The long goodbye: the rise and fall of flavodoxin during plant evolution

Abstract

Ferredoxins are electron shuttles harbouring iron-sulfur clusters that connect multiple oxido-reductive pathways in organisms displaying different lifestyles. Some prokaryotes and algae express an isofunctional electron carrier, flavodoxin, which contains flavin mononucleotide as cofactor. Both proteins evolved in the anaerobic environment preceding the appearance of oxygenic photosynthesis. The advent of an oxygen-rich atmosphere proved detrimental to ferredoxin owing to iron limitation and oxidative damage to the iron-sulfur cluster, and many microorganisms induced flavodoxin expression to replace ferredoxin under stress conditions. Paradoxically, ferredoxin was maintained throughout the tree of life, whereas flavodoxin is absent from plants and animals. Of note is that flavodoxin expression in transgenic plants results in increased tolerance to multiple stresses and iron deficit, through mechanisms similar to those operating in microorganisms. Then, the question remains open as to why a trait that still confers plants such obvious adaptive benefits was not retained. We compare herein the properties of ferredoxin and flavodoxin, and their contrasting modes of expression in response to different environmental stimuli. Phylogenetic analyses suggest that the flavodoxin gene was already absent in the algal lineages immediately preceding land plants. Geographical distribution of phototrophs shows a bias against flavodoxin-containing organisms in iron-rich coastal/freshwater habitats. Based on these observations, we propose that plants evolved from freshwater macroalgae that already lacked flavodoxin because they thrived in an iron-rich habitat with no need to back up ferredoxin functions and therefore no selective pressure to keep the flavodoxin gene. Conversely, ferredoxin retention in the plant lineage is probably related to its higher efficiency as an electron carrier, compared with flavodoxin. Several lines of evidence supporting these contentions are presented and discussed.

Keywords: Electron transfer; environmental stress; evolution; ferredoxin; flavodoxin; iron limitation..

Fig. 1.

Comparative structures of Anabaena ferredoxin and flavodoxin. (A) Ribbon diagrams of Fd (left) and Fld (right) showing interactions of the prosthetic groups at the active sites. The Fe–S cluster of Fd is represented as red-orange balls, and the FMN of Fld as red sticks. The side chains of the binding cysteines of Fd and of the stacking aromatic amino acids of Fld are coloured as black sticks. (B) Fd and Fld can be compared on the basis of their surface electrostatic potentials. The left and right panels show the Anabaena Fd and Fld, respectively, as Cα-traces. Prosthetic groups are highlighted. The two proteins are aligned according to the procedure derived by Ullmann et al. (2000). In the central picture, the structures are superimposed, showing co-localization of the redox centres. For clarity reasons, the vectors corresponding to the dipolar moments (black arrows) are drawn from positive to negative, opposite to conventional representations.

Fig. 2.

Proposed model for the protective mechanism of Fld in plastids of transgenic plants. A transmission electron micrograph of a chloroplast is used to illustrate the places (thylakoids or stroma) where the various reactions are expected to occur. The situation in WT plants under normal growth conditions is shown in the top left panel, with Fd acting as the regular shuttle between the PETC and electron-accepting processes of the chloroplast. Stress conditions in WT lines (bottom left) lead to Fd down-regulation, NADP⁺ exhaustion, and ROS build-up. Most central metabolic routes, including the Calvin cycle, are inhibited, whereas a few, such as respiration, are unaffected or even induced (not shown). Fld expression in stressed plants (top right) bypass Fd limitation, reactivating electron delivery to metabolic sinks. Simultaneous expression of Fld plus a soluble FNR (sFNR) in stressed plants (bottom right) further improves NADP(H) turnover and distribution of reducing equivalents to productive routes, resulting in increased tolerance to environmental hardships. T, thylakoids; S, stroma; ox, oxidized; red, reduced. Other abbreviations are given in the text.

Fig. 3.

An evolutionary framework of Fld distribution among photosynthetic eukaryotes. A simplified phylogenetic tree was constructed using data from Bowman (2013) and Keeling (2013). Circle sizes represent the absolute number of species analysed in each branch, whereas colours indicate the fraction of species containing (blue) or not (purple) the fld gene.

Fig. 4.

The flavodoxin gene was lost along the evolution of Viridiplantae during the colonization of terrestrial habitats by streptophyte algae. The hypothesis assumes that plants originated from freshwater streptophytes which already lacked Fld. Cyanobacteria and algae thriving in iron-deficient oceanic environments usually contained the fld gene, whereas coastal and freshwater counterparts evolved in a habitat in which iron was plentiful and bioavailable, and the role of Fld as a backup of Fd was presumably not required. Typical cyanobacteria, algae, and plants are depicted schematically. The negative correlation between iron levels and Fld presence is shown in the bottom. They are intended to represent tendencies and do not depict actual iron/Fld contents.