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Phytoplankton carbon fixation gene (RuBisCO) transcripts and air-sea CO2 flux in the Mississippi River plume

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Phytoplankton carbon fixation gene (RuBisCO) transcripts and air-sea CO2 flux in the Mississippi River plume
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  ORIGINAL ARTICLE Phytoplankton carbon fixation gene(RuBisCO) transcripts and air-sea CO 2  fluxin the Mississippi River plume David E John 1 , Zhaohui A Wang 1 , Xuewu Liu 1 , Robert H Byrne 1 , Jorge E Corredor 2 , Jose´  M Lo´ pez 2 , Alvaro Cabrera 2 , Deborah A Bronk 3 , F Robert Tabita 4 and John H Paul 1 1 College of Marine Science, University of South Florida, St Petersburg, FL, USA;  2 Department of MarineSciences, University of Puerto Rico, Recinto Universitario de Mayagu¨ez, Mayagu¨ez, Puerto Rico;  3 Department of Physical Sciences, Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point,VA, USA and   4 Department of Microbiology, The Ohio State University, Columbus, OH, USA River plumes deliver large quantities of nutrients to oligotrophic oceans, often resulting insignificant CO 2  drawdown. To determine the relationship between expression of the major gene incarbon fixation (large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO) andCO 2  dynamics, we evaluated  rbcL  mRNA abundance using novel quantitative PCR assays,phytoplankton cell analyses, photophysiological parameters, and  p  CO 2  in and around theMississippi River plume (MRP) in the Gulf of Mexico. Lower salinity (30–32) stations were dominatedby  rbcL  mRNA concentrations from heterokonts, such as diatoms and pelagophytes, which were atleast an order of magnitude greater than haptophytes,  a - Synechococcus   or high-light  Prochlor- ococcus  . However,  rbcL  transcript abundances were similar among these groups at oligotrophicstations (salinity 34–36). Diatom cell counts and heterokont  rbcL  RNA showed a strong negativecorrelation to seawater  p  CO 2 . While  Prochlorococcus   cells did not exhibit a large difference betweenlow and high  p  CO 2  water,  Prochlorococcus rbcL  RNA concentrations had a strong positivecorrelation to  p  CO 2 , suggesting a very low level of RuBisCO RNA transcription among Prochlorococcus   in the plume waters, possibly due to their relatively poor carbon concentratingmechanisms (CCMs). These results provide molecular evidence that diatom/pelagophyte productiv-ity is largely responsible for the large CO 2  drawdown occurring in the MRP, based on the co-occurrence of elevated RuBisCO gene transcript concentrations from this group and reducedseawater  p  CO 2  levels. This may partly be due to efficient CCMs that enable heterokont eukaryotessuch as diatoms to continue fixing CO 2  in the face of strong CO 2  drawdown. Our work represents thefirst attempt to relate  in situ   microbial gene expression to contemporaneous CO 2  flux measurementsin the ocean. The ISME Journal   (2007)  1,  517–531; doi:10.1038/ismej.2007.70; published online 30 August 2007 Subject Category:  geomicrobiology and microbial contributions to geochemical cycles Keywords:  phytoplankton productivity; carbon dynamics; diatoms;  Prochlorococcus  ; nutrient plumes;RNA Introduction Perhaps the least understood components of theglobal carbon cycle are those that involve the coastalocean. About half of the approximately 115pg of carbon fixed by autotrophs annually is taken in bymarine organisms (Behrenfeld  et al  ., 2001). It isestimated that about 30% of anthropogenic CO 2 emissions are absorbed into the oceans (Sabine et al  ., 2004) and the ocean margins have recently been shown to take up about 20% of this anthro-pogenic CO 2  (Thomas  et al  ., 2004). Coastal seas,estuaries and river plumes are a fundamental part of the global carbon cycle because they link terrestrial,oceanic and atmospheric carbon reservoirs. River-dominated ocean margins are the most importantclass of margins in terms of their impact on carbonsequestration (Green  et al  ., 2006). In addition, river- born nutrients yield the highest rates of primaryproduction in the biosphere (Dagg  et al  ., 2004).The Mississippi is the Earth’s seventh largest river by discharge, and its outflow into the Gulf of Mexicocreates a plume of elevated phytoplankton abun-dance in the stratified and otherwise oligotrophicGulf waters. While Mississippi River water enteringthe Gulf of Mexico has very high dissolved inorganiccarbon (DIC) concentrations and is believed to be a Received 18 April 2007; revised 9 July 2007; accepted 9 July 2007;published online 30 August 2007Correspondence: DE John, College of Marine Science, Universityof South Florida, 140 7th Ave. S, St Petersburg, FL 33701, USA.E-mail: djohn@marine.usf.edu The ISME Journal (2007) 1,  517–531 &  2007 International Society for Microbial Ecology All rights reserved 1751-7362/07  $ 30.00 www.nature.com/ismej  source of atmospheric CO 2 , the region of mixingwith oceanic water fosters high levels of primaryproductivity and inorganic carbon uptake, resultingin an area of estimated CO 2  influx to the surfaceocean (Cai, 2003; Green  et al  ., 2006; Lohrenz andCai, 2006). Evidence indicates maximum phyto-plankton biomass and primary production occurs atintermediate salinities (20–30) in the MississippiRiver plume (MRP), while productivity is light-limited in very low salinity water and nutrient-limited at the edges and outside the plume, whereseawater CO 2  partial pressure (  p CO 2 ) increases andapproaches equilibrium with the atmosphere (Loh-renz  et al  ., 1999). Productivity rates of the MRP areamong the highest of river-influenced continentalshelf systems (Cai, 2003). Phytoplankton bloomscomposed largely of chain-forming diatoms have been documented (Bode and Dortch, 1996), particu-larly in the low-salinity plume water. Picoplanktonsuch as  Synechococcus  and picoeukaryotes, whilegenerally still in greater abundance than largerphytoplankton in the plume, appear to becomerelatively more prominent in regions with salinityaround 30, as plume waters mix with the Gulf of Mexico, while  Prochlorococcus  spp. are present inhigh numbers outside the plume (salinity  B 35) inoligotrophic Gulf of Mexico waters ( Jochem, 2003;Liu  et al  ., 2004; Wawrik and Paul, 2004). From a molecular standpoint, by far the mostcommon mode of inorganic carbon entry into the biosphere is via the enzyme ribulose-1,5-bispho-sphate carboxylase/oxygenase (RuBisCO). RuBisCOsequences are somewhat conserved across evolutionyet still exhibit sufficient variation to enablephylogenetic discrimination. Most phytoplanktoncontain the form I type of RuBisCO, includingmarine  a -cyanobacteria such as  Synechococcus and  Prochlorococcus  (form IA), chlorophytes and b -cyanobacteria (form IB), and chromophytic algaesuch as diatoms and prymnesiophytes (form ID)(Tabita, 1995, 1999) Although regulation of RuBisCOactivity is complex (Hartman and Harpel, 1994),most phytoplankton actively transcribe their largesubunit genes ( rbcL  ) daily, making  rbcL   mRNA agood molecular indicator of carbon fixation poten-tial (Pichard  et al  ., 1996; Paul  et al  ., 2000b). Muchwork has focused on the measurement of   rbcL  mRNA transcript abundances to gain informationabout the occurrence and phylogenetic diversity of active carbon-fixing organisms in the marine envir-onment (Wyman  et al  ., 2000; Paul  et al  ., 2000a;Wawrik  et al  ., 2003, 2004; Corredor  et al  ., 2004). Ourprevious work on RuBisCO gene expression andphytoplankton dynamics in the Gulf of Mexico hasrevealed elevated form IA  rbcL   transcript andprimary productivity associated with offshore ordistal plume environments (Paul  et al  ., 2000a;Wawrik  et al  ., 2003, 2004). However, this work haslargely excluded the most biologically active watersof the MRP immediately to the south and west of thedelta, where other research has demonstrated  p CO 2 gradients that favor carbon flux to surface waterswhereby the plume may act as a carbon sink.The analysis of   rbcL   transcript abundances isimportant for giving not only an indication of presence but also relative  rbcL   gene expressionlevels from phytoplankton populations sampled.Following development of a quantitative reversetranscription PCR method for quantifying  rbcL  mRNA (Wawrik  et al  ., 2002), we have recentlydescribed a suite of quantitative reverse trans-cription PCR assays to quantify  rbcL   RNA from Synechococcus , high-light  Prochlorococcus , hapto-phytes, and the heterokont group from the pre-viously described assay which encompassesdiatoms, pelagophytes, pinguiophytes and dictyo-chophytes (silicoflagellates) ( John and Paul, 2007).These groups were targeted initially because  rbcL  mRNA clone library analysis from the Gulf of Mexico identified diatoms, prymnesiophytes,  a - Synechococcus , and  Prochlorococcus  to be thedominant-active carbon fixers in plume and oligo-trophic waters (Wawrik  et al  ., 2003; Wawrik andPaul, 2004).The inverse correlation between phytoplankton biomass (as chlorophyll- a ) and surface CO 2  levels inocean margin environments has been demonstratedrecently in a coastal area of the Mediterranean Sea(Huertas  et al  ., 2005). Still, data on the relationship between phytoplankton communities and  p CO 2 levels of the surface oceans are lacking, particularlywith respect to which members of the phytoplank-ton community can be the most important ineffecting CO 2  drawdown. In the current study, weaddressed the question of which major phyto-plankton groups are present and active with respectto CO 2  dynamics in the MRP and northern Gulf of Mexico. We employed a number of analyses,including  rbcL   mRNA quantification using newreal-time PCR assays and form-specific hybridi-zation probes, phytoplankton cell abundances byflow cytometry and microscopic counts, primaryproductivity measurements, and size fractionation of samples along with underway and discrete inorgan-ic carbon measurements to answer this question. Methods RNA sampling and   rbcL  gene transcript quantification Sampling was performed aboard the  R/V Pelican in July 2005. For mRNA, seawater samples (450–750ml) were collected from 3m depth using anelectric submersible pump (Rule, White Plains, NY,USA). Phytoplankton were filtered onto 0.45 m mDurapore HVLP filters (Millipore, Billerica, MA,USA) and filters were stored in 2ml polypropylenecryotubes previously filled with 0.3ml of muffled200 m m low-protein-binding zirconium oxide grind-ing beads (OPS Diagnostics, Bridgewater, NJ, USA)and 750 m l RLT buffer (Qiagen, Valencia, CA, USA)with 10 m l/ml  b -mercaptoethanol (Sigma, St Louis,  rbcL  mRNA and  p CO 2  in the Mississippi River plume DE John  et al 518 The ISME Journal  MI, USA). Samples were frozen and stored in liquidnitrogen for the duration of the cruise, and stored at  80 1 C upon return to the lab until analysis.RNA purification was performed in the lab usingthe RNEasy extraction kit (Qiagen) on a vacuummanifold with on-column DNA digestion usingRNAse-free DNAse (Qiagen) according to manufac-turer’s instructions. Columns were rinsed twiceusing 750 m l RPE buffer rather than the recom-mended 500 m l to ensure adequate removal of guanidinium-containing buffers RW1 and RLT fromthe sides and ledge in the columns. Columns werecentrifuged at 16100 g   for 2min to dry followingRPE rinses. RNA was eluted using 50 m l roomtemperature RNAse-free H 2 O. Purified RNA wasdiluted up to 10-fold to reduce the possibility of PCRinhibition by compounds co-purified with the RNA.PCR oligonucleotides employed were as reportedpreviously ( John and Paul, 2007; Supplementarydata). Reactions were performed using the TaqmanOne-step RT-PCR master mix kit (Applied Biosys-tems, Foster City, CA, USA) on an ABI Prism 7700Sequence Detector real-time PCR instrument (Ap-plied Biosystems). PCR reactions were composed of primers at concentrations of 400n M  each, withprimers pooled in assays with multiple primers(thereby giving total concentrations ranging from400n M  to 2 m M , depending on the assay), probes atconcentrations of 125n M  for single-probe assays and75n M  each for two-probe assays (150n M  total),1.25 m l Multiscribe reverse transcriptase (from Taq-man kit), 25 m l 2  Taqman master mix, 10 m l tem-plate in RNAse-free H 2 O, and the balance to 50 m lwith RNAse-free H 2 O. Thermocycling conditions forheterokonts were as follows: 45 1 C hold for 30min,95 1 C hold for 10min, then 40 cycles of 95 1 C for 20s,52 1 C for 60s and 72 1 C for 60s; haptophytes, high-light  Prochlorococcus  and  Synechococcus  wereperformed at 45 1 C hold for 30min, 95 1 C hold for10min, then 40 cycles of 95 1 C for 20s, 54 1 C for 60sand 72 1 C for 60s.Data were analyzed using the Sequence DetectionSystems software version 1.9 (Applied Biosystems).Standard curve reaction templates were createdfrom our clone libraries of   rbcL   genes (Wawrik andPaul, 2004) and reactions consisted of a pool of three in vitro  transcripts from the target clade. Standardcurves encompassing five orders of magnitude weregenerated for each PCR run. Results were inter-preted in terms of mass of transcript RNAs madeusing the Quant-iT Ribogreen kit (Invitrogen, Carls- bad, CA, USA). Each RNA extraction was analyzedfor all four assays by performing reactions in twogroups according to annealing temperature andholding the extracted RNA on ice during the interimwhile the first round of PCR was running. Averagevalues for the respective groups were calculatedusing data from two separate samples.RNA extraction and sample analyses for dot-blothybridization were performed as has been describedpreviously (Wawrik  et al  ., 2003; Corredor  et al  .,2004). Briefly, RNA was purified using RNeasycolumns and dot blots were quantitatively analyzedusing antisense  35 S-labeled probes for form IA, formIB and form ID  rbcL.  Quantitative standard curveswere created from dilutions of sense  in vitro transcripts made from the respective probe templatesequence. Standard curve dilutions were blotted inthe same manner as environmental samples andprobed with respective riboprobes along withenvironmental sample blots. Samples were analyzedin duplicate. Productivity and photophysiology  For photophysiology analysis, samples taken inTeflon-lined Niskin bottles were immediately trans-ferred to 1-liter light-shielded, acid-washed poly-ethylene bottles. Samples of 650ml were spikedwith 0.108mCi of   14 C-bicarbonate (Amersham Bio-sciences, Piscataway, NJ, USA). Aliquots (40ml) of spiked water were transferred to 40-ml borosilicateEPA vials and incubated in a photosynthetronapparatus (CHPT Mfg, Georgetown, DE, USA) at  insitu  temperature and at irradiances ranging from 0(dark sample) to 1000 m Em  2 s  1 . Time-zero sample blanks were immediately filtered before commence-ment of incubation. Following incubation (1–2h),samples were sequentially filtered onto 2 and 0.2 m m25-cm membrane filters and treated with 250 m l 10%HCl to drive off unfixed [ 14 C]bicarbonate. After 24h,10ml of scintillation fluid was added, and sampleradioactivity was determined by liquid scintillationcounting in the channels ratio mode. The resultingdata were plotted in PE (productivity vs irradiance)curves. The biomass (chlorophyll- a ) normalizedphotosynthetic parameters  a B (light-limited slope), P  Bmax  (light saturated rate) and  b B (photoinhibitionslope) were computed using the exponential for-mulation of  Platt  et al  . (1990). Samples for chlor-ophyll- a  analysis (200ml) were sequentially filteredthrough 2 and 0.2 m m membrane filters, frozen inliquid nitrogen and then ground in 5ml 90%acetone solution in a Potter–Elvejhm grinder usinga glass fiber filter to assist in cell disruption.Fluorescence analysis was performed following themethod of  Welschmeyer (1994). Carbonate chemistry analyses Shipboard CO 2 -parameter analyses were performedusing an automated flow-through multiparameterinstrument (Wang  et al  ., 2007). The instrument takescomplete measurement of air  p CO 2 , seawater  p CO 2 ,DIC and pH every 7min. Detailed procedures of thespectrophotometric measurements of these carbonparameters have been presented previously (Byrne et al  ., 2002; Wang  et al  ., 2007). Briefly for  p CO 2 , aninternal alkalinity standard with a sulfonephthaleinindicator (phenol red) is enclosed inside a liquidcore waveguide made of Teflon AF 2400 capillarytubing (DuPont, Wilmington, DE, USA), which  rbcL  mRNA and  p CO 2  in the Mississippi River plume DE John  et al 519 The ISME Journal  forms the long pathlength spectrophotometric cellduring measurements. CO 2  samples are directed toflow surrounding the liquid core waveguide, whichalso serves a CO 2 -permeable membrane. The inter-nal standard indicator solution reaches CO 2  equili- brium with sample across the liquid corewaveguide, solution pH is measured with a spectro-photometer, and  p CO 2  is then calculated. Theinternal indicator solution is renewed for eachmeasurement.  p CO 2  measurements are calibratedagainst known  p CO 2  gas standards. For DIC mea-surements, sampled seawater is first acidified using3 N  HCl to convert all inorganic carbon species toCO 2 , which is subsequently measured using bromo-cresol purple as the indicator. DIC measurements arecalibrated with Certified Reference Material (CRM)from Dr AG Dickson at the Scripps Institution of Oceanography (La Jolla, CA, USA). All measure-ments and calibrations are conducted at a constanttemperature (25 1 C) using a water thermostat. Thesystem is also equipped with a pressure gauge formeasurement of atmospheric pressure and a CTD formeasurements of temperature and salinity of watersamples. The entire system and its measurementsequence are fully automatic and controlled by a PCthrough a custom interface. The measurementprecisions are  7 1 m atm for water and atmospheric  p CO 2 , and 7 2 m molkg  1 for DIC.During field measurements, seawater sampleswere pumped into the ship using an on-board waterpump with an inlet about 3m below the surface. Themultiparameter CO 2  system withdrew water fromthe underway stream through a peristaltic pump.Atmospheric sample air was pumped from the frontof the ship about 10m above sea surface and wasdirected to the underway CO 2  system. Results of seawater  p CO 2  measured at 25 1 C were corrected tothe field temperature based on thermodynamiccalculation of the carbonate system. During thesurvey, calibrations of   p CO 2  and DIC were con-ducted occasionally to assure that no drift occurred.For hydrographic casts and time series monitoring,discreet samples of DIC were collected in stopperedglass bottles free of headspace and preserved withsaturated HgCl 2 . These DIC samples were subse-quently measured on board using the underway CO 2 system described above. Phytoplankton cell counts Samples for phytoplankton cell counts were con-centrated by reverse filtration (Dodson and Thomas,1978) using 1 m m pore-sized polycarbonate filters.Typically, 400ml was concentrated to approxi-mately 25ml of which 5ml was counted on a Zeissinverted microscope following the procedures out-lined by Hasle (Dodson and Thomas, 1978). Theentire chamber was counted at 100   for largerspecies while two to four chamber transects werecounted at 400   for smaller species. Speciesidentifications were based on descriptions in theTomas manual of phytoplankton identification(Tomas, 1997). Samples for flow cytometry wereprocessed in the lab of Lisa A Campbell at Texas Aand M University according to protocols describedpreviously (Campbell, 2001). Diel drift study  A Lagrangian drift study was performed to measurediel changes in  rbcL   transcript abundances and theother parameters. A drogued drift buoy was de-ployed and followed for over 24h. Samples from 3mwere taken every 4h as described above for RNAanalyses and photophysiological parameters. Sur-face water  p CO 2  and DIC were also measured usingthe underway CO 2  system described above. Results Surface seawater characteristics in study region Locations of sampling stations are shown in Figure 1and Table 1. Station 1 was just off the Florida Shelf in oligotrophic waters (not shown in Figure 1).Stations 3, 4 and 6 were in the area of greatest riverinfluence, evidenced by satellite ocean color, sali-nity and chlorophyll- a . Stations 6 and 7A–F werepart of a Lagrangian study, thus were rather close inproximity. The salinity of sampling stations rangedfrom 30.1 to 35.9 (Table 1). Satellite-estimatedchlorophyll content of surface water within thesampling area (Figure 1a) indicates highly produc-tive waters were present at the time of sampling.Seawater DIC and  p CO 2  for all stations ranged from1911 to 2031 m molkg  1 and 188–437 m atm, respec-tively; both generally increased with salinity,although the station with lowest  p CO 2  (Station 4)did not have lowest DIC concentrations. Atmo-spheric  p CO 2  over sampling stations (corrected to100% humidity) was fairly constant at 370 7 3 m atm.Underway carbon measurements were used to chartspatial dynamics of surface water  p CO 2  (Figure 1b).Low seawater  p CO 2 , well below atmospheric levels,was observed in the plume region, resulting in anarea of estimated CO 2  drawdown (Stations 3–7).Conversely, in the oligotrophic waters outside theMRP, a small degree of CO 2  efflux was observed(Stations 1, 2 and 8).These parameters allow the categorization of sampling stations into two basic surface marineregimes (Figure 2): (1) a ‘plume’ area characterized by lower salinity from 30 to 32, surface  p CO 2  fromunder 200 to just over 300 m atm and CO 2  flux intothe surface ocean from the atmosphere, elevatedchlorophyll- a , and elevated maximum photosyn-thetic rate ( P  max ); and (2) the open, oligotrophic Gulf of Mexico with salinity over 34,  p CO 2  over 400 m atm,and much lower chlorophyll- a  and  P  max  measure-ments. The size distribution of photosynthetic cells,indicated by chlorophyll- a  content, also varied between these two regimes (Figure 2b); larger cells  rbcL  mRNA and  p CO 2  in the Mississippi River plume DE John  et al 520 The ISME Journal  ( 4 2 m m) contained the bulk of total chlorophyll- a within the plume, while outside it the photosyn-thetic biomass was dominated by picoplankton o 2 m m. Phytoplankton cell numbers Two types of phytoplankton cell count data wereobtained: picophytoplankton ( Prochlorococcus ,  Sy-nechococcus  and picoeukaryotes) were enumerated by flow cytometry, from both the o 2 m m and whole(unfiltered) size fractions, while microphytoplank-ton such as diatoms, dinoflagellates,  Trichodes-mium  and microflagellates (which were notdifferentiated into auto- or heterotrophic organisms,see Methods) were counted by light microscopyfrom preserved samples. Figure 3 shows micro-plankton and picoplankton (whole fraction) countsas a function of surface water  p CO 2 .  Synechococcus cells were in greatest abundance in the plume, andranged from about 1  10 8 l  1 to a peak of over6  10 8 l  1 at Station 7F at the end of the diel study. Synechococcus  cell abundance in high  p CO 2 , oligo-trophic waters was  o 3% of the maximum abun-dance we measured (Figure 3a).  Prochlorococcus were more numerous far outside the plume (Stations        L      a       t       i       t      u       d      e 28.828.628.428.2 7F65432 -89.9 -89.7 -89.5 -89.3 -89.1 -88.9 -88.7 -88.5 -88.3 -88.1 -87.9 p  CO 2  flux (mmol/m 2 )/day)Longitude181410620-2-6-10-14-18-22-26-30-34 Figure 1  ( a ) Composite SeaWiFS image of estimated surface chlorophyll- a  concentrations during cruise dates in July 2005. Samplingstations are shown, with 1 oligotrophic station out of view (Station 1). Station 7   was not sampled for data reported here. (  b ) EstimatedCO 2  flux (based on Wanninkhof parameterization (Wanninkhof, 1992)) over the MRP area, showing sampling sites and cruise trackwithin or near the plume. The negative sign of numbers in the lower panel indicates the direction of   p CO 2  flux is from atmosphere tosurface ocean. Two sampling sites far outside the MRP are not shown (Stations 1 and 8). MRP, Mississippi River plume.  rbcL  mRNA and  p CO 2  in the Mississippi River plume DE John  et al 521 The ISME Journal
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