Assessing nutrient limitation of Prochlorococcus in the North Pacific subtropical gyre by using an RNA capture method

It has been hypothesized that the planktonic community of the North Pacific subtropical gyre (NPSG) underwent a ‘‘domain shift’’ in the early 1980s in which phytoplankton of the domain Eukarya were supplanted by phytoplankton of the domain Bacteria, primarily Prochlorococcus. P limitation of eukaryotic phytoplankton was implicated as the causative chemical factor in the domain shift, and we sought to investigate the current nutrient limitation status of Prochlorococcus, now 2 decades since this event. We measured ribonucleic acid (RNA) synthesis rates by NPSG plankton at Station ALOHA in 33PO 3{ 4 tracer incubations and found that RNA synthesis was the single largest biochemical sink for dissolved P, accounting for about half of the total PO 3{ 4 uptake. We also found that NH z4 stimulated RNA synthesis but that PO 3{ 4 did not, which suggested N limitation of plankton growth. We developed a new RNA capture procedure, termed radioisotope-based tracking of RNA synthesis by hybridization and capture (RIBOTRACE), to measure RNA synthesis rates by Prochlorococcus exclusively. Data from this procedure showed that NH z4 stimulated RNA synthesis by Prochlorococcus and confirmed that Prochlorococcus was N limited and not P limited. Our RIBOTRACE data do not necessarily refute the domain shift hypothesis, but suggest that any critical period of P limitation required for the domain shift must have subsided and given way to the N-limiting conditions that existed previously. The productivity, abundance, and community composition of plankton in subtropical ocean gyres is inextricably linked to the supply rate, concentration, and chemical composition of dissolved nutrients, and vice versa. But large-scale changes in atmospheric and ocean physics can perturb these linkages. During the last half century the North Pacific subtropical gyre (NPSG) has exhibited major perturbations that, in turn, undoubtedly affected the role of this biome, the world’s largest, in the global cycles of carbon and nutrients. Drawing upon long-term observations from the NPSG, Venrick et al. (1987) argued that changes in climate in the 1970s led to a doubling of phytoplankton biomass. Long supposed to be an N-limited system (Perry and Eppley 1981), Karl and colleagues later linked enhanced production in the NPSG during the early 1980s to enhanced N2 fixation by cyanobacteria (Karl et al. 1995, 1997; Dore et al. 2002). The chemical consequence of this shift was a drawdown in dissolved P (Karl and Tien 1997; Karl et al. 2001b), and the primary beneficiaries of this were cyanobacteria of the genus Prochlorococcus (Karl et al. 2001a). It was suggested that Prochlorococcus, though incapable of N2 fixation, supplanted the previous community of phytoplankton dominated by eukaryotes (e.g., diatoms) because they were better adapted for growth at lower dissolved P concentrations (Karl et al. 2001a). Thus it was hypothesized that this ‘‘domain shift’’ was the ecological response to the progression of the NPSG from a N-limited to a P-limited ecosystem (Karl et al. 1995). We sought to better understand the status of this progression, and to assess whether the population of Prochlorococcus in the NPSG had itself now—almost 2 decades since the domain shift—become P limited. Our approach was to measure changes in the P-based growth rates of Prochlorococcus in incubations supplemented with tracer-level 33PO 3{ 4 and amended with either dissolved inorganic N (DIN) or PO 3{ 4 . Although some strains of Prochlorococcus can interact directly with the organic reservoir of dissolved P, the preferred form is clearly PO 3{ 4 (Moore et al. 2005) and we expected that if Prochlorococcus were truly P limited, then additions of PO 3{ 4 would stimulate growth rates. 1 Corresponding author ([email protected]). Acknowledgments We thank D. Karl for providing access to his laboratories after the HOT cruises, for his comments on a draft of this paper, and for his continued encouragement of our studies of planktonic P physiology in the NPSG. K. Björkman, T. Gregory, D. Sadler, and the other HOT program personnel provided essential logistical support and data. We thank D. Stahl, M. Hullar, and V. Armbrust for instruction and advice during the development of the RIBOTRACE method. We particularly acknowledge B. MacGregor for publishing a clear and thorough description of her robust RNA isolation method. Two anonymous reviewers provided exceptionally thorough and helpful reviews of this paper. A.H.D. was supported by ONR N0014-00-1-0751 and by the Biotechnology Investigations Ocean Margins Program, Office of Biological and Environmental Research, U.S. Department of Energy. B.V.M. was supported by ONR N0014-06-1-0134, the Postdoctoral Program at Woods Hole Oceanographic Institution (WHOI) with funding provided by the Watson Chair to D. Repeta, and the Gryce B. Kerr and Penzance Endowed fund in support of Assistant Scientists at WHOI. Limnol. Oceanogr., 53(1), 2008, 78–88 E 2008, by the American Society of Limnology and Oceanography, Inc.