Cell-specific detection of phosphorus stress in Trichodesmium from the Western North Atlantic

The low phosphorus concentrations observed in the western North Atlantic and the western Central Atlantic suggest that phosphorus bioavailability may limit Trichodesmium productivity and N2 fixation. However, the degree to which the concentration and composition of the total phosphorus pool affects actual bioavailability is poorly understood. To better examine how phosphorus bioavailability may constrain primary production and marine N2 fixation, we have developed a molecular diagnostic tool for quickly detecting the phosphorus status of the diazotroph Trichodesmium. This diagnostic method uses a commercially available substrate to fluorescently tag cells expressing the phosphate-regulated enzyme, alkaline phosphatase. Using this diagnostic tool, we were able to distinguish phosphorus-replete from phosphorusstressed Trichodesmium populations along a transect from Grand Bahama Island to Bermuda in November 2000. The biogeochemical cycles of phosphorus (P) and other biolimiting elements are tightly linked to marine primary production. In particular, the degree to which P may limit marine primary production and concomitant carbon export has been extensively debated. Over geologic time, biological fixation of atmospheric N2, which has no P counterpart, should eventually force marine systems into a state of P limitation (Redfield 1958). This contradicts the traditional views of many researchers, developed from nutrient addition experiments, that nitrogen (N) or iron (Fe) can limit phytoplankton biomass at a given point in space and time (Ryther and Dunstan 1971; Martin et al. 1994). In the western North Atlantic, it has been suggested that the Fe supply may control N versus P limitation through modulation of N2 fixation by the diazotrophic cyanobacterium Trichodesmium. This is thought to be due to the relatively high Fe concentrations required by Trichodesmium for N2 fixation (Wu et al. 2000; Berman-Frank et al. 2001). However, recent work in the western Central Atlantic has indicated that reduced rates of N2 fixation by Trichodesmium are not affected by Fe inputs but rather correlate with P limitation (Sanudo-Wilhelmy et al. 2001). Fitting with this observation is the finding that the P concentration is exceedingly low in both areas of the Atlantic relative to other oligotrophic regimes, such as the North Pacific. For example, mean dissolved inorganic phosphate (DIP) concentrations in the western North Atlantic are ,1029 mol L21, whereas in the North Pacific DIP is typically . 1028 mol L21 (Wu et al. 2000). Although these chemical concentrations suggest that P is likely to limit productivity in the western North Atlantic, the geochemistry of P is complex, and the degree to which P is bioavailable for phytoplankton growth depends on its speciation. In the surface of oligotrophic waters, organic P (DOP) often comprises a significant proportion of the total P (Jackson and Williams 1985; Orrett and Karl 1987; Karl 1999; Wu et al. 2000). Although DIP is directly available to phytoplankton like cyanobacteria, the degree to which the complex constituents of the DOP pool are bioavailable is poorly understood, especially for different species in diverse field populations (Cembella et al. 1984a,b). A way to circumvent these interpretive challenges is to directly assay the P status of phytoplankton in a manner that is sensitive to both the DIP and DOP supply. A common approach to studying P physiology in phytoplankton is to monitor alkaline phosphatase (PhoA) activity (discussed in Cembella et al. 1984a). This scavenging enzyme is typically induced to allow cells to acquire inorganic phosphate from different organic P sources in the water column (Kuenzler and Perras 1965). In organisms in which PhoA activity has been studied in detail, it has been shown to respond to intracellular inorganic phosphate pools (e.g., Wanner 1996). Thus the expression and activity of the enzyme is sensitive to both bioavailable DIP and DOP. Herein we have applied a technique for detecting in situ PhoA activity in Trichodesmium that uses enzyme-labeled fluorescence (ELF) (González-Gil et al. 1998). With this diagnostic tool we were able to specifically identify P stress in Trichodesmium from the western North Atlantic. Methods—Axenic cultures of Trichodesmium erythraeum IMS 101 and T. thiebautii were grown as described elsewhere in P-replete medium (8 3 1026 mol L21) (Webb et al. 2001). P stress was induced by inoculating Trichodesmium colonies into media where P had been omitted. The cultures were harvested when the biomass was reduced compared with that of the P-augmented control, the timing of which depended on inoculum (typically 6–10 d). Field populations and chemical data were collected during a cruise to the western North Atlantic in November of 2000 (see Table 1 for coordinates). Colonies were obtained from surface water by towing a 130.0-mm net. Isolated colonies were washed twice in 0.2-mm filter-sterilized seawater and gently filtered onto 5.0-mm polycarbonate filters for storage at 2208C prior to analyses. To assay for P stress, Trichodesmium colonies were isolated from field samples or cultures and transferred to PolyL Lysine microscopy slides. The colonies were incubated with 70% ethanol for 30 min at 48C and then rinsed in Trichodesmium three-quarters–strength seawater base medium