Arabidopsis FNRL protein is an NADPH-dependent chloroplast oxidoreductase resembling bacterial ferredoxin-NADP+ reductases.

Plastidic ferredoxin-NADP+ oxidoreductases (FNRs; EC:1.18.1.2) together with bacterial type FNRs (FPRs) form the plant-type FNR family. Members of this group contain a two-domain scaffold that forms the basis of an extended superfamily of flavin adenine dinucleotide (FAD) dependent oxidoreductases. In this study, we show that the Arabidopsis thaliana At1g15140 [Ferredoxin-NADP+ oxidoreductase-like (FNRL)] is an FAD-containing NADPH dependent oxidoreductase present in the chloroplast stroma. Determination of the kinetic parameters using the DCPIP NADPH-dependent diaphorase assay revealed that the reaction catalysed by a recombinant FNRL protein followed a saturation Michaelis-Menten profile on the NADPH concentration with kcat = 3.2 ± 0.2 s-1 , KmNADPH = 1.6 ± 0.3 µM and kcat /KmNADPH = 2.0 ± 0.4 µM-1 s-1 . Biochemical assays suggested that FNRL is not likely to interact with Arabidopsis ferredoxin 1, which is supported by the sequence analysis implying that the known Fd-binding residues in plastidic FNRs differ from those of FNRL. In addition, based on structural modelling FNRL has an FAD-binding N-terminal domain built from a six-stranded ß-sheet and one α-helix, and a C-terminal NADP+ -binding α/ß domain with a five-stranded ß-sheet with a pair of α-helices on each side. The FAD-binding site is highly hydrophobic and predicted to bind FAD in a bent conformation typically seen in bacterial FPRs.

Downregulation of TAP38/PPH1 enables LHCII hyperphosphorylation in Arabidopsis mutant lacking LHCII docking site in PSI.

Redox-regulated reversible phosphorylation of the light-harvesting complex II (LHCII) controls the excitation energy distribution between photosystem (PS) II and PSI. The PsaL and PsaH subunits of PSI enable the association of pLHCII to PSI. Here, we show that the failure of the psal mutant to dock pLHCII to PSI induces excessive phosphorylation of LHCII, primarily due to a marked downregulation of the TAP38/PPH1 phosphatase occurring at post-transcriptional level. TAP38/PPH1 is shown to be associated with megacomplex that contains both photosystems in a light- and LHCII-PSII core-phosphorylation-dependent manner. It is suggested that proper megacomplex-related association of TAP38/PPH1 protects it against degradation.

Posttranslational Modifications of Chloroplast Proteins: An Emerging Field.

Posttranslational modifications of proteins are key effectors of enzyme activity, protein interactions, targeting, and turnover rate, but despite their importance, they are still poorly understood in plants. Although numerous reports have revealed the regulatory role of protein phosphorylation in photosynthesis, various other protein modifications have been identified in chloroplasts only recently. It is known that posttranslational N(α)-acetylation occurs in both nuclear- and plastid-encoded chloroplast proteins, but the physiological significance of this acetylation is not yet understood. Lysine acetylation affects the localization and activity of key metabolic enzymes, and it may work antagonistically or cooperatively with lysine methylation, which also occurs in chloroplasts. In addition, tyrosine nitration may help regulate the repair cycle of photosystem II, while N-glycosylation determines enzyme activity of chloroplastic carbonic anhydrase. This review summarizes the progress in the research field of posttranslational modifications of chloroplast proteins and points out the importance of these modifications in the regulation of chloroplast metabolism.

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.

Depletion of leaf-type ferredoxin-NADP(+) oxidoreductase results in the permanent induction of photoprotective mechanisms in Arabidopsis chloroplasts.

Arabidopsis thaliana contains two photosynthetically competent chloroplast-targeted ferredoxin-NADP(+) oxidoreductase (FNR) isoforms that are largely redundant in their function. Nevertheless, the FNR isoforms also display distinct molecular phenotypes, as only the FNR1 is able to directly bind to the thylakoid membrane. We report the consequences of depletion of FNR in the F(1) (fnr1 × fnr2) and F(2) (fnr1 fnr2) generation plants of the fnr1 and fnr2 single mutant crossings. The fnr1 × fnr2 plants, with a decreased total content of FNR, showed a small and pale green phenotype, accompanied with a marked downregulation of photosynthetic pigment-protein complexes. Specifically, when compared with the wild type (WT), the quantum yield of photosystem II (PSII) electron transport was lower, non-photochemical quenching (NPQ) was higher and the rate of P700(+) re-reduction was faster in the mutant plants. The slight over-reduction of the plastoquinone pool detected in the mutants resulted in the adjustment of the reactive oxygen species (ROS) scavenging systems, as both the content and de-epoxidation state of xanthophylls, as well as the content of α-tocopherol, were higher in the leaves of the mutant plants when compared with the WT. The fnr1 fnr2 double mutant plants, which had no detectable FNR and possessed an extremely downregulated photosynthetic machinery, survived only when grown heterotrophically in the presence of sucrose. Intriguingly, the fnr1 fnr2 plants were still capable of sustaining the biogenesis of a few malformed chloroplasts.

Nodularin uptake and induction of oxidative stress in spinach (Spinachia oleracea).

The bloom-forming cyanobacterium Nodularia spumigena produces toxic compounds, including nodularin, which is known to have adverse effects on various organisms. We monitored the primary effects of nodularin exposure on physiological parameters in Spinachia oleracea. We present the first evidence for the uptake of nodularin by a terrestrial plant, and show that the exposure of spinach to cyanobacterial crude water extract from nodularin-producing strain AV1 results in inhibition of growth and bleaching of the leaves. Despite drastic effects on phenotype and survival, nodularin did not disturb the photosynthetic performance of plants or the structure of the photosynthetic machinery in the chloroplast thylakoid membrane. Nevertheless, the nodularin-exposed plants suffered from oxidative stress, as evidenced by a high level of oxidative modifications targeted to various proteins, altered levels of enzymes involved in scavenging of reactive oxygen species (ROS), and increased levels of α-tocopherol, which is an important antioxidant. Moreover, the high level of cytochrome oxidase (COX II), a typical marker for mitochondrial respiratory protein complexes, suggests that the respiratory capacity is increased in the leaves of nodularin-exposed plants. Actively respiring plant mitochondria, in turn, may produce ROS at high rates. Although the accumulation of ROS and induction of the ROS scavenging network enable the survival of the plant upon toxin exposure, the upregulation of the enzymatic defense system is likely to increase energetic costs, reducing growth and the ultimate fitness of the plants.

Drought stress-induced upregulation of components involved in ferredoxin-dependent cyclic electron transfer.

Linear photosynthetic electron transfer, consisting of both Photosystem (PS) II and PSI, converts light energy into the chemical forms ATP and NADPH, whereas PSI cyclic electron transfer (CET) is exclusively involved in ATP synthesis. In the chloroplasts of higher plants, there are two partially redundant CET routes. The ferredoxin (FD) or ferredoxin-plastoquinone reductase (FQR)-dependent route cycles electrons from PSI to plastoquinone via ferredoxin (FD), while in the NDH-dependent route, NADPH donates electrons to the NDH-complex for reduction of the plastoquinone pool. In the present study, we show that drought stress induces transcriptional and translational upregulation of the PGR5 and PGRL1 genes, which are the only characterized components of the FQR-dependent CET thus far. In contrast, the expression of the NDH-H gene, a representative of the NDH-complex, did not differ between the drought-stressed and the control plants. The overall expression level of the ferredoxin-NADP(+)-oxidoreductase (FNR) genes increased upon drought stress, with a concomitant release of FNR from the thylakoid membrane. Moreover, drought stress accelerated the rate of P700(+) re-reduction, which may indicate induction of CET. Responses of the PSAE, FD and PSAD gene families upon drought stress are also described.

Comparative analysis of leaf-type ferredoxin-NADP oxidoreductase isoforms in Arabidopsis thaliana.

Physiological roles of the two distinct chloroplast-targeted ferredoxin-NADP(+) oxidoreductase (FNR) isoforms in Arabidopsis thaliana were studied using T-DNA insertion line fnr1 and RNAi line fnr2. In fnr2 FNR1 was present both as a thylakoid membrane-bound form and as a soluble protein, whereas in fnr1 the FNR2 protein existed solely in soluble form in the stroma. The fnr2 plants resembled fnr1 in having downregulated photosynthetic properties, expressed as low chlorophyll content, low accumulation of photosynthetic thylakoid proteins and reduced carbon fixation rate when compared with wild type (WT). Under standard growth conditions the level of F(0)'rise' and the amplitude of the thermoluminescence afterglow (AG) band, shown to correlate with cyclic electron transfer (CET), were reduced in both fnr mutants. In contrast, when plants were grown under low temperatures, both fnr mutants showed an enhanced rate of CET when compared with the WT. These data exclude the possibility that distinct FNR isoforms feed electrons to specific CET pathways. Nevertheless, the fnr2 mutants had a distinct phenotype upon growth at low temperature. The fnr2 plants grown at low temperature were more tolerant against methyl viologen (MV)-induced cell death than fnr1 and WT. The unique tolerance of fnr2 plants grown at low temperature to oxidative stress correlated with an increased level of reduced ascorbate and reactive oxygen species (ROS) scavenging enzymes, as well as with a scarcity in the accumulation of thylakoid membrane protein complexes, as compared with fnr1 and WT. These results emphasize a critical role for FNR2 in the redistribution of electrons to various reducing pathways, upon conditions that modify the photosynthetic capacity of the plant.