The fate of primary production, whether rapidly exported from the photic zone, transferred up the trophic web, or recycled efficiently in the surface layer, depends primarily on the kind of phytoplankton that assimilate the carbon. High new production is associated with large cells, and most blooms are characterized by dominance of a single species of large or protected cells (e.g., diatoms, coccolithophorids). Episodic upwelling events typify conditions in which diatoms are able to bloom, and in which a few species usually dominate. Although escape (usually temporary) from grazing may be the ultimate reason that bloom biomass accumulates, the ability of diatoms to grow rapidly when nitrate becomes available must be a factor in allowing that escape.
This project will investigate the basis of the diatom strategy by focusing on the regulation of genes involved in nitrate uptake and transport in eukaryotic phytoplankton. By evaluating gene expression in response to the changing nitrogen availability of a classic upwelling cycle in Monterey Bay, California, the investigators will elucidate the genetic and physiological basis of the largely predictable outcome of diatom dominance in such systems. This work builds on the rich literature of oceanographic and field research into nitrogen uptake dynamics in natural populations, and on the great body of research on phytoplankton biochemistry and nutrient kinetics. The novel aspect of the proposed research is the addition of the genetic level of regulation and response. The time is right for this project because of the very recent acquisition of gene sequences for nitrate reductase and nitrate transporter genes in diatoms, and recent methodological progress on assays for gene expression. Quantitative PCR (Q-PCR) and quantitative reverse transcriptase PCR (Q-RT-PCR) will be used to quantify gene copy number and mRNA levels for nitrate reductase and transporter genes in several ecotypes of diatoms, E. huxleyi and marine chlorophytes. Initial work has demonstrated the power of these approaches and already has led to insights into different strategies of eukaryotic phytoplankton. In addition, preliminary results from a microarray containing several dozen different nitrate reductase and rubisco gene probes has allowed the investigators to determine the relative abundance of discrete ecotypes of phytoplankton and, independently, to assess the relative level of gene expression for each type. Thus the investigators have developed the initial tools necessary for investigation of genetic regulation of phytoplankton response to nutrient availability. Culture experiments will be used to characterize the sensitivity, resolution and time scale of the assays before application to field samples. In parallel with further assay development and optimization, the project investigators will conduct an upwelling experiments in which the assays for gene expression and cell activity will be deployed in conjunction with 15N tracer incubation and size fraction experiments to measure N transformations. This field exercise will be conducted in a dock incubation using Monterey Bay water; over the time course of a simulated upwelling event, the expression of reductase and transporter genes specific to certain ecotypes (empirically defined on the basis of sequence similarity from field samples) of the major groups (diatoms, E. huxleyi, chlorophytes) will be quantified. The results of the study will allow a mechanistic understanding of the plankton physiologies that are responsible for the reliable dominance of diatoms in upwelling regimes.
The broader impacts of this study will develop from the efforts made by the PIs, post doc and students supported by the project to involve undergraduates in the research and to enrich their teaching and training opportunities by this experience. Plans are outlined to involve undergraduates in senior thesis research related to both field and lab aspects of the project. The interdisciplinary nature of this work lends itself to attracting students from various departments (geosciences, molecular biology, ecology and evolutionary biology).
