Phospholipid synthesis rates in the eastern subtropical South Pacific Ocean

Phospholipid synthesis rates in the eastern subtropical South Pacific Ocean B. A. S. Van Mooy, T. Moutin, S. Duhamel, P. Rimmelin, and F. Van Wambeke Department of Marine Chemistry and Geochemistry, Wood Hole Oceanographic Institution, MS #4, Wood Hole, MA 02543, USA Laboratoire d’Océanographie et de Biogéochimie, UMR-CNRS 6535, Case 901, Centre d’Océanologie de Marseille, Université de la Méditerranée – Campus de Luminy, 13 288 Marseille cedex 9, France Laboratoire de Microbiologie, Géochimie et Ecologie Marines, UMR-CNRS 6117, Case 901, Centre d’Océanologie de Marseille, Université de la Méditerranée – Campus de Luminy, 13 288 Marseille Cedex 9, France Received: 17 July 2007 – Accepted: 6 August 2007 – Published: 20 August 2007 Correspondence to: B. A. S. Van Mooy (bvanmooy@whoi.edu)


Introduction
Cell membrane lipids form the interface between a cell and its environment, and, as such, house many of the enzyme and transporter systems that are required to harvest energy and material from the environment.Microbial picoplankton (i.e.plankton <2 µm) have higher cell surface area to cell volume ratios than larger plankton, and therefore we would expect cell membrane lipids to be a particularly important biochem-Introduction

Conclusions References
Tables Figures

Back Close
Full Screen / Esc Printer-friendly Version

Interactive Discussion
EGU ical component of picoplankton.The picoplanktonic community dominates the surface waters of the open ocean (e.g.Cho and Azam, 1990) and is composed of diverse populations of picoeukaryotes, cyanobacteria and heterotrophic bacteria (e.g.Campbell and Vaulot, 1993;Cavender-Bares et al., 2001).Membrane lipids generally compose 15 to 25% of the carbon in planktonic cells (Wakeham et al., 1997), and, as such, their synthesis constitutes a substantial fraction of overall community anabolism and carbon demand.The synthesis of one class of membrane lipids, the phospholipids, creates an additional burden to planktonic cells because it creates a demand for nutrient phosphorus.In the surface waters of the North Pacific subtropical gyre (NPSG) the synthesis of phospholipids was shown to consume 18 to 28% of the PO 3− 4 taken up by the total planktonic community (Van Mooy et al., 2006).However, the demand for phosphorus created by phospholipid synthesis does not appear to be equally distributed among the various types of plankton that compose the total planktonic community.For example, the synthesis of phospholipids constitutes less than a few percent of overall phosphorus demand by Prochlorococcus, the picocyanobacterium that dominates the phytoplanktonic community of the NPSG and South Pacific subtropical gyre (SPSG) (Bj örkman et al., 2000;Campbell et al., 1994;Van Mooy et al., 2006;Grob et al., 2007).In addition to phospholipids, cyanobacterial membranes also contain abundant glycolipids and sulfolipids (Wada and Murata, 1998).Moreover, these organisms appear to have the ability to substitute sulfolipids for phospholipids when nutrient phosphorus concentrations are low in the environment (Benning, 1998).The observation that sulfolipids were the most abundant membrane lipid in the surface waters of the NPSG, led Van Mooy et al. (2006) to hypothesize that cyanobacteria employ few phospholipids and that heterotrophic bacteria were the organisms primarily responsible for phospholipid synthesis.
We sought to examine the importance of phospholipid synthesis under a range of biological and chemical conditions, with the intention of refining our understanding of the biological origin of phospholipids and the role of phospholipid synthesis within the overall upper ocean phosphorus cycle.We encountered such a range of conditions on ), which was a transect between the hyperoligotrophic waters of SPSG and the nutrient-rich waters upwelled off Chile.We quantified phospholipid synthesis rates at several depths at twelve different stations, and compared these data to chlorophyll fluorescence, primary production rates, and heterotrophic bacterial production rates.These data add further support to the hypothesis that phospholipids are primarily of heterotrophic bacterial origin, and as such, phospholipid synthesis rates are a molecular proxy for the relative role of heterotrophic bacteria in the upper ocean phosphorus cycle.

Sampling
Data in this paper were obtained during the second leg of the BIOSOPE cruise between Easter Island and Concepcion, Chile aboard the R/V L' Atalante (Fig. 1; Claustre et al., 20071 ).Seawater was collected using a Niskin bottle at depths corresponding to either 100, 50, 15, 3, 1 or 0.3% of surface photosynthetically available radiation (PAR).

Incubations
We collected 250 ml samples of seawater directly from the Niskin bottle into acidwashed polycarbonate bottles.These bottles were then spiked with 0.37 MBq of 33 PO 3− 4 (Amersham), which was an amendment of approximately 130 pmol L −1 of 33 PO 3− 4 and was less than 1% of ambient concentrations of PO

Phospholipid extraction and analysis
Following filtration, the membranes were immediately immersed in a glass centrifuge tube containing a Bligh and Dyer (1959) extraction mixture, which consisted of 1.5 ml of dichloromethane, 3 ml of methanol and 1.2 ml of 0.1 X phosphate buffered saline (PBS) solution and stored overnight at -20 • C. The following day, 1.5 ml of dichloromethane and 1.5 ml MilliQ water (Millipore) were added.The samples were centrifuged and the lower organic phase, containing the phospholipids, was recovered.These total phospholipid extracts were used to determine the 33 PO 3− 4 incorporation rates into the total phospholipids: 500 µL of the extract was mixed with 10 mL of UltimaGold scintillation cocktail (PerkinElmer) in a 20 ml polyethylene scintillation vial.The 33 P radioactivity was determined on a Tricarb scintillation counter (Packard) using standard methods (Duhamel et al., 2006).

Calculations
The steady state production rates of phospholipids were determined as follows: Where P is the hourly production rate of phospholipids (pmol P L −1 h −1 ), A is the 33 P radioactivity of the phospholipid extract (dpm), S is the specific 33 P radioactivity of

EGU
Phosphate concentrations were reported by Moutin et al. (2007).Water from the same Niskin bottles was also used to determine the total PO 3− 4 incorporation rates using standard methods (Moutin et al., 2007).Contribution of phospholipid production rates to the total PO 3− 4 incorporation rates were expressed as percent by taking the quotient of the two values and multiplying by 100.Only one phospholipid synthesis rate measurement was made at each depth, except at 30 • S 98 • W where samples were conducted in triplicate and average error was found to be 6.2%.Data were plotted using Ocean Data View software (Schlitzer, R., Ocean Data View, http://odv.awi-bremerhaven.de,2004).

Additional measurements
Chlorophyll fluorescence data were obtained in situ using a Chelsea Aquatracka MkIII fluorometer and are expressed in relative units.Heterotrophic bacterial production (BP) hourly rates were determined using 3 H-leucine incubation and standard microcentrifuge-based protocols as described by Van Wambeke et al. (2007).Primary production (PP) rates were determined as hourly dissolved inorganic carbon uptake rates into particulate material using H 14 CO − 3 incubations as described by Duhamel et al. (2007).Rates of BP, PP and phospholipid synthesis were determined from waters sampled during the same cast and usually from the same Niskin bottles.

Results
Phospholipid synthesis rates ranged from 1 pmol P L −1 h −1 in the SPSG to >200 pmol P L −1 h −1 off the coast of Chile (Fig. 2a).In general, the phospholipid synthesis rates were relatively constant in the hyperoligotrophic waters west of 98

EGU
When expressed as a percentage of total PO 3− 4 incorporation (Fig. 2b), the phospholipid synthesis rates ranged from 4% to 23% of the total PO 3− 4 incorporation rate, with the average being 14±5% (mean ± s.d.; n=48).Highest percentages were observed in the upper mixed layer of the SPSG, while the lowest rate percentages were observed off the coast of Chile.There was a deep minimum layer in these rate percentages that gradually shoaled between 105 • W and 90 • W; east of this, minimum values were observed in a layer centered at about 50 m depth with slightly higher percentages above and markedly higher percentages below.This pattern closely resembled that of chlorophyll fluorescence (Fig. 2c), which showed a maximum layer that shoaled from west to east and centered at about 50 m east of 90 • W.

Discussion
During the second leg of the BIOSOPE cruise we encountered a broad range of oceanographic conditions and this was reflected in the phospholipid synthesis rates that we measured, which spanned nearly two orders of magnitude (Fig. 2a).As expected, the rates increased from west to east in conjunction with increasing rates of biomass production (Van Wambeke et al., this issue).The phospholipid synthesis rates in the SPSG were generally ≤5 pmol P L −1 h −1 , which is an order of magnitude slower than observed in the NPSG where the rates ranged from 40 to 120 pmol P L −1 h −1 (Van Mooy and Devol, 20072 ; Van Mooy et al., 2006).Thus, phospholipid synthesis rates in the SPSG reflected the extreme hyperoligotrophic conditions in this region (Claustre et al., 2007 1 ).When viewed as a percentage of total PO 3− 4 incorporation, it is clear that phospholipid synthesis was a major component of the upper ocean phosphorus cycle across the entire transect (Fig. 2).However, this was particularly true in the near-surface wa-Introduction

Conclusions References
Tables Figures

Back Close
Full Screen / Esc Printer-friendly Version

Interactive Discussion
EGU ters of SPSG, were values in excess of 15% were consistently observed.So despite the fact that overall phospholipid synthesis rates were an order of magnitude slower in the SPSG than in the NPSG, the relative percentages that phospholipid synthesis contributed to total PO 3− 4 incorporation were very similar to those in the NPSG, which were also in excess of 15% (Van Mooy et al., 2006).It has been argued that heterotrophic bacteria are the primary source of phospholipids in the NPSG (Van Mooy et al., 2006), and we suggest here that the common percentage of phospholipid synthesis in the NPSG and SPSG reflects the common planktonic community structure of these environments, where heterotrophic bacteria compose the majority of total planktonic cells (Bj örkman et al., 2000;Campbell et al., 1997;Grob et al., 2007).
We observed a layer where phospholipid synthesis contributed <15% of total PO 3− 4 incorporation, which coincided with a layer of maximum chlorophyll fluorescence (Figs.2b, c).At the station at 92 • W, chlorophyll fluorescence was highest between 50 and 100 m depth, and Grob et al. ( 2007) observed maximums in the abundances of picoeukaryotes and Prochlorococcus in this same depth interval.This low contribution of phospholipid synthesis to total PO43-incorporation coupled with the abundance of Prochlorococcus suggests that production by phytoplankton contributed disproportionately less to phospholipid synthesis than heterotrophic bacteria.In culture studies, cyanobacteria have been shown to use very little of total PO 3− 4 incorporation for phospholipid synthesis (Cuhel and Waterbury, 1984;Van Mooy et al., 2006).Cyanobacteria and picoeukaryotes are also often very rich in membrane lipids that do not contain phosphorus, such as sulfolipids, glycolipids, and betaine lipids (e.g.Bell and Pond, 1996;Kato et al., 1996;Van Mooy et al., 2006;Wada and Murata, 1998).In the NPSG, RNA synthesis accounted for about half of total PO 3− 4 incorporation and, as such, was a much larger biochemical sink for PO 3− 4 than phospholipid synthesis (Van Mooy and Devol 2 ; Van Mooy et al., 2006).Thus we interpret the minimum layer in percent phospholipid synthesis as a layer where nucleic acid synthesis by phytoplankton dilutes phospholipid synthesis by heterotrophic bacteria.Further support for this interpretation comes from the data from the below the chlorophyll maximum: due to

Conclusions References
Tables Figures

Back Close
Full Screen / Esc

Printer-friendly Version
Interactive Discussion

EGU
the decrease in PP with depth the relative contribution of heterotrophic bacteria to total biomass production would presumably be greater below the chlorophyll maximum than at shallower depths, and, indeed, phospholipid synthesis contributes a greater percentage of total PO 3− 4 uptake below the chlorophyll maximum (Fig. 2b).If phytoplankton did indeed make a disproportionately smaller contribution to phospholipid synthesis than heterotrophic bacteria, then we would expect the percent contribution of phospholipid synthesis to total PO 3− 4 uptake to have been lowest where phytoplankton contributed the most to overall biomass production.To determine whether this was true, we examined the relationship between the ratio of PP to BP (PP:BP) and the percent contribution of phospholipid synthesis to total PO 3− 4 incorporation by plotting these data versus one another (Fig. 3); all of these parameters are methodologically independent.We performed a linear regression of the data and found that nearly half of the variance (r 2 =0.49;P<0.01) in the percent contribution of phospholipid synthesis to total PO 3− 4 incorporation could be explained by the variance in PP:BP.This is a remarkable relationship considering the immense diversity in the populations of both phytoplankton and heterotrophic bacteria across this nearly 3000 km transect.Furthermore, the relationship is not driven simply by data from the uniquely hyperoligotrophic SPSG portion of the transect, since data points from west of 98 • W span almost the entire range of values.The y-intercept, where PP:BP is zero, predicts that heterotrophic bacteria alone dedicate 21.1±8.3%(mean ± s.d.) of total PO 3− 4 incorporation to phospholipid synthesis.In contrast, we regressed the data with an inverse first order equation (r 2 =0.46;P<0.01; not shown) to represent the approach to infinite PP:BP, and the percent contribution of phospholipid synthesis to total PO 3− 4 incorporation by phytoplankton alone was predicted to be 7.2±7.7%.This analysis supports our hypothesis that heterotrophic bacteria are a more important source of phospholipid synthesis than phytoplankton.Admittedly, the population differences of the endmember communities probably impacts the estimates of percent phospholipid synthesis by phytoplankton and heterotrophic bacteria.For example, the highest ratios of PP:BP occur east of 98 • W outside of the hyperoligotrophic gyre and outside of the area domi-  Grob et al., 2007), which could explain why the estimate of 7.2% from the regression is so much higher than the 0.4% observed in axenic strains of Prochlorococcus (Van Mooy et al., 2006).Furthermore, PO 3− 4 was present in abundance at every station and depth that we examined and was never the nutrient that limited either phytoplanktonic or bacterial production (Bonnet, 2007; Van Wambeke 3 ), thus, it is reasonable to expect that phytoplankton were free to employ optimal proportions of phospholipids versus glycolipids and sulfolipids in their membranes.It is important to recognize that the "sulfolipid-phospholipid substitution hypothesis" of Benning (1998) predicts that phytoplankton would have the biochemical motivation to synthesize even fewer phospholipids under conditions where phosphorus is the limiting nutrient, such as in the Mediterranean Sea (Moutin et al., 2002;Thingstad et al., 2005).
Given that heterotrophic bacteria were responsible for the majority of phospholipid synthesis we can use the synthesis rate data to constrain the role of this group of organisms within the overall phosphorus cycle observed during the BIOSOPE cruise.First, it is clear that heterotrophic bacteria play an very important role in the phosphorus cycle of the SPSG, which agrees with Duhamel et al. (this issue) who found that the fraction of plankton <0.6 µm were responsible for the about a third of PO 3− 4 incorporation.Second there were two patches where phospholipid synthesis contributed <7.5% of total PO 3− 4 uptake (Fig. 2b) and these represent areas where phytoplankton production played a more dominant role in the cycling of phosphorus.This type of information has important implications for understanding the distribution of phytoplanktonic vs. bacterial sinks for PO We found that phospholipid synthesis was a major component of the upper ocean phosphorus cycle during the BIOSOPE cruise.Phospholipid synthesis made the greatest percentage contribution to total PO 3− 4 incorporation in the surface waters of the SPSG, and the least in the upwelled waters off of Chile.Furthermore, there was a layer in minimum phospholipid synthesis percentage values that corresponded to the layer of maximum chlorophyll concentrations.Regression analyses showed a strong relationship between the variance in phospholipid percentages and variance in PP:BP.These analyses also predicted that heterotrophic bacteria may utilize more than a fifth of their total PO 3− 4 incorporation for the synthesis of phospholipids, while phytoplankton may utilize only several percent.Thus phospholipid synthesis is a term in the phosphorus cycle that is dominated by heterotrophic bacteria, and the distribution of the phospholipid synthesis rate during the BIOSOPE cruise confirmed that heterotrophic bacteria play a very important role in the phosphorus cycle of the SPSG.

Fig. 1 .
Fig. 1.Map of stations where phospholipid synthesis rates were determined during the second leg of the BIOSOPE cruise.