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BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

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Ferredoxin/flavodoxin-NADPH reductases (FPRs) catalyze the reversible electron transfer between NADPH and ferredoxin/flavodoxin. The Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes contains two isoenzymes, FPR1ver3 and FPR2ver3. Absorption spectra of these FPRs revealed typical features of flavoproteins, consistent with the use of FAD as a prosthetic group. Spectral differences indicate distinct electronic arrangements for the flavin in each enzyme. Steady-state kinetic measurements show that the enzymes display catalytic efficiencies in the order of 1-6 µm-1·s-1, although FPR1ver3 exhibited higher kcat values compared to FPR2ver3. When flavodoxinver3 was used as a substrate, both reductases exhibited dissimilar behavior. Moreover, only FPR1ver3 is induced by oxidative stimuli, indicating that the polyextremophile Ver3 has evolved diverse strategies to cope with oxidative environments.

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Foliar development involves successive phases of cell proliferation and expansion that determine the final leaf size, and is characterized by an early burst of reactive oxygen species generated in the photosynthetic electron transport chain (PETC). Introduction of the alternative PETC acceptor flavodoxin in tobacco chloroplasts led to a reduction in leaf size associated to lower cell expansion, without affecting cell number per leaf. Proteomic analysis showed that the biogenesis of the PETC proceeded stepwise in wild-type leaves, with accumulation of light-harvesting proteins preceding that of electron transport components, which might explain the increased energy and electron transfer to oxygen and reactive oxygen species build-up at this stage. Flavodoxin expression did not affect biogenesis of the PETC but prevented hydroperoxide formation through its function as electron sink. Mature leaves from flavodoxin-expressing plants were shown to contain higher levels of transcripts encoding components of the proteasome, a key negative modulator of organ size. Proteome profiling revealed that this differential accumulation was initiated during expansion and led to increased proteasomal activity, whereas a proteasome inhibitor reverted the flavodoxin-dependent size phenotype. Cells expressing plastid-targeted flavodoxin displayed lower endoreduplication, also associated to decreased organ size. These results provide novel insights into the regulation of leaf growth by chloroplast-generated redox signals, and highlight the potential of alternative electron shuttles to investigate the link(s) between photosynthesis and plant development.

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Flavodoxins are electron carrier flavoproteins present in bacteria and photosynthetic microorganisms which duplicate the functional properties of iron-sulphur containing ferredoxins and replace them under adverse environmental situations that lead to ferredoxin decline. When expressed in plant chloroplasts, flavodoxin complemented ferredoxin deficiency and improved tolerance to multiple sources of biotic, abiotic and xenobiotic stress. Analysis of flavodoxin-expressing plants grown under normal conditions, in which the two carriers are present, revealed phenotypic effects unrelated to ferredoxin replacement. Flavodoxin thus provided a tool to alter the chloroplast redox poise in a customized way and to investigate its consequences on plant physiology and development. We describe herein the effects exerted by the flavoprotein on the function of the photosynthetic machinery. Pigment analysis revealed significant increases in chlorophyll a, carotenoids and chlorophyll a/b ratio in flavodoxin-expressing tobacco lines. Results suggest smaller antenna size in these plants, supported by lower relative contents of light-harvesting complex proteins. Chlorophyll a fluorescence and P700 spectroscopy measurements indicated that transgenic plants displayed higher quantum yields for both photosystems, a more oxidized plastoquinone pool under steady-state conditions and faster plastoquinone dark oxidation after a pulse of saturating light. Many of these effects resemble the phenotypes exhibited by leaves adapted to high irradiation, a most common environmental hardship faced by plants growing in the field. The results suggest that flavodoxin-expressing plants would be better prepared to cope with this adverse situation, and concur with earlier observations reporting that hundreds of stress-responsive genes were induced in the absence of stress in these lines.

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Flavodoxins are small electron transfer proteins containing flavin mononucleotide (FMN) as a prosthetic group, which play an important role during oxidative stress or iron limitation. The aims of this study were the identification and characterization of flavodoxins in the model aromatic-degrader Paraburkholderia xenovorans LB400 and the analyses of their protective effects during oxidative stress induced by paraquat and H2O2. Two genes (BxeA0278 and BxeB0391) encoding flavodoxins (hereafter referred to as fldX for flavodoxin from P. xenovorans), were identified at the LB400 major and minor chromosome. Genomic context of the flavodoxin-encoding genes showed genes encoding membrane proteins, transporters, and proteins involved in redox processes and biosynthesis of macromolecules. A secondary structure prediction of both LB400 flavodoxins showed the characteristic flavodoxin structure of five ß-sheets intercalated with five α-helices. FldX1 contains a loop intercalated in the fifth ß-strand, which indicates that it belongs to the long-chain flavodoxins, whereas FldX2 is a short-chain flavodoxin. A phylogenetic analysis of 73 flavodoxins from 43 bacterial genera revealed eight clusters (I-VIII), while FldX1 and FldX2 grouped separately within a long-chain and a short-chain flavodoxin clades. FldX1 and FldX2 were overexpressed in P. xenovorans. Interestingly, the strain overexpressing the long-chain flavodoxin FldX1 (p2-fldX1) showed a faster growth in glucose than the control strain. The recombinant strain overexpressing the long-chain flavodoxin FldX1 (p2-fldx1) exposed to paraquat (20 mM) possessed lower susceptibility to growth inhibition on plates and higher survival in liquid medium than the control strain. The strains overexpressing the flavodoxins FldX1 and FldX2 showed higher survival during exposure to 1 mM paraquat (>95%) than the control strain (68%). Compared to the control strain, strains overexpressing FldX1 and FldX2 showed lower lipid peroxidation (>20%) after exposure to 1 mM paraquat and a lower protein carbonylation (~30%) after exposure to 1 mM H2O2 was observed. During exposure to paraquat, strain p2-fldx1 downregulated the katG4, hpf, trxB1 and ohr genes (> 2-fold), whereas strain p2-fldx2 upregulated the oxyR and ahpC1 genes (> 2-fold). In conclusion, the flavodoxins FldX1 and FldX2 of P. xenovorans LB400 conferred protection to cells exposed to the oxidizing agents paraquat and H2O2.

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The development of oxygenic photosynthesis by primordial cyanobacteria ~2.7 billion years ago led to major changes in the components and organization of photosynthetic electron transport to cope with the challenges of an oxygen-enriched atmosphere. We review herein, following the seminal contributions as reported by Jaganathan et al. (Functional genomics and evolution of photosynthetic systems, vol 33, advances in photosynthesis and respiration, Springer, Dordrecht, 2012), how these changes affected carriers and enzymes at the acceptor side of photosystem I (PSI): the electron shuttle ferredoxin (Fd), its isofunctional counterpart flavodoxin (Fld), their redox partner ferredoxin-NADP+ reductase (FNR), and the primary PSI acceptors F x and F A/F B. Protection of the [4Fe-4S] centers of these proteins from oxidative damage was achieved by strengthening binding between the F A/F B polypeptide and the reaction center core containing F x, therefore impairing O2 access to the clusters. Immobilization of F A/F B in the PSI complex led in turn to the recruitment of new soluble electron shuttles. This function was fulfilled by oxygen-insensitive [2Fe-2S] Fd, in which the reactive sulfide atoms of the cluster are shielded from solvent by the polypeptide backbone, and in some algae and cyanobacteria by Fld, which employs a flavin as prosthetic group and is tolerant to oxidants and iron limitation. Tight membrane binding of FNR allowed solid-state electron transfer from PSI bridged by Fd/Fld. Fine tuning of FNR catalytic mechanism led to formidable increases in turnover rates compared with FNRs acting in heterotrophic pathways, favoring Fd/Fld reduction instead of oxygen reduction.

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Crop yield reduction due to salinity is a growing agronomical concern in many regions. Increased production of reactive oxygen species (ROS) in plant cells accompanies many abiotic stresses including salinity, acting as toxic and signaling molecules during plant stress responses. While ROS are generated in various cellular compartments, chloroplasts represent a main source in the light, and plastid ROS synthesis and/or elimination have been manipulated to improve stress tolerance. Transgenic tobacco plants expressing a plastid-targeted cyanobacterial flavodoxin, a flavoprotein that prevents ROS accumulation specifically in chloroplasts, displayed increased tolerance to many environmental stresses, including drought, excess irradiation, extreme temperatures and iron starvation. Surprisingly, flavodoxin expression failed to protect transgenic plants against NaCl toxicity. However, when high salt was directly applied to leaf discs, flavodoxin did increase tolerance, as reflected by preservation of chlorophylls, carotenoids and photosynthetic activities. Flavodoxin decreased salt-dependent ROS accumulation in leaf tissue from discs and whole plants, but this decline did not improve tolerance at the whole plant level. NaCl accumulation in roots, as well as increased osmotic pressure and salt-induced root damage, were not prevented by flavodoxin expression. The results indicate that ROS formed in chloroplasts have a marginal effect on plant responses during salt stress, and that sensitive targets are present in roots which are not protected by flavodoxin.

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Oxidative stress and iron limitation represent the grim side of life in an oxygen-rich atmosphere. The versatile electron transfer shuttle ferredoxin, an iron-sulfur protein, is particularly sensitive to these hardships, and its downregulation under adverse conditions severely compromises survival of phototrophs. Replacement of ferredoxin by a stress-resistant isofunctional carrier, flavin-containing flavodoxin, is a widespread strategy employed by photosynthetic microorganisms to overcome environmental adversities. The flavodoxin gene was lost in the course of plant evolution, but its reintroduction in transgenic plants confers increased tolerance to environmental stress and iron starvation, raising the question as to why a genetic asset with obvious adaptive value was not kept by natural selection. Phylogenetic analyses reveal that the evolutionary history of flavodoxin is intricate, with several horizontal gene transfer events between distant organisms, including Eukarya, Bacteria, and Archaea. The flavodoxin gene is unevenly distributed in most algal lineages, with flavodoxin-containing species being overrepresented in iron-limited regions and scarce or absent in iron-rich environments. Evaluation of cyanobacterial genomic and metagenomic data yielded essentially the same results, indicating that there was little selection pressure to retain flavodoxin in iron-rich coastal/freshwater phototrophs. Our results show a highly dynamic evolution pattern of flavodoxin tightly connected to the bioavailability of iron. Evidence presented here also indicates that the high concentration of iron in coastal and freshwater habitats may have facilitated the loss of flavodoxin in the freshwater ancestor of modern plants during the transition of photosynthetic organisms from the open oceans to the firm land.

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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.

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