eventually by a cessation of cell division. The addition of 10 M Na2SeO3 to nutrient enriched artificial seawater resulted in excellent growth of T. pseudonana and only a slight inhibition of growth occurred at Na2SeO3 concentrations of 10 T and 10 S M. By contrast, Na2SeO4 failed to support growth of T. pseudonana when supplied at concentrations less than 10 X M and the growth rate at this concentration was only one quarter of the maximum growth rate. The addition of 10 T and 10 S M Na2SeO4 to the culture medium was toxic and cell growth was completely inhibited. Eleven trace elements were tested for their ability to replace the selenium requirement by this alga and all were without effect. In selenium-deficient and selenium-starved cultures of T. pseudonana cell volume increased as much as 10-fold as a result of an increase in cell length (along the pervalvar axis) but cell width was constant. It is concluded that selenium is anmore » indispensable element for the growth of T. pseudonana and it should be included as a nutrient enrichment to artificial seawater medium when culturing this alga.« less

Dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS) biological cycling rates were determined during SERIES, a mesoscale iron-fertilization experiment conducted in the high-nutrient low-chlorophyll (HNLC) waters of the northeast subarctic Pacific. The iron fertilization resulted in the rapid development of a nanoplankton assemblage that persisted for 11 days before abruptly crashing. The nanoplankton bloom was followed by a diatom bloom, accompanied by an important increase in bacterial abundance and production. These iron-induced alterations of the plankton assemblage coincided with changes in the size and biological cycling of the DMSP and DMS pools. The initial nanoplankton bloom resulted in increases in particulate DMSP (DMSPp; 77–180 nmol L−1), dissolved DMSP (DMSPd; 1–24 nmol L−1), and biological gross (0.11–0.78 nmol L−1 h−1) and net (0.04–0.74 nmol L−1 h−1) DMS production rates. During the nanoplankton bloom, DMSPd consumption by bacteria exceeded their sulfur demand and the excess sulfur was probably released as DMS, consistent with the high gross DMS production rates observed during that period. The crash of the nanoplankton bloom was marked by the rapid decline of DMSPp, DMSPd, and gross DMS production to their initial values. Following the crash of the nanoplankton bloom, bacterial production and estimated sulfur demand reached transient maxima of 9.3 μg C L−1 d−1 and 14.2 nmol S L−1 d−1, respectively. During this period of high bacterial production, bacterial DMSPd consumption was also very high (6 nmol L−1 h−1), but none of the consumed DMSPd was converted into DMS and a net biological DMS consumption was measured. This transient period initiated a rapid decrease in DMS concentrations inside the iron-enriched patch, which persisted during the following diatom bloom due to low biological gross and net DMS production that prevented the replenishment of DMS. Our results show that the impact of Fe fertilization on DMS production in HNLC waters result from a complex interplay between the dynamics of the algal blooms and their influence on bacterial DMSP and DMS metabolism.

Diatoms are responsible for 40% of ocean production and are strongly limited by the lack of iron salts in the sea. The shortage of this important nutrient may explain a surprising discovery: the oceanic diatom Thalassiosira oceanica uses a copper-containing plastocyanin for electron transport. All other chlorophyll c-containing taxa, including coastal diatoms, use the iron-containing cytochrome c6 instead. The use of these metalloproteins matches the availabilities of copper and iron in the ocean, and this new discovery suggests that copper is a potentially important nutrient in the open sea. The supply of some essential metals to pelagic ecosystems is less than the demand, so many phytoplankton have slow rates of photosynthetic production and restricted growth1. The types and amounts of metals required by phytoplankton depends on their evolutionary history2 and on their adaptations to metal availability3,4, which varies widely among ocean habitats. Diatoms, for example, need considerably less iron (Fe) to grow than chlorophyll-b-containing taxa2, and the oceanic species demand roughly one-tenth the amount of coastal strains5,6,7. Like Fe, copper (Cu) is scarce in the open sea, but notably higher concentrations of it are required for the growth of oceanic than of coastal isolates8. Here we report that the greater Cu requirement in an oceanic diatom, Thalassiosira oceanica, is entirely due to a single Cu-containing protein, plastocyanin, which—until now—was only known to exist in organisms with chlorophyll b and cyanobacteria. Algae containing chlorophyll c, including the closely related coastal species T. weissflogii, are thought to lack plastocyanin and contain a functionally equivalent Fe-containing homologue, cytochrome c6 (ref. 9). Copper deficiency in T. oceanica inhibits electron transport regardless of Fe status, implying a constitutive role for plastocyanin in the light reactions of photosynthesis in this species. The results suggest that selection pressure imposed by Fe limitation has resulted in the use of a Cu protein for photosynthesis in an oceanic diatom. This biochemical switch reduces the need for Fe and increases the requirement for Cu, which is relatively more abundant in the open sea.

We measured the isotopic composition and accumulation of particulate organic matter (POM) and the uptake of carbon (C) and nitrogen (N) in an early bloom of the most productive recurring polynya of the Arctic Ocean. The estimated compensation irradiance at the onset of the bloom was similar to the average for the North Atlantic Ocean, implying that shallow mixing was of critical importance for the bloom’s early initiation. Planktonic POM had a much lower d 13 C than ice POM, suggesting that ice-algae contributed little to the pelagic biomass. The overall isotopic fractionation of pelagic N during bloom development was consistent with in situ diatom growth under saturating irradiance and limiting NO . Soon after the ice cleared, rapid physiological changes induced an order of 2 3 magnitude increase in the C and NO uptake capacity of diatoms, leading to very high f ratios (NO uptake : total 2 2 3 3 N uptake). Most of the NO taken up appeared in the POM, so that little net release of reduced N occurred during 2 3 the period of active growth. Given the tight coupling between photosynthesis and NO uptake under N limitation, 2 3 the magnitude of primary production in the Arctic Ocean is expected to respond to changes in N supply.

Centric diatoms isolated from open ocean environments require higher concentrations of Cu for growth than their coastal counterparts. In artificial seawater medium containing ,1 nmol L 21 Cu, three coastal species maintained near maximum rates of growth, but the oceanic clones were unable to survive. Copper limitation was more severe in the diatoms grown in low- than in high-Fe seawater, suggesting that Cu and Fe were interacting essential resources. The interactive effect was in part the result of a Cu requirement for Fe transport. Thalassiosira weissflogii and Thalassiosira oceanica had lower Fe quotas and slower rates of Fe uptake when [Cu] was reduced in the medium. Brief exposure of Cu-limited cells to 10 nmol L 21 Cu increased the instantaneous Fe uptake rate by 1.5 times in T. oceanica. Steady-state uptake rates of both species at high, growth-saturating concentrations of Fe were also Cu dependent. Oceanic species appeared to have an additional Cu requirement that was independent of Fe acquisition and likely responsible for their higher requirements compared to coastal species. Evidence for the importance of Cu in natural communities of phytoplankton was obtained from an incubation experiment performed in the Fe-limited basin of the Bering Sea. Addition of 2 nmol L 21 Cu doubled the phytoplankton net growth rate compared to the untreated controls and, in the presence of extra Fe, increased the growth rate compared to the samples amended with Fe alone. The results suggest that Cu may be an important micronutrient for phytoplankton growth in low-Fe regions of the sea because of its role in Fe acquisition. Paradoxically, oceanic diatoms may be more susceptible to the effects of low Cu concentrations than coastal species.

We grew Thalassiosira weissflogii to steady state over a range of Fe-limiting conditions with nitrate or ammonium as the N source. Nitrate-dependent cells had faster Fe-uptake rates, contained significantly higher intracellular Fe quotas, and grew faster than cells cultivated with NH4 1 when Fe was most limiting. Under these conditions, carbon (C) : chlorophyll a ratios and the minimum fluorescence yield per chlorophyll a increased, but N source had no effect on either parameter. The ratio of variable to maximum fluorescence (F v F ) declined little with Fe limitation 21 m even when T. weissflogii was grown at 25% of its maximum rate (mmax). C : N ratios were higher in nitrate than in ammonium-grown cells and were constant at all Fe levels. Protein was independent of Fe and N, and amino acids were lowest in cells using NO . The P content of T. weissflogii (mol P L 21 cell volume) increased by 1.5 times as 2 3 Fe became most limiting to growth, causing N : P and C : P ratios to decline significantly. The elemental stoichiometry for Fe-limited new production of T. weissflogii (0.25mmax) was thus 70C : 10N : 5.9Si : 1P : 0.00074Fe (by mols) compared with 97C : 14N : 4.7Si : 1P : 0.029Fe for nutrient-replete conditions. Uptake rate ratios of NO : PO showed the same dependence on Fe as the cellular N : P quotas, decreasing as [Fe] decreased. Iron 2 32 34 limitation influenced the elemental composition of this marine diatom and will alter the assimilation ratios of C, N, and P in the high nitrate, low chlorophyll regions of the sea.

We have discovered that coastal and oceanic diatoms require more manganese (Mn) to grow in iron (Fe) -deficient than in Fe-sufficient seawater. At low inorganic concentrations, like those of the open sea, Fe and Mn can thus colimit Thalassiosira pseudonana and T. oceanica so that maximum rates of cell division are achieved only when both resources are added simultaneously to cultures. Colimited diatoms amended with either Fe or Mn alone show unique physiological responses, which implies that the observed interaction between Fe and Mn is not caused by a substitution of one metal for the other. Iron deficiency increases the Mn quota of T. pseudonana by three times compared with controls and enhances the production of reactive oxygen species by 1.7 times in T. weissflogii. Both diatoms respond to this oxidative stress by increasing the activities of the antioxidant enzyme superoxide dismutase (SOD). The Mn content of the SODs increases by 1.8 to 2.8 times when Fe is limiting, which suggests that the SODs contain Mn and may account for part of the observed increase in the Mn quota. Such an increased biochemical requirement may elevate the Mn content of low Fe diatoms, and possibly other phytoplankton, resulting in high Mn: Fe ratios in surficial particulate matter in Fe-limited regions of the sea.

Pathogenic gram-negative bacteria use specific receptors to transport ferric siderophore complexes across their outer membrane during iron (Fe)-limited growth. Receptors such as these have not yet been characterized in oceanic heterotrophic bacteria. We examined four species of g-proteobacteria for the presence of Fe-siderophore receptors with the use of a nondenaturing polyacrylamide gel electrophoresis binding assay and the siderophore ferrioxamine B (FB) labeled with 55 Fe. Small-subunit rRNA sequence analysis assigned these bacteria to the genera Pseudoalteromonas and Alteromonas. Two oceanic species, Pseudoalteromonas haloplanktis (Neptune) and Alteromonas macleodii (Jul88), which were shown previously to transport and assimilate Fe bound to FB during growth, synthesized an outer-membrane FB receptor under Fe-limiting conditions. Only low concentrations of the receptors were detected in these bacteria when they were grown with high concentrations of Fe. The FB receptor of P. haloplanktis (Neptune) had an apparent molecular mass of 79 kDa and an externally oriented binding site. The molecular mass of the receptor of A. macleodii (Jul88) was 100 kDa. No FB receptors were detected by our methods in two coastal species, Pseudoalteromonas rubra(LMG1) and Pseudoalteromonas piscicida(PWF3). P. haloplanktis (Neptune) and A. macleodii (Jul88) also bound 55 Fe-ferrichrome, a trihydroxamate siderophore like FB. Binding assays conducted with 115 nmol L 2 15 5 Fe-FB in the presence of increasing concentrations of desferrioxamine B showed a progressive decrease in the amount of 55 Fe-FB bound by the receptor protein, suggesting strong affinity of the receptor for the Fe-free siderophore. Our results provide the first demonstration of Fe-siderophore receptors in oceanic heterotrophic bacteria. Heterotrophic bacteria have evolved Fe(III) transport systems that enable them to grow in environments containing extraordinarily low concentrations of Fe. When Fe is scarce, many of these organisms excrete low‐molecular mass Febinding compounds, called siderophores, that bind free Fe(III) in solution. The Fe-siderophore complex attaches to its cognate receptor on the outer membrane of the cell and is subsequently internalized (Braun and Killman 1999; Ratledge and Dover 2000). Some bacterial receptors recognize more than one siderophore, not necessarily of the same structural type (e.g., FhuE of E. coli) (van der Helm 1998). Many bacteria express receptors for siderophores released by other species or for Fe sources contained in their hosts (Braun et 1

New, net, and diatom production in the North Water were estimated during May to July 1998 from in vitro measurements of nitrate uptake and mesoscale temporal changes in the inventories of nitrate, silicate, oxygen, and inorganic carbon (DIC). Sampling stations were divided into two domains according to the position of the dominant water types: the silicate-rich Arctic water (SRAW) and Baffin Bay Water (BBW). BBW dominated in the southeast and was associated with relatively shallow upper mixed layers (UMLs) and weak horizontal advection. A phytoplankton bloom started in late April in BBW and grew slowly over 7 weeks, during which time the build-up of particulate organic nitrogen and carbon accounted for ca. 80% of the nitrate and DIC deficit, respectively. Over half of the new production (1.37 g C m −2 d −1 ) during this period was attributed to wind-driven replenishment of nitrate in the euphotic zone. The bloom culminated when seasonally declining winds and rising temperatures severed the UML from the deep nutrient reservoir. The same change in weather induced ice melt, stratification, and bloom development in northern SRAW, which had previously been characterized by deep UMLs. Collectively, the results imply that the timing and magnitude of blooms in the North Water are controlled by a succession of oceanic and climatic forcings. New C production in the North Water during April to July (1.11 g C m −2 d −1 ) was an order of magnitude higher than in adjacent waters and up to 8 times higher than in the Northeast Water polynya. As much as 80% of this production was mediated by diatoms >5 μm, suggesting potentially high and efficient C transfer to the herbivorous food web and deep waters.

Thalassiosira spp., large-chain forming centric diatoms, typically dominate the biomass during phytoplankton blooms in the North Water Polynya (76 to 79°N, centred on ca. longitude 75°W), which is the largest recurring polynya in the Canadian Arctic. We used an experimental method based on semi-continuous cultures to investigate mechanisms responsible for bloom mainte- nance and associated changes in microbial food web constituents. We compared 2 treatments: (1) a new nutrient system in which the cultures were partially enriched every 2 d with nutrient-rich sea- water from depth to simulate horizontal or vertical advection, and (2) a recycled nutrient system in which large particles (> 2.0 µm) were partially removed every 2 d to simulate grazing and sinking losses without nutrient replacement. The experiment lasted 8 d. In the new nutrient treatment, large diatoms, particularly Thalassiosira spp. and to a lesser extent Chaetoceros spp., consumed the added nutrients and continued to dominate production and biomass of the protist community. The total eukaryotic community production in the 'recycled' community shifted to one dominated by dino- flagellates and ciliates in the absence of diatom growth. These 2 end points corresponded to 2 types of communities observed in the North Water Polynya in June 1998. Net production rates for viruses and bacteria were not significantly different between treatments. These results demonstrate the importance of advective processes in maintaining a prolonged diatom bloom. An underlying micro- bial food web dominated by large (> 20 µm) ciliates and dinoflagellates was able to maintain similar rates of net production respective of new versus recycled nutrient supply. Dominance of the protist communities by large cells under both conditions is likely to favour the sustained high productivity of zooplankton and megafauna that characterize the North Water Polynya.

Cyanobacteria are globally important primary producers that have an exceptionally large iron requirement for photosynthesis. In many aquatic ecosystems, the levels of dissolved iron are so low and some of the chemical species so unreactive that growth of cyanobacteria is impaired. Pathways of iron uptake through cyanobacterial membranes are now being elucidated, but the molecular details are still largely unknown. Here we report that the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 contains three exbB-exbD gene clusters that are obligatorily required for growth and are involved in iron acquisition. The three exbB-exbDs are redundant, but single and double mutants have reduced rates of iron uptake compared with wild-type cells, and the triple mutant appeared to be lethal. Short-term measurements in chemically well-defined medium show that iron uptake by Synechocystis depends on inorganic iron (Fe′) concentration and ExbB-ExbD complexes are essentially required for the Fe′ transport process. Although transport of iron bound to a model siderophore, ferrioxamine B, is also reduced in the exbB-exbD mutants, the rate of uptake at similar total [Fe] is about 800-fold slower than Fe′, suggesting that hydroxamate siderophore iron uptake may be less ecologically relevant than free iron. These results provide the first evidence that ExbB-ExbD is involved in inorganic iron uptake and is an essential part of the iron acquisition pathway in cyanobacteria. The involvement of an ExbB-ExbD system for inorganic iron uptake may allow cyanobacteria to more tightly maintain iron homeostasis, particularly in variable environments where iron concentrations range from limiting to sufficient.

Three species of photosynthetic flagellates capable of phagotrophy (mixotrophic species) were tested for their abilities to use inorganic iron colloids for growth. Ochromonas sp., Chrysochromulina ericina (a coastal strain), and C. ericina (an oceanic strain) were grown in iron-free seawater supplemented with 1 mM Fe-ferrihydrite (amorphous hydrous ferric oxide), magnetite (Fe3O4)/maghemite (g-Fe2O3), hematite (a-Fe2O3), or goethite (a-FeOOH). Desferrioxamine B, an iron-binding siderophore, was used to reduce the concentration of dissolved iron in the colloid-amended media, and none of the flagellates were able to use its iron complex as an iron source under the conditions of the experiments. Both strains of Chrysochromulina grew at 35%‐70% of their maximum rates with goethite, hematite, and magnetite/maghemite but were unable to use ferrihydrite. Ochromonas grew well with ferrihydrite but could not use any of the other forms. Thalassiosira oceanica (clone 1003) and Thalassiosira pseudonana (clone 3H), diatoms that could only take up dissolved forms of iron, were unable to use any of the colloids tested. The mechanism of iron acquisition by the flagellates appeared to involve ingestion of the iron colloids, because bacteria resident in the cultures were too iron poor to be a significant source of iron and were unable to use the iron contained in the colloids themselves. Variations in the sizes of the colloids were hypothesized to account for differences in their availability, independent of colloid chemical stability. The results provide the first strong evidence for direct use (i.e., without prior dissolution) of colloidal iron by mixotrophic phytoplankton and document a new pathway of iron acquisition that may be important for their survival in low-iron waters of the sea.

Thalassiosira oceanica (Hustedt) Hasle et Heimdal (clone 1003) attained rapid rates of growth in low Fe seawater containing the siderophore ferrioxamine B (FeDFB) as the sole Fe source. Short-term rates of Fe uptake were 109 times faster than those predicted from the equilibrium concentration of inorganic Fe, suggesting that FeDFB was the substrate for the Fe transport system. An extracellular reduction step, mediated by a cell surface reductase, preceded Fe transport from FeDFB and was induced under Fe limitation. The half-saturation constant for the reduction was 0.68 μM. Iron reduction rates were two times faster than uptake rates, so that the activities of the reductase and the transporter were tightly coupled. The rates of Fe reduction of a number of Fe chelators, including synthetic organic ligands (nitrilotriacetate, diethylenetriaminepentaacetate, and EDTA) and fungal siderophores (desferrioxamine B and desferrioxamine E), were inversely proportional to the ratio of the stability constants of their Fe(III) and Fe(II) complexes and varied by a factor of two times, like the redox potentials of the Fe complexes. Platinum (II), a known inhibitor of Fe reductase activity, appeared to reduce the rates of Fe uptake from FeDFB but not from inorganic complexes. The results suggested that reoxidation of Fe(II) produced by reduction may be a necessary part of the Fe internalization reaction. Ferric reductase could be relevant to phytoplankton nutrition in the open sea where organic Fe complexes dominate the dissolved speciation and where the concentration of inorganic Fe is limiting.

Under Fe-limiting conditions, nitrate (NO3 2 )-grown marine diatoms have higher intracellular Fe requirements, but divide as fast or faster than ammonium (NH4 1 )-grown cells by maintaining faster steady-state Fe uptake rates. Here we report that Thalassiosira oceanica, clone 1003, possesses an Fe reductase that reduces Fe(III) bound to a variety of organic ligands, including the siderophore desferrioxamine B (DFB), a high affinity, Fe(III)-specific ligand. Reduction is mediated extracellularly and is induced by Fe deficiency. Cellular rates of Fe(III) reduction are significantly faster in NO 3 2 - than in NH4 1 -grown cultures suggesting a link with N metabolism. At subsaturating Fe concentrations, short- and long-term Fe uptake rates are also significantly faster in NO3 2 - than in NH4 1 -grown cells. The results suggest that when Fe is limiting, faster rates of reduction of organically bound Fe(III) by phytoplankton promote faster rates of Fe transport and growth. The implications of these findings could be significant for understanding phytoplankton Fe nutrition in oceanic waters where organic complexation dominates the speciation of Fe. We hypothesize that the reductive Fe transport pathway may enable phytoplankton to directly utilize Fe bound to strong organic ligands in the sea. Iron plays a catalytic role in many biochemical reactions as a cofactor of enzymes and proteins involved in chlorophyll synthesis, detoxification of reactive oxygen species, respiratory and photosynthetic electron transport, and N assimilation. Changes in the activity of these reactions or their replacement by functionally equivalent Fe-deficient pathways can greatly influence cellular Fe requirements of organisms. Because the NO3 2 assimilatory pathway is highly Fe dependent, utilization of NO3 2 by marine centric diatoms (Thalassiosira spp.) imparts a higher metabolic demand for Fe than the use of NH4 1 . The demand for Fe is such that the Fe quotas (Fe : C) of NO3 2 -grown cells are 1.8 times higher than those of phytoplankton using NH4 1 (Maldonado and Price 1996). Despite the higher Fe requirement for NO3 2 assimilation, under moderately Fe-limiting conditions (ca. 0.7‐0.85 m/mmax), growth rates of NO3 2 -amended cultures are not slower than those of NH4 1 -amended ones (Maldonado and Price 1996). Nitrate-grown cells apparently compensate for their extra Fe requirement by sustaining faster steadystate Fe uptake rates (Maldonado and Price 1996). The

The marine diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle was grown over a wide range of irradiances and temperatures in both NO3 - and NH4 + -amended seawater containing high Fe. Maximum growth rates were not affected by N source, but the initial slope of the growth irradiance curve was significantly greater for the NH4 + -grown cells. Light-limited phytoplankton con- tained 4 times more Fe (µmol Fe mol -1 C) than light-sufficient cells, and at intermediate irradiances had the highest chlorophyll a concentrations (g C g -1 chl a). Iron quotas increased as irradiance was reduced because growth rates declined by 20-fold, whereas steady-state Fe transport rates declined by only a factor of 6. Accumulation of Fe was apparently regulated by a light-induced physiological requirement. Similar reductions in growth rate with declining temperature elicited only minor changes in Fe quota, and the increase in Fe quota, was observed only at the 2 lowest temperatures. At irradiance levels between 37 and 150 µmol photons m -2 s -1 , the relative increase in Fe quota exactly matched the relative increase predicted from the change in abundance of PSI and PSII reported to occur during photoacclimation of T. weissflogii. Subsequent increases in Fe quota at low light may represent additional changes to the photosynthetic apparatus or accumulation of Fe in a non-metabolic pool. The results demonstrate that the Fe content of phytoplankton is influenced by ambient light and imply that the extent of photoacclimation of natural populations may be con- strained by the availability of Fe.

Experiments were conducted along a coastal-oceanic transect in the NE subarctic Pacific to examine acquisition of organically complexed Fe by autotrophic and heterotrophic plankton. During short-term experiments, plankton took up Fe bound to the siderophores desferrioxamine B and E, microbial Fe chelates with a high affinity for Fe. Uptake occurred in all size fractions: 0.2–1, 1–3, and >3 μm. Heterotrophic bacteria had higher Fe : C ratios (1.5 to 2 times) than phytoplankton, and accounted for 70±8% of the total Fe uptake by the community (mol Fe ml −1 h −1 ). This latter result was partially explained by the higher C biomass of bacteria, but was not related to their productivity. Carbon-specific uptake rates of Fe were also faster (1.6±1.5 times) in bacteria than phytoplankton. When the rates were normalized per cell surface area, however, phytoplankton were observed to transport Fe at a rate more than 30 times that of bacteria. Large phytoplankton greater than 3 μm reduced Fe bound to organic ligands extracellularly. Their Fe : C ratios and rates of uptake and reduction of organically bound Fe were very similar at all stations along the transect and were characteristic of Fe-stressed phytoplankton. A strong seasonal trend of Fe uptake and reduction was apparent. The results suggest that heterotrophic bacteria are responsible for a large fraction of dissolved Fe uptake and that the indigenous plankton of the subarctic Pacific are able to acquire Fe bound to strong organic ligands, the predominant form of dissolved Fe in the sea.

Phytoplankton acclimate to low irradiance by increasing their cellular demand for Fe, to allow synthesis of additional light-harvesting pigments and Fe-containing redox proteins involved in photosynthesis. In the open NE subarctic Pacific, Fe concentrations limit primary productivity and irradiances may be suboptimal, particularly during winter. Phytoplankton thus may be unable to fulfill their extra Fe requirements for growth under these low-light conditions and become effectively co-limited. We tested this hypothesis by manipulating Fe and light in in vitro experiments at OSP (Ocean Station PAPA, 50°N 145°W) during winter 1997. The results show that metabolic rates, growth, and photosynthetic parameters of phytoplankton are enhanced in winter by increasing either irradiance or Fe. The greatest response occurs when Fe and light are amended concomitantly, confirming that the community is indeed co-limited by both resources. Analysis of environmental conditions (i.e. incident irradiance, mixed layer depth and Fe concentrations) in winter at OSP reveals that they are similar to those observed in the austral spring and fall at three sites in the Southern Ocean. Extrapolating our experimental field results to the Southern Ocean illustrates that co-limitation by light and Fe also may play an important role in regulating phytoplankton growth in this region.

Recent studies demonstrate that dissolved iron in seawater is bound to strong organic complexes that have stability constants comparable to those of microbial iron chelates. We examined iron acquisition by seven strains of heterotrophic marine bacteria from a number of siderophore‐iron complexes, including desferrioxamine B (DFB) and marine siderophores partially purified from iron-limited cultures. Hydroxamate siderophores were detected in the supernatants of four strains, one of which also produced a catechol. All strains transported iron bound to siderophores regardless of whether or not they produced their own, and the majority took up iron bound to DFB. Uptake rates of Fe siderophores were similar among iron-limited strains and among ligands. Transport of FeDFB by strain Neptune was enhanced 20 times by iron limitation, whereas uptake of unchelated iron (Fe9) did not saturate at the highest concentration tested and was not regulated by the iron nutritional status of the cells. The half-saturation constant for uptake of FeDFB by Neptune was 15 nM, the lowest reported for an Fe siderophore in any microorganism. Iron uptake by the catechol-producing strain, LMG1, differed markedly in two respects from the other strains: LMG1 could not take up iron bound to DFB; furthermore, transport of Fe9 by iron-limited LMG1 was 10 times faster than the other strains and was upregulated 46 times compared to Fe-sufficient cells. Experimental evidence suggests that iron transport by LMG1 may be mediated by surface-associated catechol siderophores that scavenge inorganic ferric species as well as iron bound to weaker complexes, such as EDTA (ethylenediaminetetraacetic acid). The combined results of the study highlight the importance of siderophores in iron transport by heterotrophic marine bacteria and suggest, by inference, that bacteria may rely on siderophores to acquire iron in situ.

Prokaryotic microbes play a critical role in oceanic Fe cycling. They contain most of the biogenic Fe in offshore waters and are responsible for a large portion of the Fe uptake by the plankton community. In the subarctic North Pacific, surface populations of heterotrophic species assimilate more than 50% of the dissolved Fe and thus compete directly with phytoplankton for this limiting resource. In oligotrophic tropical and subtropical waters, photosynthetic bacteria become more important in Fe cycling as the number of unicellular cyanobacteria increases and the nitrogen-fixing Trichodesmium, which contains most of the biogenic Fe in the mixed layer, becomes abundant. Like their terrestrial counterparts, heterotrophic and phototrophic marine bacteria produce Fe-binding siderophores that are involved in Fe acquisition. Evidence exists that bacteria may modify Fe chemistry in the sea through the production of these ligands and thereby play a significant role in regulating production of eukaryotic phytoplankton.

Some classes of marine phytoplankton are believed to be more tolerant of high concentrations of trace metals than others, but the results of experimental tests of this hypothesis are ambiguous. Eleven species of phytoplankton representing five classes were grown in Aquil medium containing Cd concentrations between 10−8 and 10−5 M ([Cd2+]= 10−9.85 to 10−6.84 M), and growth rates and intracellular concentrations of Cd, C, N, and S were measured. The mean Cd2+ concentration (pCd50) that reduced the growth rate of each species to 50% of its maximum varied by 2.5 orders of magnitude, from 10−6.23 for Emiliania huxleyi to 10−8.79 for Synechococcus sp. Taxonomic trends in Cd resistance were not apparent in these data. Cadmium quotas (mol Cd·L−1 cell volume) were lowest in species of Bacillariophyceae (ANOVA, P < 0.001), suggesting that they might regulate Cd transport differently than other taxa. Cellular S:C molar ratios increased in four of seven phytoplankton grown at high pCd (7.37–6.84) compared to low Cd ion concentrations (no added Cd), a result of increases in S·L−1 cell volume. Nitrogen:carbon molar ratios were also higher in Cd-exposed phytoplankton, as changes in N and S were highly correlated (r= 0.98, P < 0.0001). In two species that were examined, S:C ratios increased as a linear function of increasing Cd concentration. The results demonstrate large variability in Cd resistance among phytoplankton that is primarily a function of interspecific differences in Cd detoxification.

Under conditions of iron stress, many organisms replace the common iron-sulfur redox protein ferredoxin with flavodoxin, a functionally equivalent, non-iron-containing protein. These 2 proteins have been proposed to be indicators of iron nutritional status in marine phytoplankton, but little is known of their expression and regulation. This study characterized their expression by: testing 17 marine phytoplankton isolates from 4 different algal classes for their ability to induce flavodoxin under iron limitation, determining the effect of ecologically relevant limiting factors (other than iron) on flavodoxin expression using the marine centric diatom Thalassiosira weissflogii as a model organism, and examining, in detail, the relationship between iron availability and relative ferredoxin/flavodoxin abundance again using T weissflogii as a model. In the organisms examined, the most common response (1 of 17 isolates ) to iron limitation was induction of flavodoxin and suppression of ferredoxin expression. The remaining 5 isolates, largely of coastal origin, were never observed to produce flavodoxin. These 5 non-inducing organisms have been shown to have high intrinsic Fe requirements for growth and should therefore not present a problem for field measurements of ferredoxin and flavodoxin in iron-poor areas. Expression of flavodoxin in T. weissflogii was found to be specific to iron limitation, and was not induced by nitrate, phosphate, silicate, zinc or light deficiency. The prevalence of the flavodoxin response and its insensitivity to other limiting factors support its use as an indicator of the presence of iron limitation. Iron regulation of relative ferredoxin and flavodoxin abundance (the Fd index) and iron availability was examined in greater detail by measuring the Fd index in T. weissflogii grown over a range of limiting iron concentrations. The relationship between Fd index and growth rate (a proxy for iron availability) is composed of 2 distinct regions. In the first region, at low growth rates, ferredoxin is un

Iron is unique among biologically essential trace metals in having a higher particulate than dissolved concentration in ocean surface waters1. Uptake of dissolved iron is generally considered to be the norm for phytoplankton, as even the smallest iron-bearing particles are unavailable for transport into cells2,3. But the oceanic dissolved fraction is so small, and the particulate fraction so inert2, that phytoplankton production is limited by a dearth of available iron in some regions4. Here we use incubation experiments to show that Ochromonas sp., a common photosynthetic flagellate from the Pacific Ocean, can obtain iron directly in particulate form, by ingesting bacteria. Iron acquisition is highly efficient; Ochromonas assimilates 30% of the ingested ration, acquiring a high intracellular iron concentration and maintaining a significantly faster growth rate than when iron is provided in the dissolved phase. Phytoplankton capable of such phagotrophy (so-called mixotrophic species) may thus be able to assimilate iron in both particulate and dissolved forms in the ocean. Moreover, when iron availability is limited, the iron ‘cost’ of growth is diminished because Ochromonas derives a greater fraction of its energy from the bacteria. Analysis of standing stocks and clearance rates of plankton in the equatorial Pacific shows that the iron flux through mixotrophic flagellates can amount to 35–58% of the total Fe uptake by the entire autotrophic community. Our results suggest that the phagotrophic ingestion of bacteria may be an effective adaptive strategy for photosynthetic organisms to obtain iron for growth in iron-limited regions of the sea.

Phytochelatin, an intracellular metal-binding polypeptide synthesized in eucaryotic algae in response to metals such as Cd and Cu, was measured in particulate samples collected from the equatorial Pacific. The concentrations in these samples (normalized to total particulate chl a ) were unexpectedly high compared to laboratory culture data and were on average slightly more than in coastal areas where the metal concentrations are typically much greater. In part, the high field concentrations can be explained by the low cellular concentrations of chlorophyll a resulting from very low ambient Fe, but laboratory experiments provide a possible explanation for the rest of the difference. At low concentrations of inorganic Cd (Cd′=3 pM), increasing amounts of phytochelatin were induced by decreasing Zn concentrations in the culture medium of two diatoms: Thalassiosira weissflogii , a coastal species, and T. parthenaia , an isolate from the equatorial Pacific. In all previous studies, phytochelatin production has been directly correlated with increasing metal concentrations. Decreasing Co also resulted in higher phytochelatin concentrations in T. weissflogii and Emiliania huxleyi . Replicating the field concentrations of Zn, Co, and Cd in the laboratory results in cellular concentrations (amol -1 cell) that are very similar to those estimated for the field. Contrary to the expectation that high metal concentrations in the equatorial upwelling would cause elevated phytochelatin concentrations, there was no increase in phytochelatin concentrations from 20° S to 10° N—near surface samples were roughly the same at all stations. Also, most of the depth profiles had a distinct subsurface maximum. Neither of these features is readily explained by the available Zn and Cd data. Incubations with additions of Cd and Cu performed on water sampled at four separate stations induced significantly higher concentrations of phytochelatins than those in controls in a majority of the samples. The differences between metal additions and controls were greater within the upwelling zone, where water presumably has had less time to accumulate biogenic complexing agents. However, the uniformity of phytochelatin concentrations in and out of the upwelling region suggests that the phytoplankton or the biota are successfully maintaining a tight control over the trace metal chemistry both intra- and extracellularly across a wide range of oceanic conditions.

This work was funded by the Natural Sciences and Engineering Research Council of Canada and the McGill University Faculty of Graduate Studies and Research. Z.C. was supported by a fellowship from the Fonds pour la Formation de Chercheurs et l’Aide a la Recherche (Quebec).

The interaction between Fe requirements and N metabolism in centric diatoms was investigated to determine whether use of nitrate (NO3-) imparts a higher cellular Fe demand for growth than use of ammonlunl (NHdC), and thus reduces fitness under Fe deficiency. SIX species of the genus Thalassiosira from a variety of habitats were examined. Coastal and central gyre representat~ves grew faster in Fe-sufficient media containing NH,', but isolates from the equatorial Pacific, an oceanic highnutrient, low-biomass region, achieved maximum rates with NO3Iron quotas ranged from 26 to 102 pm01 Fe mol-' C and were not affected in a predictable manner by N source or habitat. Relative growth rates were diminished in Fe-deficient media, particularly in coastal species which grew at less than 25% of thelr maximum rates (pmdr). All oceanic species maintained fast rates of growth (0.8 p,,,) under the same Fe-limiting conditions, despite having 4 times less intracellular Fe than the coastal species. Fe:C ra t~os of Fe -de f~c~en t Thalassios~ra spp. ranged from 0.7 to 14 pm01 mol'] and were significantly greater (by 1.8 times) in all species when N O 3 was the N source (p < 0.05). Steady-state Fe uptake rates were also faster in NO3dependent cells a t low Fe. Nitrogen source had different effects on Fe-limited growth rates. Surprisingly, T oceanica (clone 1003) and T weissflogii grew faster with NO3even though higher Fe requirements for use of oxid~zed N were expected to reduce d~vision rates relative to NH,+-grown cells. When total Fe concentrations In the medlum were decreased to 1 nM, growth rates of T oceanjca (clone 1003) decreased to 0.2 p,,, and were s~gn~ficant ly faster (25%) in NHdt than in NO3--amended media. Under these more stressful Fe-limiting conditions, Fe quotas were the same in cells cultured in both N-based media. Our results thus demonstrate that phototrophic phytoplankton require significantly more cellular Fe to grow on NO3than NH,' Nitrate-grown cells are able to obtain this extra Fe, even when Fe is limiting, suggesting that Fe acquisition 1s somehow linked to NO,metabolism. Under severe Fe deficiency, however, NO,utilization reduces division rates compared to NHdt, because cells are unable to fulfil1 their extra Fe requ~rements .

Al (10 μM) and F (50 μM) added singly or in combination have no effect on growth of Chlamydomonas reinhardtii in acid or alkaline media containing organic or inorganic P. Cell surface phosphatase activity, however, is increased in glucose-1-phosphate-amended cultures exposed to Al, but only at low pH. This result is an indirect effect caused by Al binding to the organic P, which inhibits organic P utilization and induces phosphatase synthesis in C. reinhardtii as a compensatory response. Phosphatase activity is inversely correlated with lake pH, suggesting that such a mechanism may operate in nature. Al thus appears to enhance phosphatase activity in acidified waters by impeding dissolved organic P catabolism.

In coastal and shelf waters, substantial external inputs of iron come from riverine sources and bottom sediments, leading to markedly higher dissolved and particulate iron concentrations. Much of the particulate iron in nearshore waters is inorganic and processes that solubilize this reservoir, making it accessible to phytoplankton, are especially relevant (see below). The greater iron inputs to coastal and shelf regions compared to the open ocean are accompanied by high iron requirements of neritic phytoplankton species (Brand et al., 1983; Sunda et al., 1991). These systems therefore might be strongly influenced as the comparatively rich iron resource is diminished by phytoplankton blooms. The single largest reservoir of iron in the surface waters of HNLC regions may be the biota itself. New evidence indicates that this biological pool of iron is recycled on the time scale of days, much like N and P (Hutchins et al., 1993). This “input” of regenerated iron to surface waters is estimated to be more than an order of magnitude greater than the external supply rate of iron (Bruland, this meeting; Morel, this meeting) and may largely satisfy the iron-demand of phytoplankton in these systems. This view is supported by recent results of Price et al. (1994) showing that iron uptake rates of plankton in the equatorial Pacific are sufficient to entirely turn over the dissolved iron pool within half a day or less. Presently, there is no indication of the chemical forms of this regenerated iron or whether these forms are directly reassimilated by phytoplankton. B. What are the sinks of iron? The removal of iron from surface waters is fairly well constrained within a geochemical (i.e. mean residence time) perspective, however, the mechanisms and dynamics of this removal is not well understood. Mechanisms for removing iron from surface waters include: * sorption and precipitation, 0 biological assimilation, aggregation of inorganic or organic colloids, and . sinking of mineral and biogenic particles. While much of the particulate iron introduced via rivers, sediment resuspension, or as mineral aerosols will be removed by settling, ascertaining the underlying basis for the removal of “dissolved” iron forms is much more difficult. In regions with a high sinking flux of inorganic mineral particles (e.g. in some coastal and well mixed shelf waters), dissolved iron may be removed abiotically by sorption to surfaces of these particles. Similarly, sinking organic particles also can scavenge soluble iron from surface waters (Morel and Hudson, 1985). Dissolved iron also is “removed” via direct assimilation by phytoplankton. The subsequent sinking of live cells or fecal matter will transport a portion of this biogenic iron from surface waters. In addition to direct assimilation and sorption onto sinking (mineral and biogenic) particles, iron may sorb to colloidal organic matter which is abundant in surface waters (Wells and Goldberg, 1992, 1994). The stability of this colloidal phase is a topic of much dispute (Honeyman and Santschi, 1989; Bauer et al., 1992; Moran and Buesseler, 1992; Wells and Goldberg, 1993), but the extremely large colloidal surface area combined with the particle reactive nature of iron suggests that aggregation of organic colloids could be important for removing iron. Significant unresolved issues regarding iron removal include (1) identifying the specific mechanisms of iron sorption to abiotic and biotic sinking particles, which will shed light on how changes in iron speciation may affect this removal pathway, (2) changes in the iron “export efficiency” of the assimilation pathway with shifts in primary productivity or species assemblage, and (3) the abundance and reactivity of iron in the colloidal reservoir. The relative importance of abiotic, bioM.L. Wells et al.iMarine Chemistry 48 (199.5) 157-182 163 tic and colloid aggregation removal processes will vary from regime to regime and with season. (2) What are the chemical speciation and forms of iron among the soluble, colloidal and particulate fractions, including the rates and mechanisms of transformations among these forms? There are some large gaps in our knowledge of iron chemistry in seawater. The development of trace metal clean techniques for seawater collection and analysis over the past decade (Bruland et al., 1979; Gordon et al., 1982; Landing and Bruland, 1987) has given us reliable profiles for iron in the traditional categories of particulate (> 0.4 pm) and “dissolved” (< 0.4 pm) fractions; however, the chemical forms of iron within these fractions has been largely speculative. For example, there now is evidence that iron exists, at least partially, in the small colloidal phase (Wells and Goldberg, 1991; Wu and Luther, 1994; Powell and Landing, abstract) which is included in operationally defined “dissolved” fractions. Determining how iron is partitioned among these phases, and among various chemical forms within each phase, is central to understanding iron speciation in seawater.