Elke Ströher

Dynamic plastid redox signals integrate gene expression and metabolism to induce distinct metabolic states in photosynthetic acclimation in Arabidopsis

Plants possess acclimation responses in which structural reconfigurations adapt the photosynthetic apparatus to fluctuating illumination. Long-term acclimation involves changes in plastid and nuclear gene expression and is controlled by redox signals from photosynthesis. The kinetics of these signals and the adjustments of energetic and metabolic demands to the changes in the photosynthetic apparatus are currently poorly understood. Using a redox signaling system that preferentially excites either photosystem I or II, we measured the time-dependent impact of redox signals on the transcriptome and metabolome of Arabidopsis thaliana. We observed rapid and dynamic changes in nuclear transcript accumulation resulting in differential and specific expression patterns for genes associated with photosynthesis and metabolism. Metabolite pools also exhibited dynamic changes and indicate readjustments between distinct metabolic states depending on the respective illumination. These states reflect reallocation of energy resources in a defined and reversible manner, indicating that structural changes in the photosynthetic apparatus during long-term acclimation are additionally supported at the level of metabolism. We propose that photosynthesis can act as an environmental sensor, producing retrograde redox signals that trigger two parallel adjustment loops that coordinate photosynthesis and metabolism to adapt plant primary productivity to the environment.

Mitochondrial Malate Dehydrogenase Lowers Leaf Respiration and Alters Photorespiration and Plant Growth in Arabidopsis

Malate dehydrogenase (MDH) catalyzes a reversible NAD+-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO2 assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO2 assimilation/intercellular CO2 curves at low CO2, and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.

The redox-sensitive transcription factor Rap2.4a controls nuclear expression of 2-Cys peroxiredoxin A and other chloroplast antioxidant enzymes

BackgroundThe regulation of the chloroplast antioxidant capacity depends on nuclear gene expression. For the 2-Cys peroxiredoxin-A gene (2CPA) a cis-regulatory element was recently characterized, which responds to photosynthetic redox signals.ResultsIn a yeast-one-hybrid screen for cis-regulatory binding proteins, the transcription factor Rap2.4a was isolated. Rap2.4a controls the transcript abundance of the prominent chloroplast antioxidant enzyme through binding to the CGCG core of a CE3-like element. Rap2.4a activity is regulated by dithiol/disulfide transition of regulatory cysteinyl residues and subsequent changes in the quaternary structure. The mid-point redox potential of Rap2.4a activation is -269 mV (pH 7.0).ConclusionThe redox sensitivity of Rap2.4a establishes an efficient switch mechanism for redox control of nuclear gene activity of chloroplast antioxidants, in which Rap2.4 is a redox-sensor and a transducer of redox information.

Multiple Redox and Non-Redox Interactions Define 2-Cys Peroxiredoxin as a Regulatory Hub in the Chloroplast

In plants, the highly abundant 2-cysteine peroxiredoxin (2-CysPrx) is associated with the chloroplast and involved in protecting photosynthesis. This work addresses the multiple interactions of the 2-CysPrx in the chloroplast, which depend on its redox state. Transcript co-regulation analysis showed a strong linkage to the peptidyl-prolyl-cis/trans isomerase Cyclophilin 20-3 (Cyp20-3) and other components of the photosynthetic apparatus. Co-expression in protoplasts and quantification of fluorescence resonance energy transfer (FRET) efficiency in vivo confirmed protein interactions of 2-CysPrx with Cyp20-3 as well as NADPH-dependent thioredoxin reductase C (NTRC), while thioredoxin x (Trx-x) did not form complexes that could enable FRET. Likewise, changes in FRET of fluorescently labeled 2-CysPrx in vitro and in vivo proved redox dependent dynamics of 2-CysPrx. Addition of Cyp20-3 to an in vitro peroxidase assay with 2-CysPrx had no significant effect on peroxide reduction. Also, in the presence of NTRC, addition of Cyp20-3 did not further enhance peroxide reduction. In addition, 2-CysPrx functioned as chaperone and inhibited aggregation of citrate synthase during heat treatment. This activity was partly inhibited by Cyp20-3. As a new interaction partner of decameric 2-CysPrx, photosystem II could be identified after chloroplast fractionation and in pull-down assays after reconstitution. In summary, the data indicate a dynamic function of plant 2-CysPrx as redox sensor, chaperone, and regulator in the chloroplast with diverse functions beyond its role as thiol peroxidase.

The redox imbalanced Mutants of Arabidopsis Differentiate Signaling Pathways for Redox Regulation of Chloroplast Antioxidant Enzymes

A network of enzymatic and nonenzymatic antioxidants protects chloroplasts from photooxidative damage. With all enzymatic components being nuclear encoded, the control of the antioxidant capacity depends on chloroplast-to-nucleus redox signaling. Using an Arabidopsis (Arabidopsis thaliana) reporter gene line expressing luciferase under control of the redox-sensitive 2-cysteine peroxiredoxin A (2CPA) promoter, six mutants with low 2CPA promoter activity were isolated, of which five mutants show limitations in redox-box regulation of the 2CPA promoter. In addition to 2CPA, the transcript levels for other chloroplast antioxidant enzymes were decreased, although a higher oxidation status of the ascorbate pool, a higher reduction state of the plastoquinone pool, and an increased oxidation status of the 2-Cys peroxiredoxin pool demonstrated photooxidative stress conditions. Greening of the mutants, chloroplast ultrastructure, steady-state photosynthesis, and the responses to the stress hormone abscisic acid were wild type like. In the rosette state, the mutants were more sensitive to low CO2 and to hydrogen peroxide. Comparison of gene expression patterns and stress sensitivity characterizes the mutants as redox imbalanced in the regulation of nuclear-encoded chloroplast antioxidant enzymes and differentiates redox signaling cascades.

The biological roles of glutaredoxins.

Grxs (glutaredoxins) are small ubiquitous redox enzymes. They are generally involved in the reduction of oxidative modifications using glutathione. Grxs are not only able to reduce protein disulfides and the low-molecular-mass antioxidant dehydroascorbate, but also represent the major enzyme class responsible for deglutathionylation reactions. Functional proteomics, including interaction studies, comparative activity measurements using heterologous proteins and structural analysis are combined to provide important insights into the crucial function of Grxs in cellular redox networks. Summarizing the current understanding of Grxs, with a special focus on organelle-localized members across species, genus and kingdom boundaries (including cyanobacteria, plants, bacteria, yeast and humans) lead to two different classifications, one according to sequence structure that gives insights into the diversification of Grxs, and another according to function within the cell that provides a basis for assessing the different roles of Grxs.

The dynamic thiol–disulphide redox proteome of the Arabidopsis thaliana chloroplast as revealed by differential electrophoretic mobility

The dynamics of the thiol-disulphide redox proteome is central to cell function and its regulation. Altered mobility of proteins in the oxidized and reduced state allows the MS-based identification of those thiol-disulphide proteins that undergo major conformational changes. A proteomic approach was taken with thylakoid-bound, luminal and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-less stromal subproteome fractions of the chloroplast from Arabidopsis thaliana. Among the 49 verified polypeptides were 22 novel redox proteins, previously not reported as being part of the redox proteome. Among the redox-affected proteins were PsbA (D1), PsaA1 and PsaF, chloroplast monodehydroascorbate reductase and also the Deg1 protease. Recombinant Deg1 and Deg2 revealed redox dependence of their proteolytic activity. The data provide new insights into the redox network of the chloroplast.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

Unlike thioredoxins, glutaredoxins are involved in iron-sulfur cluster assembly and in reduction of specific disulfides (i.e. protein-glutathione adducts), and thus they are also important redox regulators of chloroplast metabolism. Using GFP fusion, AtGrxC5 isoform, present exclusively in Brassicaceae, was shown to be localized in chloroplasts. A comparison of the biochemical, structural, and spectroscopic properties of Arabidopsis GrxC5 (WCSYC active site) with poplar GrxS12 (WCSYS active site), a chloroplastic paralog, indicated that, contrary to the solely apomonomeric GrxS12 isoform, AtGrxC5 exists as two forms when expressed in Escherichia coli. The monomeric apoprotein possesses deglutathionylation activity mediating the recycling of plastidial methionine sulfoxide reductase B1 and peroxiredoxin IIE, whereas the dimeric holoprotein incorporates a [2Fe-2S] cluster. Site-directed mutagenesis experiments and resolution of the x-ray crystal structure of AtGrxC5 in its holoform revealed that, although not involved in its ligation, the presence of the second active site cysteine (Cys32) is required for cluster formation. In addition, thiol titrations, fluorescence measurements, and mass spectrometry analyses showed that, despite the presence of a dithiol active site, AtGrxC5 does not form any inter- or intramolecular disulfide bond and that its activity exclusively relies on a monothiol mechanism.

Redox-Dependent Regulation of the Stress-Induced Zinc-Finger Protein SAP12 in Arabidopsis thaliana

The stress-associated protein SAP12 belongs to the stress-associated protein (SAP) family with 14 members in Arabidopsis thaliana. SAP12 contains two AN1 zinc fingers and was identified in diagonal 2D redox SDS-PAGE as a protein undergoing major redox-dependent conformational changes. Its transcript was strongly induced under cold and salt stress in a time-dependent manner similar to SAP10, with high levels after 6 h and decreasing levels after 24 and 48 h. The transcript regulation resembled those of the stress marker peroxiredoxin PrxIID at 24 and 48 h. Recombinant SAP12 protein showed redox-dependent changes in quaternary structure as visualized by altered electrophoretic mobility in non-reducing SDS polyacrylamide gel electrophoresis. The oxidized oligomer was reduced by high dithiothreitol concentrations, and also by E. coli thioredoxin TrxA with low dithiothreitol (DTT) concentrations or NADPH plus NADPH-dependent thioredoxin reductase. From Western blots, the SAP12 protein amount was estimated to be in the range of 0.5 ng mug(-1) leaf protein. SAP12 protein decreased under salt and cold stress. These data suggest a redox state-linked function of SAP12 in plant cells particularly under cold and salt stress.

Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis

Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T-DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co-expressed with a number of genes encoding mitochondrial proteins, including SDH1-1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin-adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein-bound flavin-adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro-respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.