Abstract
Oceanic phytoplankton are known to fix CO2 primarily through the action of the enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase). The amino acid and nucleotide sequence of the large subunit of this enzyme have been conserved across the evolution of the chlorophytic plants (from cyanobacteria to higher plants via green algae) with approximately 80 and 70 % homology at the amino acid and nucleotide levels respectively. To understand the molecular regulation of this enzyme in phytoplankton, we have measured levels of the RuBPCase large subunit (rbcL) mRNA and DNA, in combination with rates of photosynthetic CO2 fixation, autofluorescent cell counts, and chlorophyll a in natural phytoplankton communities of Tampa Bay (Flonda, USA) and the southeastern Gulf of Mexico. We measured rbcL mRNA, rbcL DNA, and the ratio of rbcL mRNA to rbcL DNA by extracting RNA and DNA and probing the extracts with the Synechococcus PCC 6301 rbcL gene as a probe. Additionally, rbcL mRNA was amplified from certain samples using a reverse transcriptase-linked polymerase chain reaction procedure. In a transect from Tampa Bay seaward, levels of rbcL mRNA decreased 3to 8-fold from the estuarine environment to the offshore environment, and followed similar trends as photosynthetic CO2 fixation, picocyanobacterial counts, and chlorophyll a. In an offshore vertical profile, the subsurface maximum in the rbcL mRNA/DNA ratio coincided with the 60 m maximum in photosynthetic assimilation rates. In a die1 study using 150 1 of offshore water in a deck-top incubator, the rbcL mRNA.DNA ratio was nearly an order of magnitude greater (76 ng mRNA ng-' DNA) during the light period than in the dark (17 ng mRNA ng-' DNA). Likewise, the photosynthetic rate (PB) at constant illun~ination was highest during daylight hours (6.2 pg C pg-' chl a h-') and lowest at night (1.9 pg C pg-' chl a h-'). These results indicate that (1) carbon fixation in oceanic phytoplankton may be transcriptionally regulated at the level of the RuBPCase gene and (2) that the quantitatlon of particular target mRNAs and DNAs is an effective means to study the regulation of conserved gene functions in natural water column microbial populations.
Highlights
The enzyme ribulose-1,5-bisphosphate carboxylase/ oxygenase (E.C. 4.1.1.39, RuBPCase) is the major carboxylating enzyme in the phytoplankton of the world's oceans (Descolas-Gros & Fontugne 1985, Beardall 1989, Colman 1989).Knowledge of the in situ regulation of this enzyme will improve our understanding of the mechanisms controlling primary production in the oceans.'Addressee for correspondenceO Inter-Research 1993In all phytoplankton and multicellular plants, RuBPCase exists as a multimeric enzyme comprised of large and small subunits in equal proportion (L8S8)
The results of sampling of surface waters at stations in the Gulf of Mexico along a nearshore-offshore transect for RuBPCase large subunit (rbcL) Messenger RNA (mRNA) and DNA, chl a, carbon fixation and autofluorescent cell counts appear in Figs. 2, 3 & 4 respectively
A sample was collected at the offshore Stn 8 and probed for the expression of the catechol dioxygenase gene ( x y l E ) as a control for nonspecific hybridization as in Pichard & Paul (1993).No xylE hybridization was found with RNA extracts from this station. rbcL mRNA concentrations ranged from 185.8 to 0.2 ng I-', with highest values in nearshore waters, lowest in offshore waters (Fig. 2 )
Summary
The enzyme ribulose-1,5-bisphosphate carboxylase/ oxygenase (E.C. 4.1.1.39, RuBPCase) is the major carboxylating enzyme in the phytoplankton of the world's oceans (Descolas-Gros & Fontugne 1985, Beardall 1989, Colman 1989).Knowledge of the in situ regulation of this enzyme will improve our understanding of the mechanisms controlling primary production in the oceans.'Addressee for correspondenceO Inter-Research 1993In all phytoplankton and multicellular plants, RuBPCase exists as a multimeric enzyme comprised of large and small subunits in equal proportion (L8S8). Ser. 101: 55-65, 1993 terms of RuBPCase large subunits Within this group RuBPCase is approximately 85 % similar at the amino acid level, but only 55% sirmlar to cyanobacteria, green algae and higher plants (Newman & Cattolico 1987, Douglas et al 1990, Kostrzewa et al 1990, Hwang & Tabita 1991).While diatoms, dinoflagellates, cryptomonads, and other 'non-green' algae are present in offshore marine waters (Vargo 1984, Siege et al 1990), the most abundant phytoplankters in the oligotrophic ocean are the 'green' lineage-type picophytoplankton (Waterbury et al 1986). These Synechococcus-like picoplankton (103to 104cells ml-l) and free-living prochlorophytes (up to 105 cells ml-l) are responsible for up to 80 % of the water column primary production (Takahashi & Hori 1984, Itturiaga & Mitchell 1986, Chisholm et al 1988)
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