ferredoxin-NADP Reductase Research Articles

Variations between the photosynthetic properties of elite and landrace Chinese rice cultivars revealed by simultaneous measurements of 820 nm transmission signal and chlorophyll a fluorescence induction

The difference between the photosynthetic properties of elite and landrace Chinese rice cultivars was studied, using chlorophyll a fluorescence induction (mostly a monitor of Photosystem II activity) and I820 transmission signal (mostly a monitor of Photosystem I activity) to identify potential photosynthetic features differentiating these two groups, which show different degrees of artificial selection and grain yields. A higher fluorescence (related to PSII) IP rise phase and a lower P700+ (related to PSI) accumulation were observed in the elite cultivars as compared to the landraces. Using these data, together with simulation data from a kinetic model of fluorescence induction, we show that the high IP rise phase and the low P700+ accumulation can be a result of transient block on electron transfer and traffic jam on the electron acceptor side of PSI under a high [NADPH]/[NADP+] ratio. Considering that the ferredoxin NADP+ reductase (FNR) transcript levels of XS134 (a representative elite cultivars) remains unaffected during the first few minutes of light/dark transition compared to Q4145 (a representative landrace cultivars), which shows a strong decline during the same time range, we propose that the FNR of elite cultivars may take more time to be inactivated in darkness. During this time the FNR enzyme can continue to reduce NADP+ molecules, leading to initially high [NADPH]/[NADP+] ratio during OJIP transient. These data suggested a potential artificial selection of FNR during the breeding process of these examined elite rice cultivars.

Ferredoxin, in conjunction with NADPH and ferredoxin-NADP reductase, transfers electrons to the IscS/IscU complex to promote iron–sulfur cluster assembly

Fe–S cluster biogenesis is an essential pathway coordinated by a network of protein–protein interactions whose functions include desulfurase activity, substrate delivery, electron transfer and product transfer. In an effort to understand the intricacies of the pathway, we have developed an in vitro assay to follow the ferredoxin role in electron transfer during Fe–S cluster assembly. Previously, assays have relied upon the non-physiological reducing agents dithionite and dithiothreitol to assess function. We have addressed this shortcoming by using electron transfer between NADPH and ferredoxin-NADP-reductase to reduce ferredoxin. Our results show that this trio of electron transfer partners are sufficient to sustain the reaction in in vitro studies, albeit with a rate slower compared with DTT-mediated cluster assembly. We also show that, despite overlapping with the CyaY protein in binding to IscS, Fdx does not interfere with the inhibitory activity of this protein. We suggest explanations for these observations which have important consequences for understanding the mechanism of cluster formation. Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Enhancement of photophysical and photosensitizing properties of flavin adenine dinucleotide by mutagenesis of the C-terminal extension of a bacterial flavodoxin reductase.

The role of the mobile C-terminal extension present in Rhodobacter capsulatus ferredoxin-NADP(H) reductase (RcFPR) was evaluated using steady-state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C-terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild-type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD-binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge-transfer quenching of (1) FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet- and triplet-state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified.

Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser.

Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question.

Comparative proteomic analysis of tobacco expressing cyanobacterial flavodoxin and its wild type under drought stress

Tobacco plants expressing cyanobacterial flavodoxin (Fld) show enhanced tolerance to a wide range of abiotic stresses including drought, temperature and UV. The mechanisms of adaptation to stress conditions under Fld expression are largely unknown. Here, we applied comparative proteomic analysis to uncover the changes in the proteome profile of Fld-expressing plants in response to drought stress. Using high-resolution two-dimensional gel electrophoresis, we were able to detect 930 protein spots and compare their abundance. We found changes up to 1.5 fold for 52 spots under drought in transgenic and/or wild type plants. Using combined MALDI-TOF/TOF and ESI-Q/TOF analysis 39 (24 in wild type, 11 in transgenic, and 4 in both) drought-responsive proteins (DRPs) could be identified. The majority of DRPs are known to be involved in photosynthesis, carbohydrate and energy metabolism, amino acid and protein synthesis and processing, and oxidative stress responses. Among candidate DRPs, the abundance of remurin, ferredoxin-NADP reductase, chloroplast manganese stabilizing protein-II, phosphoglycerate mutase, and glutathione S-transferase decreased in drought stressed Fld-tobacco while S-formylglutathione hydrolase and pyridoxine biosynthesis protein abundance increased. In wild type plants, drought caused a reduction of proteins related to carbohydrate metabolism. These results suggest that the stress tolerance conferred by Fld expression is strongly related to control mechanisms regarding carbohydrate and energy metabolism as well as oxidative stress responses.

Posttranslational modifications of FERREDOXIN-NADP+ OXIDOREDUCTASE in Arabidopsis chloroplasts.

Rapid responses of chloroplast metabolism and adjustments to photosynthetic machinery are of utmost importance for plants' survival in a fluctuating environment. These changes may be achieved through posttranslational modifications of proteins, which are known to affect the activity, interactions, and localization of proteins. Recent studies have accumulated evidence about the crucial role of a multitude of modifications, including acetylation, methylation, and glycosylation, in the regulation of chloroplast proteins. Both of the Arabidopsis (Arabidopsis thaliana) leaf-type FERREDOXIN-NADP(+) OXIDOREDUCTASE (FNR) isoforms, the key enzymes linking the light reactions of photosynthesis to carbon assimilation, exist as two distinct forms with different isoelectric points. We show that both AtFNR isoforms contain multiple alternative amino termini and undergo light-responsive addition of an acetyl group to the α-amino group of the amino-terminal amino acid of proteins, which causes the change in isoelectric point. Both isoforms were also found to contain acetylation of a conserved lysine residue near the active site, while no evidence for in vivo phosphorylation or glycosylation was detected. The dynamic, multilayer regulation of AtFNR exemplifies the complex regulatory network systems controlling chloroplast proteins by a range of posttranslational modifications, which continues to emerge as a novel area within photosynthesis research.

Comparative transcriptional study on the hydrogen evolution of marine microalga Tetraselmis subcordiformis

It's the first comparative transcriptome study of Tetraselmis subcordiformis' H2 production with carbonyl cyanide m-chlorophenylhydrazone (CCCP) by de novo RNA-seq. The global analysis of the annotated genes and pathways unveiled it a good candidate for biofuels. Focusing on H2 production, the genes coding active center subunit (HydA) and assembly subunits (HydEF and HydG) of hydrogenase proportionally expressed during the induction but stopped after illuminated; on contrast, the gene of the active centre stabilization subunit (Hyd3) highly expressed during the H2 evolution, indicating its key role in the prolonged H2 production. The results also showed genes of HCP4, ppGpp phosphohydrolase and RpoD relating to the hydrogenase expression regulation. On the energy aspect, genes of CF-ATPases and ferredoxin-NADP reductase in chloroplast down-regulated while Mito-ATPase in mitochondria and pyruvate metabolism pathway up-regulated during the H2 production, consisting with our previous reports on physiological level.

Teaching the Z-Scheme of electron transport in photosynthesis: a perspective.

This paper deals with how Govindjee taught the Z-Scheme of electron transport in oxygenic photosynthesis at Ravenshaw University, Cuttack, Odisha, India, in 2014, in a unique and highly effective fashion-using students to act as molecules, representing the entire electron transport chain from water to nicotinamide adenine dinucleotide phosphate (NADP(+)). It culminated in a show by B.Sc. students in the garden of the Department of Botany, Ravenshaw University. The first author (PKM) personally acted as Ferredoxin NADP Reductase (FNR) catalyzing the reduction of NADP(+) to NADPH, taking electrons from reduced ferredoxin at the end of Photosystem I. On the other hand, the Q-cycle was played by M.Sc. students, who acted as molecules running this ingenious cycle that produces extra protons. An interesting event was when a student, acting as a herbicide, who was dressed like a devil (fierce looking, in black clothes with a sword; "Yamaraj: The God of Death", as he called himself), stopped all reactions by throwing out QB, the second plastoquinone molecule of Photosystem II, and that too aggressively, taking its position instead. The second author was the major organizer of the Z-scheme show. We provide here a basic background on the process, a bit on Govindjee's teaching, and some selected pictures from the drama played in March, 2014 at Ravenshaw University. Here, we also recognize the teacher Govindjee for his ingenious and fun-filled teaching methods that touched the hearts and the souls of the students as well as the teachers of Ravenshaw University. He was rated as one of the most-admired teachers of plant biology at our university.

Functional and phylogenetic divergence of fungal adenylate-forming reductases.

A key step in fungal L-lysine biosynthesis is catalyzed by adenylate-forming L-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized L-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.

A comparison of wild and cultivated strawberries for nitrogen uptake and reduction

The strawberry (Fragaria × ananassa) requires substantial amounts of nitrogen to produce reasonable yields. Wild strawberries (e.g. F. virginiana) grow in soils in the absence of supplemental nitrogen and may possess desirable genes, alleles or expression patterns that support efficient nitrogen uptake and assimilation. The F. virginiana accessions and cultivated materials were used to monitor the accumulation kinetics of five nitrate responsive genes (nitrate transporter, NRT1.1; nitrate reductase, NIA1; nitrite reductase, NiR; ferredoxin NADP reductase, FNR; and transketolase, TK) along with nitrate reductase activity. The results revealed expression patterns that were reminiscent of those observed in Arabidopsis. Wild strawberries exhibited higher transcript accumulation for all genes examined except the nitrate transporter, compared to cultivated materials. Nitrate reductase activity (NRA) did not change in roots but increased in leaves in response to nitrate. Most wild genotypes had higher NRA in aerial tissues compared to cultivated materials. Comparisons of enzyme activity to transcript accumulation kinetics suggest that available nitrate reductase is activated and not likely synthesized de novo, suggesting that some wild accessions may possess enhanced nitrogen assimilation mechanisms. These attributes may be useful to breeding programs in the design of elite plants that may make more efficient use of fertilizers.

Biochemical characterization of photosystem I-associated light-harvesting complexes I and II isolated from state 2 cells of Chlamydomonas reinhardtii.

Two photosystems, PSI and PSII, drive electron transfer in series for oxygenic photosynthesis using light energy. To balance the activity of the two photosystems under varying light conditions, mobile antenna complexes, light-harvesting complex IIs (LHCIIs), shuttle between the two photosystems during state transitions. PSI forms a complex consisting of PSI core and its peripheral light-harvesting complex (LHCI) in plants and algae. In a previous study, we isolated a PSI-LHCI-LHCII supercomplex containing both LHCI and LHCII from state 2 cells of Chlamydomonas reinhardtii. In the present study, we isolated a PSI-LHCI-LHCII supercomplex associating with more LHCII complexes under a further optimized protocol. We determined its antenna size by three independent methods and revealed that the associated LHCIIs increased the antenna size by about 70 Chls and transferred light energy to the PSI core. Uniform labeling of total cellular proteins with (14)C indicated that the PSI-LHCI-LHCII supercomplex contains 1.85 copies of LhcbM5 and CP29 and 1.29 copies of CP26. PSI-LHCI-LHCII also stably bound 0.4 copy of ferredoxin-NADP(+) oxidoreductase (FNR) that catalyzes light-induced electron transfer from PSI to NADP(+) in the presence of ferredoxin. We discuss the possible organization of these LHCIIs in the PSI-LHCI-LHCII supercomplex.

Physiological and proteomic alterations in rice (Oryza sativa L.) seedlings under hexavalent chromium stress.

Rice plants employ two strategies to cope with Cr toxicity: immobilizing Cr ions into cell walls to reduce its translocation and activating antioxidant defense to mitigate Cr-induced oxidative stress. The investigation aimed at understanding the physiological and proteomic responses of rice seedlings to hexavalent chromium (Cr(6+)) stress was conducted using two rice genotypes, which differ in Cr tolerance and accumulation. Cr toxicity (200 µM) heavily increased the accumulation of H2O2 and [Formula: see text], enhanced lipid peroxidation, decreased cell viability and consequently inhibited rice plant growth. Proteomic analyses suggest that the response of rice proteome to Cr stress is genotype- and Cr dosage-dependent and tissue specific. Sixty-four proteins, which show more than fourfold difference under either two Cr levels, have been successfully identified. They are involved in a range of cellular processes, including cell wall synthesis, energy production, primary metabolism, electron transport and detoxification. Two proteins related to cell wall structure, NAD-dependent epimerase/dehydratase and reversibly glycosylated polypeptide were greatly up-regulated by Cr stress. Their enhancements coupled with callose accumulation by Cr suggest that cell wall is an important barrier for rice plants to resist Cr stress. Some enzymes involved in antioxidant defense, such as ferredoxin-NADP reductase, NADP-isocitrate dehydrogenase, glyoxalase I (Gly I) and glutamine synthetase 1 (GS1) have also been identified in response to Cr stress. However, they were only detected in Cr-tolerant genotype, indicating the genotypic difference in the capacity of activating the defense system to fight against Cr-induced oxidative stress. Overall, two strategies in coping with Cr stress in rice plants can be hypothesized: (i) immobilizing Cr ions into cell walls to reduce its translocation and (ii) activating antioxidant defense to mitigate Cr-induced oxidative stress.

Enhanced Photosynthesis and Carbon Metabolism Favor Arsenic Tolerance in Artemisia annua, a Medicinal Plant as Revealed by Homology-Based Proteomics.

This paper provides the first proteomic evidence of arsenic (As) tolerance and interactive regulatory network between primary and secondary metabolism in the medicinal plant, Artemisia annua. While chlorophyll fluorescence and photosynthetic rate depicted mild inhibition, there was a significant enhancement in PSI activity, whole chain, ATP, and NADPH contents in 100 μM As treatments compared to the control plants. However, a decrease in the above variables was recorded under 150 μM treatments. Proteomic decoding of the survival strategy of A. annua under As stress using 2-DE followed by MALDI-MS/MS revealed a total of 46 differentially expressed protein spots. In contrast to other plants where As inhibits photosynthesis, A. annua showed appreciable photosynthetic CO2 assimilation and allocation of carbon resources at 100 μM As concentration. While an increased accumulation of ATP synthase, ferredoxin-NADP(H) oxidoreductase, and FeS-rieske proteins supported the operation of cyclic electron transport, mdr ABC transporter protein and pcs gene might be involved in As detoxification. The most interesting observation was an increased accumulation of LEAFY like novel protein conceivably responsible for an early onset of flowering in A. annua under As stress. This study not only affirmed the role of energy metabolism proteins but also identified potential candidates responsible for As tolerance in plants.

The end of the line: can ferredoxin and ferredoxin NADP(H) oxidoreductase determine the fate of photosynthetic electrons?

At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems.

NtcA is responsible for accumulation of the small isoform of ferredoxin:NADP oxidoreductase

In several cyanobacteria, petH, the gene encoding ferredoxin:NADP oxidoreductase (FNR), is transcribed from at least two promoters depending on growth conditions. Two transcripts (short and long) are translated from two different translation initiation sites, resulting in two isoforms (large and small, respectively). Here, we show that in Synechocystis PCC6803 the global transcriptional regulator NtcA activates transcription from the distal petH promoter. Modification of the NtcA-binding site prevents NtcA binding to the promoter in vitro and abolishes accumulation of the small isoform of FNR in vivo. We also demonstrate that a similar petH transcription and translation regime occurs in other cyanobacteria. The conditions under which this system operates provide hints for the function of each FNR isoform.

A STD-NMR study of the interaction of the Anabaena ferredoxin-NADP+ reductase with the coenzyme.

Ferredoxin-NADP+ reductase (FNR) catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD) techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.

Arabidopsis tic62 trol Mutant Lacking Thylakoid-Bound Ferredoxin–NADP+ Oxidoreductase Shows Distinct Metabolic Phenotype

Ferredoxin-NADP+ oxidoreductase (FNR), functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP+ was detected. Since the CO₂ fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants.

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic, Nitrate-limiting Conditions

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO2-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.

External loops at the ferredoxin-NADP+ reductase protein–partner binding cavity contribute to substrates allocation

Ferredoxin-NADP+ reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)+/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet–loop–sheet motif, loop102–114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261–269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102–114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261–269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261–269 appears a determinant to ensure reversibility in photosynthetic FNRs.

Enhanced H2 photoproduction by down-regulation of ferredoxin-NADP+ reductase (FNR) in the green alga Chlamydomonas reinhardtii

Renewable H2 photoproduction by green algae such as Chlamydomonas reinhardtii is a promising system for solar fuels. However, large-scale application of the system has lagged virtually due to lack of high H2-producing strains. We previously identified ferredoxin-NADP+ reductase (FNR) among the 105 proteins differentially expressed in Chlamydomonas during sulfur-deprived H2 photoproduction. In this work, we used an RNA interference (RNAi) approach to generate Chlamydomonas mutant strains with reduced levels of FNR. We found that fnr-RNAi strains exhibited higher rates of H2 photoproduction (2.5-fold) than wild type under sulfur-deprived condition. To elucidate the basis for this increase, we analyzed the physiological characteristics of the fnr-RNAi strains under such condition. Major changes, due to the down-regulation of FNR, included the lower rates of photosynthetic O2 evolution (44%), greater reduction of Rubisco amounts (60%) and higher rates of starch degradation (140%). These may result in an earlier onset of anaerobiosis and increased electron supply to the hydrogenases in the mutant strains. The results provide new information of FNR in regulating H2 metabolism as well as potential strains for further improvement of the organism toward application in solar-powered systems.