Analysis of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities by group-specific gene probing

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. (chrome) were used as probes at select stations to examine levels of rbcL mRNA in specific size fractions (>5 mu m, 1-5 mu m, <1 mu m) in surface waters of the mouth of Tampa Bay (estuarine), West Florida Shelf (coastal), and from the offshore Gulf of Mexico. Using DNA purified from algal isolates, we demonstrated that the cyano probe was specific for the chlorophyte/cyanobacterial RubisCO evolutionary lineage and the chrome probe was specific for the chromophyte evolutionary Lineage (diatoms, prymnesiophytes, and other non-green microalgae). For coastal/estuarine environments, both cyano and chrome rbcL mRNA was predominately confined to the >5 mu m size fraction, whereas in offshore oligotrophic environments, the cyano mRNA was associated with smaller cells (<1 mu m). Similarly, C-14 carbon fixation rates and chi a were predominately associated with the >5 mu m fraction in coastal/estuarine environments, while in offshore environments, a greater percentage was present in the <1 mu m fraction. In profiles through the euphotic zone, cyano rbcL mRNA exhibited maximal values at depths above 65 m at all stations where the waters were dominated by Synechococcus and Prochlorococcus. In contrast, chrome rbcL mRNA increased with depth from undetectable levels in surface waters to its highest levels al 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.