Quantitative analysis of iron-stress related proteins in Thalassiosira weissflogii: Measurement of flavodoxin and ferredoxin using HPLC

Abstract

The controversy surrounding the question of whether iron limits phytoplankton production in high-nutrient, low-chlorophyll regions of the world's oceans has stimulated research on numerous fronts, including the search for diagnostic indicators of phytoplankton iron nutritional status. Flavodoxin (Flv), an iron-independent protein capable of catalyzing many of the same reactions as the non-heme, iron-sulfur protein ferredoxin (Fd), is produced by certain prokaryotic and eukaryotic organisms in response to iron stress. Few eukaryotic marine phytoplankton species have been examined for their ability to synthesize Flv. primarily due to the lack of a suitable analytical method. In response to this need, a high performance liquid chromatography (HPLC)/photodiode array detection method was developed for the separation and identification of Flv and Fd in crude protein extracts of algal samples. The utility of this technique was then demonstrated with a study of Flv and Fd production by the diatom Thalassiosira weissflogii, a eukaryotic marine phytoplankton species. Flv was detected in iron- limited T. weissflogii at levels about 20-fold greater (by molecules) than in iron- replete cells. Although constitutive expression of this protein under iron- sufficient conditions was observed, actual levels were very low. By comparison, the iron-containing protein Fd was abundant in iron- replete cells but undetectable in iron-limited cells (limit of detection ca 1 μg ml-1). If Flv (by molecules) is expressed as a percentage of the combined 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 limits. Thus, the constitutive nature of Flv production by T. weissflogii does not preclude its possible use as a diagnostic indicator of iron stress. The HPLC method developed during this study, in conjunction with cellular level techniques (e.g. antibodies, nucleic acid probes), will permit a rapid, accurate assessment of phytoplankton iron nutritional status without the need for intrusive techniques such as bottle incubations. Additional laboratory and field studies are now needed to evaluate the applicability of Flv and Fd relative abundance as a diagnostic indicator of iron limitation in a broad range of species and natural phytoplankton communities.