QUANTITATIVE-ANALYSIS OF IRON-STRESS RELATED PROTEINS IN THALASSIOSIRA-WEISSFLOGII - MEASUREMENT OF FLAVODOXIN AND FERREDOXIN USING HPLC

The controversy surrounding the question of whether iron limits phytop lankton production in high-nutrient, low-chlorophyll regions of the wo rld's oceans has stimulated research on numerous fronts, including the search for diagnostic indicators of phytoplankton iron nutritional st atus. Flavodoxin (Fly), an iron-independent protein capable of catalyz ing many of the same reactions as the non-heme, iron-sulfur protein fe rredoxin (Fd), is produced by certain prokaryotic and eukaryotic organ isms in response to iron stress. Few eukaryotic marine phytoplankton s pecies have been examined for their ability to synthesize Fly, primari ly due to the lack of a suitable analytical method. In response to thi s need, a high performance liquid chromatography (HPLC)/photodiode arr ay detection method was developed for the separation and identificatio n of Fly and Fd in crude protein extracts of algal samples. The utilit y of this technique was then demonstrated with a study of Fly and Fd p roduction by the diatom Thalassiosira weissflogii, a eukaryotic marine phytoplankton species. Fly was detected in iron-limited T. weissflogi i at levels about 20-fold greater (by molecules) than in iron-replete cells. Although constitutive expression of this protein under iron-suf ficient conditions was observed, actual levels were very low. By compa rison, the iron-containing protein Fd was abundant in iron-replete cel ls but undetectable in iron-limited cells (limit of detection ca 1 pg ml(-1)). If Fly (by molecules) is expressed as a percentage of the com bined pool of Fly and Fd, [Flv/(Flv + Fd)] x 100, values fall between ca 10 and 15% (n = 6) for iron-replete cells, but are consistently 100 % under Fe-limiting conditions (n = 5), where Fd is below detection li mits. Thus, the constitutive nature of Fly production by T. weissflogi i does not preclude its possible use as a diagnostic indicator of iron stress. The HPLC method developed during this study, in conjunction w ith cellular level techniques (e.g. antibodies, nucleic acid probes), will permit a rapid, accurate assessment of phytoplankton iron nutriti onal status without the need for intrusive techniques such as bottle i ncubations. Additional laboratory and field studies are now needed to evaluate the applicability of Fly and Fd relative abundance as a diagn ostic indicator of iron limitation in a broad range of species and nat ural phytoplankton communities.