RIBULOSE-BISPHOSPHATE CARBOXYLASE GENE-EXPRESSION IN SUBTROPICAL MARINE-PHYTOPLANKTON POPULATIONS

Oceanic phytoplankton are known to fix CO2 primarily through the actio n 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 approximatel y 80 and 70 % homology at the amino acid and nucleotide levels respect ively. To understand the molecular regulation of this enzyme in phytop lankton, 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 phytoplank ton communities of Tampa Bay (Florida, 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 t he Synechococcus PCC 6301 rbcL gene as a probe. Additionally, rbcL mRN A was amplified from certain samples using a reverse transcriptase-lin ked polymerase chain reaction procedure. In a transect from Tampa Bay seaward, levels of rbcL mRNA decreased 3- to 8-fold from the estuarine environment to the offshore environment, and followed similar trends as photosynthetic CO2 fixation, picocyanobacterial counts, and chlorop hyll 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 diel study using 150 1 of offshore water in a deck-top incubator, the rbcL mRNA/DNA ratio was nearly an order of m agnitude greater (76 ng mRNA ng-1 DNA) during the light period than in the dark (17 ng mRNA ng-1 DNA). Likewise, the photosynthetic rate (P( B)) at constant illumination was highest during daylight hours (6.2 mu g C mug-1 chl a h-1) and lowest at night (1.9 mug C mug-1 chl a h-1). These results indicate that (1) carbon fixation in oceanic phytoplankt on may be transcriptionally regulated at the level of the RuBPCase gen e and (2) that the quantitation of particular target mRNAs and DNAs is an effective means to study the regulation of conserved gene function s in natural water column microbial populations.