Root-type ferredoxin-NADP+ oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses.

In Arabidopsis, two leaf-type ferredoxin-NADP+ oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP+ , while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using ß-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

Comparative analysis of thylakoid protein complexes in state transition mutants nsi and stn7: focus on PSI and LHCII.

The photosynthetic machinery of plants can acclimate to changes in light conditions by balancing light-harvesting between the two photosystems (PS). This acclimation response is induced by the change in the redox state of the plastoquinone pool, which triggers state transitions through activation of the STN7 kinase and subsequent phosphorylation of light-harvesting complex II (LHCII) proteins. Phosphorylation of LHCII results in its association with PSI (state 2), whereas dephosphorylation restores energy allocation to PSII (state 1). In addition to state transition regulation by phosphorylation, we have recently discovered that plants lacking the chloroplast acetyltransferase NSI are also locked in state 1, even though they possess normal LHCII phosphorylation. This defect may result from decreased lysine acetylation of several chloroplast proteins. Here, we compared the composition of wild type (wt), stn7 and nsi thylakoid protein complexes involved in state transitions separated by Blue Native gel electrophoresis. Protein complex composition and relative protein abundances were determined by LC-MS/MS analyses using iBAQ quantification. We show that despite obvious mechanistic differences leading to defects in state transitions, no major differences were detected in the composition of PSI and LHCII between the mutants. Moreover, both stn7 and nsi plants show retarded growth and decreased PSII capacity under fluctuating light as compared to wt, while the induction of non-photochemical quenching under fluctuating light was much lower in both nsi mutants than in stn7.

Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography.

Exploring the structure and function of protein complexes requires their isolation in the native state-a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism for the physiology of oxygenic phototrophs, and the biogenesis of membrane-bound photosynthetic complexes has traditionally been studied using this cyanobacterium. In a typical approach, the protein complexes are purified with a combination of His-affinity chromatography and a size-based fractionation method such as gradient ultracentrifugation and/or native electrophoresis. However, His-affinity purification harbors prominent contaminants and the levels of many proteins are too low for a feasible multi-step purification. Here, we have developed a purification method for the isolation of 3x FLAG-tagged proteins from the membrane and soluble fractions of Synechocystis. Soluble proteins or solubilized thylakoids are subjected to a single affinity purification step that utilizes the highly specific binding of FLAG-affinity resin. After an intensive wash, the captured proteins are released from the resin under native conditions using an excess of synthetic 3x FLAG peptide. The protocol allows fast isolation of low abundant protein complexes with a superb purity.

Chloroplast Acetyltransferase NSI Is Required for State Transitions in Arabidopsis thaliana.

The amount of light energy received by the photosynthetic reaction centers photosystem II (PSII) and photosystem I (PSI) is balanced through state transitions. Reversible phosphorylation of a light-harvesting antenna trimer (L-LHCII) orchestrates the association between L-LHCII and the photosystems, thus adjusting the amount of excitation energy received by the reaction centers. In this study, we identified the enzyme NUCLEAR SHUTTLE INTERACTING (NSI; AT1G32070) as an active lysine acetyltransferase in the chloroplasts of Arabidopsis thaliana Intriguingly, nsi knockout mutant plants were defective in state transitions, even though they had a similar LHCII phosphorylation pattern as the wild type. Accordingly, nsi plants were not able to accumulate the PSI-LHCII state transition complex, even though the LHCII docking site of PSI and the overall amounts of photosynthetic protein complexes remained unchanged. Instead, the nsi mutants showed a decreased Lys acetylation status of specific photosynthetic proteins including PSI, PSII, and LHCII subunits. Our work demonstrates that the chloroplast acetyltransferase NSI is needed for the dynamic reorganization of thylakoid protein complexes during photosynthetic state transitions.

Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei.

The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

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.

Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances.

Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation.

The extreme Albino3 (Alb3) C terminus is required for Alb3 stability and function in Arabidopsis thaliana.

MAIN CONCLUSION: The extreme Alb3 C terminus is important for Alb3 stability in a light dependent manner, but is dispensable for LHCP insertion or D1 synthesis. YidC/Oxa1/Alb3 dependent insertion of membrane proteins is evolutionary conserved among bacteria, mitochondria and chloroplasts. Chloroplasts are challenged by the need to coordinate membrane integration of nuclear encoded, post-translationally targeted proteins into the thylakoids as well as of proteins translated on plastid ribosomes. The pathway facilitating post-translational targeting of the light-harvesting chlorophyll a/b binding proteins involves the chloroplast signal recognition particle, cpSRP54 and cpSRP43, as well as its membrane receptor FtsY and the translocase Alb3. Interaction of cpSRP43 with Alb3 is mediated by the positively charged, stromal exposed C terminus of Alb3. In this study, we utilized an Alb3 T-DNA insertion mutant in Arabidopsis thaliana lacking the last 75 amino acids to elucidate the function of this domain (alb3∆C). However, the truncated Alb3 protein (Alb3∆C) proved to be unstable under standard growth conditions, resulting in a reduction of Alb3∆C to 20 % of wild-type levels. In contrast, accumulation of Alb3∆C was comparable to wild type under low light growth conditions. Alb3∆C mutants grown under low light conditions were only slightly paler than wild type, accumulated almost wild-type levels of light harvesting proteins and were not affected in D1 synthesis, therefore showing that the extreme Alb3 C terminus is dispensable for both, co- and post-translational, protein insertion into the thylakoid membrane. However, reduction of Alb3∆C levels as observed under standard growth conditions resulted not only in a severely diminished accumulation of all thylakoid complexes but also in a strong defect in D1 synthesis and membrane insertion.

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.

Looking for the Last Universal Common Ancestor (LUCA).

Genomic sequences across diverse species seem to align towards a common ancestry, eventually implying that eons ago some universal antecedent organism would have lived on the face of Earth. However, when evolution is understood not only as a biological process but as a general thermodynamic process, it becomes apparent that the quest for the last universal common ancestor is unattainable. Ambiguities in alignments are unavoidable because the driving forces and paths of evolution cannot be separated from each other. Thus tracking down life's origin is by its nature a non-computable task. The thermodynamic tenet clarifies that evolution is a path-dependent process of least-time consumption of free energy. The natural process is without a demarcation line between animate and inanimate.