Characterization of ferredoxin and flavodoxin as molecular indicators of iron limitation in marine eukaryotic phytoplankton

Abstract

Expression and regulation of the ferredoxin and flavodoxin proteins in marine phytoplankton were investigated to assess their utility as biomarkers of iron limitation. A phylogenetic survey of seventeen microalgal species showed flavodoxin induction, with accompanying ferredoxin repression, to be a common response to iron stress. A minority of organisms examined never expressed flavodoxin, a condition associated with, but not characteristic of, neritic habitats. Antibodies raised against ferredoxin and flavodoxin from Thalassiosira weissflogii proved to be mono- and diatom-specific, respectively. Flavodoxin induction responded specifically to iron limitation and not to nitrogen, phosphorus, silicate, zinc or light deficiency. In iron-limited T. weissflogii, relative cellular ferredoxin and flavodoxin content (Fd index) varied with growth rates above ~50%μmax and was not affected by growth on either nitrate or ammonium as a sole nitrogen source. Below ~50%μmax, ferredoxin was absent. This variation with severity of stress and specificity to iron limitation make the Fd index an excellent choice as an indicator of iron limitation. HPLC measurement of ferredoxin and flavodoxin during the IronExII mesoscale enrichment experiment detected a strong flavodoxin signal but no significant ferredoxin synthesis, despite increases in chlorophyll and photosynthetic efficiency (Fv/Fm) observed by others. The absence of ferredoxin and the persistence of flavodoxin suggested that iron addition released the phytoplankton from iron starvation but was insufficient to completely relieve physiological iron limitation. Laboratory experiments demonstrated that a pennate diatom clone isolated from the IronExII bloom expressed both flavodoxin and ferredoxin and could alter its protein expression in about one day, further supporting the conclusion of continued iron limitation during IronExII. During IronExII, Fd index was uniformly zero while Fv/Fm increased from 0.26 to 0.56. In contrast, a laboratory iron addition experiment showed little change in Fv/Fm when the Fd index increased from 0.5-0.9. A conceptual model of the covariation of Fv/Fm and Fd index describes a complementary relationship between the two measures. Model results suggest that photochemical systems are affected by iron limitation only after cellular adaptive capacity, in the form of ferredoxin, is exhausted.