How cells function in the face of low internal P resources and very low external P supplies has been investigated in recent years, using a variety of alternative techniques that overcome the problem of how to quantify chemically the small amounts of determinand present. For instance, Falkner et al. (1989) applied force-flow functions, derived by Thellier (1970), to demonstrate that the typical external concentrations of phosphate below which cells of Cyanobacteria fail to balance their minimal maintenance requirements indeed fall within the range 1-50 nmol L-1 (0.03-1.5 | g P L-1). In the application of Aubriot et al. (2000), the importance of the affinity of uptake mechanisms and of the opportunism to invoke them in the face of erratic supplies was especially emphasised. Hudson et al. (2000) applied a radiobioas-say technique which was also able to demonstrate that the amount of phosphorus in the medium supporting active phytoplankton populations can fall less than 10 nmol L-1 (i.e. <10-8 M), without necessarily impairing productivity.
In another line of investigation, it has been shown that some strains of Cyanobacteria are able to maintain full growth down to external concentrations of 100 nmol P L-1 (~3 |g L-1), without producing any of the regulator proteins that signal the activity state of the transport system to the controlling operons (Mann, 1995; Scanlan and Wilson, 1999). As suggested in Section 4.2.2, the presence of regulator proteins is indicative of incipient cell starvation, triggering the appropriate intracellular defensive reactions. Work on the bacterium Vibrio (Kjelle-berg et al., 1993) showed that symptoms include a sharp slowdown in cell growth, following an abrupt deceleration in the rates of protein synthesis. Assembly of macromolecules is halted by the action of synthesis inhibitors, followed by the reorganisation of the cell components and the adjustment of the fatty-acid content of the membranes to resist lysis. In turn, these actions are followed by a decline in the rate of respiration and other metabolic activity.
Central to these reactions are the transducing signals. Certain nucleotides are known to increase in response to falling nitrogen concentration and amino-acid synthesis. One of these, guanosine 3',5 -bipyrophosphate (ppGpp), is generated in nitrogen-starved E. coli (Gentry et al., 1993) and Vibrio (Kjelleberg et al., 1993). Homologues to these are found in cyanobacterial cells experiencing a sharp reduction in photon flux (Mann, 1995). Incipient starvation and ribosomal stalling are thought to lead to ppGpp synthesis and, thence, to the communication of starvation. Mann's group was able to grow plank-tic cyanobacteria in media in which phosphorus concentrations fell to <0.1 |M (i.e. less than 3 |g P L-1) before compounds like ppGpp began to appear in the cells. This is strongly suggestive of the probability that cells do not experience phosphorus shortages in media containing MRP concentrations greater than this.
Finally, in this context, the emerging technique of using fluorimetric labelling to detect the intracellular transients induced by incipient nutrient starvation (the so-called NIFT, nutrient-induced fluorescent transients) has been applied to microalgae grown under P-replete and P-deficient conditions to identify the reactivity of the cells. According to the experiments of Beardall et al. (2001), NIFT responses were wholly lacking in each of four species of freshwater microalgae in media containing >0.13 |M (4 |g P L-1).
These various threads lead to a strong consensus that phosphorus availability does not limit phytoplankton activity and growth before the MRP concentration in the medium falls almost to the limits of conventional analytical detection. At this point, phytoplankton may draw on internal reserves such that activity is not immediately suppressed by lower external concentrations. Even at <0.1 |M, it is not the concentration of phosphorus that is critical so much as the capacity of the intracellular storage and the affinity of the biological uptake mechanism for the small amounts of bioavailable phosphorus being turned over in the system (Hudson et al., 2000).
Two other mechanisms for contending with MRP limitation of metabolic activity are available to certain species of phytoplankton. The first involves the production of extracellular phos-phatases. Many freshwater species, in fact, produce alkaline phosphatases which liberate phosphate from organic solutes that can then be absorbed by the alga (Cembella et al., 1984). They are produced in response to external MRP deficiency, almost as soon as it develops (Healey, 1973). In the past, phosphatase activity has been considered to be indicative of phosphorus limitation (Rhee, 1973). There is little doubting the fact that phosphorus thus sequestered increases the resource availability to the cell. For phosphatase production to be able to offer any survival advantage, however, the phosphatase must be retained at or close to the cell surface (Turpin, 1988). Phos-phatase activity might then raise significantly the ability of algae to tolerate chronically P-deficient conditions. There is little evidence to suggest that phosphatase production does much to enhance the growth dynamics of assemblages, or any component species, when inorganic phosphorus sources are effectively exhausted (Reynolds, 1992a).
The second mechanism involves the phagotrophic ingestion of organic particles, including especially other organisms such as bacteria. As indicated earlier (Section 3.4.4), those photosynthetic organisms capable of supplementing or, perhaps, fulfilling their requirements for nutrients and carbon by ingesting organic particulates are called mixotrophs. The best-known examples come from among the dinoflagellates (marine and freshwater Gymno-diniales and Gonyaulacales) and from among the Chromulinales, including Ochromonas (Riemann et al., 1995; Geider and Maclntyre, 2002). Certain pigmented cryptomonads are reputedly mixotrophic (Porter et al., 1985): this need not be surprising insofar as the phagotrophic abilities of the typically colourless cryptomonad genera (such as Katablepharis and Cyathomonas) have long been recognised (Klaveness, 1988). As a source of phosphorus, bacterivory and phagotrophy offer a rich alternative to scarce dissolved inorganic sources and, unlike phosphatase secretion, the available resource would seem to be less restricted. However, a low-phosphorus environment pervades all its trophic levels: bacterivory is not a sustainable alternative to deficient MRP if the bacteria are themselves simultaneously P-limited. Mixotrophy is particularly beneficial as a supplementary source of nutrient in those (generally smaller) water bodies that receive inputs of terrestrial organic matter) but are otherwise quite oligotrophic (Riemann et al., 1995).
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