Marine phytoplankton fix carbon dloxide primarily through the action of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the first enzyme in the Calvin Cycle. Although the regulation of this enzyme has been studied in algal cultures and higher plants, little is known regarding RubisCO regulation in natural phytoplankton populations. To determine if natural communities of phytoplankton utilize transcriptional regulation to control RubisCO expression, we investigated the diel relationship between '" carbon fixation and RublsCO large subunit (rbcL) transcript levels, rbcL DNA, chlorophyll a, autofluorescent cell counts and bacterial direct counts for natural communities of the southeastern Gulf of Mexico. Studies were performed with natural phytoplankton populations in Lagrangian studies or deck-top incubators as well as with a Prochlorococcus isolate in culture from the North Pacific Ocean. rbcL mRNA and DNA were detected by stringent hybridization using an antisense rbcL RNA gene probe originating from Synechococcus sp. PCC 6301. For natural communities, carbon fixation maxima (-0.1 to 0.7 pg C 1-' h-') occurred from early morning to mld afternoon, with minimal values at nlght. Peak levels of rbcL mRNA (3.6 to 22.2 ng 1-') almost always coincided with the time of maxlmum carbon fixation and were positively correlated. When natural phytoplankton population~ or the Prochlorococcus culture were maintained in continuous illuminat~on for 24 h, the same die1 pattern of RubisCO regulation was observed, implying that diel entra~ned rhythms in rbcL transcription occurred. The results thus indicate that natural phytoplankton communrties appear to regulate carbon fixatlon at least in part by transcriptional control of RubisCO synthesis and that such patterns appear to be rhythmlc in nature.

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The factors affecting the regulation of photosynthetic carbon fixation in diverse phytoplankton populations are not yet understood. To this end, we have measured the expression of the gene (rbcL) for the major carbon fixation enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase, in coastal phytoplankton populations off Cape Hatteras and in oligotrophic oceanic picoplankton near Bermuda. Using gene probes specific for the cyanobacterial/chlorophytic clade and the chromophytic clade (diatoms, chrysophytes, prymnesiophytes, and others) of Form I rbcL genes (''cyano'' and ''chromo'' probes, respectively), we have measured rbcL messenger ribonucleic acid (mRNA) levels in size-fractionated coastal waters, in a decktop diel incubator and a Lagrangian drifter study, and in vertical profiles in stratified, oligotrophic ocean water. In coastal waters influenced by estuarine plumes, an equal distribution of carbon fixation between the picoplankton and the micro/nannoplankton occurred, with cyano rbcL mRNA coinciding with Synechococcus counts in the Ͻ1-m fraction, with the majority of the chromo rbcL mRNA expression occurring in the larger sized phytoplankton fraction. In profiles of oligotrophic oceanic waters, the cyano rbcL mRNA was found in the upper water column (ϳ50-m depth) and coincided with peaks in Synechococcus counts. The chromo rbcL mRNA was concentrated at the subsurface chlorophyll a (Chl a) maximum (ϳ85 m) and corresponded to red-fluorescing cell counts, thought to be picoeucaryotes and diatoms. Photosynthetic carbon fixation and RUBISCO enzyme activity encompassed both cyano and chromo rbcL mRNA peaks, suggesting a near equal contribution to carbon fixation in the water column by these two phytoplankton clades. Both decktop diel incubator studies and a Lagrangian drifter study in coastal waters indicated cyano rbcL transcription in the morning and chromophytic rbcL transcription in the late afternoon/early evening. Thus, the two major clades of RUBISCOcontaining phytoplankton occupy separate niches in time, space, and cell size in the waters off Cape Hatteras. The factors determining such clade-specific niches may include efficiency of nutrient utilization, differences in relative carboxylase/oxygenase activity ( values) of cyano (Form IB) and chromophytic (Form ID) RUBISCOs, and differences in pigment composition/adaptation to light regimes. Additionally, we propose that chromo rbcL mRNA may be indicative of new production, whereas cyano rbcL mRNA correlates with recycled production in stratified, oligotrophic oceanic environments.Although photosynthetic carbon fixation is one of the most important biological processes occurring in the ocean, little is known concerning the molecular regulation of this process in natural phytoplankton assemblages. The enzyme responsible for the vast majority of photosynthetic carbon fixation by phytoplankton is ribulose bisphosphate carboxylase/oxygenase (RUBISCO) (Raven 1993). There is evidence for ␤-carboxylation (Descolas- Gros and Fontugne 1985;Colman 1989;C...

To understand the composition and photosynthetic carbon fixing activities of natural phytoplankton communities, we employed group-specific ribulose bisphosphate carboxylase (RubisCO) large subunit gene probes (rbcL) to examine RubisCO gene expression. The rbcL genes from Synechococcus PCC6301 (cyano) and from Cylindrotheca sp. [chromo) were used as probes at select stations to examine levels of rbcL mRNA in speciflc size fractions (>5 pnl, 1-5 pm, c 1 pm) in surface waters of the mouth of Tampa Bay (estuarine), West Florida Shelf (coastal), and from the offshore Gull of Mex~co. Using DNA purified from algal isolates, we demonstrated that the cyano probe was specific for the chlorophyte/cyanobacterial RubisCO evolutionary lineage and the chromo probe was specific for the chromophyte evolutionary lineage (dlatoms, prymnesiophytes, and other non-green microalgae). For coastal/estuarine environments, both cyano and chromo rbcL mRNA was predominately confined to the >5 pm size fraction, whereas in offshore oligotrophic environments, the cyano mRNA was associated with smaller cells ( < l pm). Similarly, '"C carbon fixation rates and chl a were predominately associated with the >5 pm fraction in coastal/estuarine environments, while in offshore environments, a greater percentage was present In the < l pm fraction. In profiles through the euphotic zone, cyano rbcL rnRNA exhibited maximal values at depths above 65 m at all stations where the waters were dominated by Synechococcus and Prochlorococcus. In contrast, chromo rbcL mRNA increased wlth depth from undetectable levels in surface waters to its highest levels at or below the subsurface chlorophyll maximum (SCM. 67 m or deeper). Carbon fixation rates were generally elevated in both surface waters and around the SCM. The SCM was dominated by chromophytic picoeucaryotes, as detected by HPLC pigment analysis and flow cytometry. Such analyses are consistent with the rbcL gene probe patterns of euphotic zones of offshore oligotrophic environments. This study demonstrates the utility of group-specific gene probes for examining the expression of carbon fixing genes in phytoplankton and is a first approach to understanding the active phytoplankton community structure and its relationship to the fixation of inorganic carbon in marine environments.

Coastal plumes of low salinity water that extend hundreds of kilometers offshore into oligotrophic waters are often found in the Gulf of Mexico. To characterize one such feature, a series of photoautotrophic activity and biomass parameters were measured at 2 stations in the eastern Gulf of Mexico, including pigments by high performance liquid chromatography (HPLC), autotrophic picoplankton abundance by flow cytometry, photoautotrophic I4C-HC03-fixation, and Ribulose-1,5-&phosphate carboxylase large subunit gene (rbcL) transcriptional activity. One sampling site (Stn 4) was in a 15 m deep, low salinity (29.8 ppt) plume 242 km west of Tampa Bay. This feature contained relatively high chlorophyll a (chl a) concentrations, carbon fixation rates, and Synechococcus cell abundance (8.6 X 104 cells rnl-l) at 3 m depth and a relatively shallow (45 m depth) subsurface chlorophyll a maximum (SCM). We also found a high level (1.1 ng I-') of Form IA rbcL mRNA in the surface water as determined by probing with a 1.1 kb Synechococcus WH7803 rbcL probe. Form 1A rbcLs have been found to occur malnly in chemosynthetic autotrophic bacteria but have recently been described in Synechococcus WH7803 as well as in Prochlorococcus GP2. In contrast, a nearby station outside of the plume (Stn 7) had a SCM at 83 m, lower chl a, Synechococcus cell counts, and carbon fixation rates in the surface waters. The amount of Form IA rbcL was only about 3 % of the concentration found in the surface waters of Stn 4. Both stations had an abundance of Prochlorococcus cells (>105 rnl-') at intermediate depths (20 to 70 m). The picoeucaryote community occurred principally below the Prochlorococcus community, coinciding with the SCM, and was composed of diatoms, pryrnnesiophytes, and pelagophytes as determined by HPLC pigment analysis. This report represents the first description of Form IA rbcL transcriptional activity in the marine environment, and indicates that Form 1A rbcLcontaining picoplankton (like Prochlorococcus GP2 and Synechococcus WH7803) may be important in the primary production of low salinity, surface water plumes of the Gulf of Mexico.