The Role of the Plant Antioxidant System in Drought Tolerance.

Water deficiency compromises plant performance and yield in many habitats and in agriculture. In addition to survival of the acute drought stress period which depends on plant-genotype-specific characteristics, stress intensity and duration, also the speed and efficiency of recovery determine plant performance. Drought-induced deregulation of metabolism enhances generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which in turn affect the redox regulatory state of the cell. Strong correlative and analytical evidence assigns a major role in drought tolerance to the redox regulatory and antioxidant system. This review compiles current knowledge on the response and function of superoxide, hydrogen peroxide and nitric oxide under drought stress in various species and drought stress regimes. The meta-analysis of reported changes in transcript and protein amounts, and activities of components of the antioxidant and redox network support the tentative conclusion that drought tolerance is more tightly linked to up-regulated ascorbate-dependent antioxidant activity than to the response of the thiol-redox regulatory network. The significance of the antioxidant system in surviving severe phases of dehydration is further supported by the strong antioxidant system usually encountered in resurrection plants.

Low CO2 induces urea cycle intermediate accumulation in Arabidopsis thaliana.

The non-proteinogenic amino acid ornithine links several stress response pathways. From a previous study we know that ornithine accumulates in response to low CO2. To investigate ornithine accumulation in plants, we shifted plants to either low CO2 or low light. Both conditions increased carbon limitation, but only low CO2 also increased the rate of photorespiration. Changes in metabolite profiles of light- and CO2-limited plants were quite similar. Several amino acids that are known markers of senescence accumulated strongly under both conditions. However, urea cycle intermediates respond differently between the two treatments. While the levels of both ornithine and citrulline were much higher in plants shifted to 100 ppm CO2 compared to those kept in 400 ppm CO2, their metabolite abundance did not significantly change in response to a light limitation. Furthermore, both ornithine and citrulline accumulation is independent from sugar starvation. Exogenous supplied sugar did not significantly change the accumulation of the two metabolites in low CO2-stressed plants, while the accumulation of other amino acids was reduced by about 50%. Gene expression measurements showed a reduction of the entire arginine biosynthetic pathway in response to low CO2. Genes in both proline biosynthesis and degradation were induced. Hence, proline did not accumulate in response to low CO2 like observed for many other stresses. We propose that excess of nitrogen re-fixed during photorespiration can be alternatively stored in ornithine and citrulline under low CO2 conditions. Furthermore, ornithine is converted to pyrroline-5-carboxylate by the action of δOAT.

Regulatory cis-elements are located in accessible promoter regions of the CAT2 promoter and affect activating histone modifications in Arabidopsis thaliana.

Catalase 2 (CAT2) plays an important role in the detoxification of hydrogen peroxide released either during photorespiration or as a consequence of biotic and abiotic stress as well as in the initiation of senescence. To date, our understanding of the regulation of CAT2 gene expression is rather poor. Chromatin immunoprecipitation experiments revealed that a wide region of the CAT2 promoter is nucleosome depleted, reflecting the ability to rapidly respond to changing environmental and stress conditions and, thus, adjusting the transcript levels of CAT2. The lowest nucleosome density was found in the region of -900 bp relative to the transcription initiation start (TIS) where two regulatory elements are located. The distance of the nucleosome depleted region to the TIS is quite unusual because the majority of nucleosome free regions are generally located in close vicinity to the 5' untranslated region. The analysis of transgenic 5' upstream deletion::gusA Arabidopsis lines showed that this region is important for the regulation of CAT2 promoter activity. To evaluate the function of the two motifs, the contribution of each element to CAT2 promoter activity was analyzed by site directed mutagenesis. The data revealed that the CAT2 promoter is regulated by the ACGT motif (Box2) rather than by the G-Box binding motif (Box1) in the vegetative phase of development. Furthermore, the presence of both Box1 and Box2 positively affected the abundance of activating histone modifications.

The 5'UTR Intron of Arabidopsis GGT1 Aminotransferase Enhances Promoter Activity by Recruiting RNA Polymerase II.

Photorespiration is essential for the detoxification of glycolate and recycling of carbon to the Calvin Benson Bassham cycle. Enzymes participating in the pathway have been identified, and investigations now focus on the regulation of photorespiration by transporters and metabolites. However, regulation of photorespiration on the gene level has not been intensively studied. Here, we show that maximum transcript abundance of Glu:glyoxylate aminotransferase 1 (GGT1) is regulated by intron-mediated enhancement (IME) of the 5' leader intron rather than by regulatory elements in the 5' upstream region. The intron is rich in CT-stretches and contains the motif TGTGATTTG that is highly similar to the IME-related motif TTNGATYTG. The GGT1 intron also confers leaf-specific expression of foreign promoters. Quantitative PCR analysis and GUS activity measurements revealed that IME of the GGT1 5'UTR intron is controlled on the transcriptional level. IME by the GGT1 5'UTR intron was at least 2-fold. Chromatin immunoprecipitation experiments showed that the abundance of RNA polymerase II binding to the intron-less construct is reduced.

Intron-Mediated Enhancement: A Tool for Heterologous Gene Expression in Plants?

Many plant promoters were characterized and used for transgene expression in plants. Even though these promoters drive high levels of transgene expression in plants, the expression patterns are rarely constitutive but restricted to some tissues and developmental stages. In terms of crop improvement not only the enhancement of expression per se but, in particular, tissue-specific and spatial expression of genes plays an important role. Introns were used to boost expression in transgenic plants in the field of crop improvement for a long time. However, the mechanism behind this so called intron-mediated enhancement (IME) is still largely unknown. This review highlights the complexity of IME on the levels of its regulation and modes of action and gives an overview on IME methodology, examples in fundamental research and models of proposed mechanisms. In addition, the application of IME in heterologous gene expression is discussed.

A simple mechanism for the establishment of C2-specific gene expression in Brassicaceae.

The transition of C3 , via C2 towards C4 photosynthesis is an important example of stepwise evolution of a complex genetic trait. A common feature that was gradually emphasized during this trajectory is the evolution of a CO2 concentration mechanism around Rubisco. In C2 plants, this mechanism is based on tissue-specific accumulation of glycine decarboxylase (GDC) in bundle sheath (BS) cells, relative to global expression in the cells of C3 leaves. This limits photorespiratory CO2 release to BS cells. Because BS cells are surrounded by photosynthetically active mesophyll cells, this arrangement enhances the probability of re-fixation of CO2 . The restriction of GDC to BS cells was mainly achieved by confinement of its P-subunit (GLDP). Here, we provide a mechanism for the establishment of C2 -type gene expression by studying the upstream sequences of C3 Gldp genes in Arabidopsis thaliana. Deletion of 59 bp in the upstream region of AtGldp1 restricted expression of a reporter gene to BS cells and the vasculature without affecting diurnal variation. This region was named the 'M box'. Similar results were obtained for the AtGldp2 gene. Fusion of the M box to endogenous or exogenous promoters supported mesophyll expression. Nucleosome densities at the M box were low, suggesting an open chromatin structure facilitating transcription factor binding. In silico analysis defined a possible consensus for the element that was conserved across the Brassicaceae, but not in Moricandia nitens, a C2 plant. Collective results provide evidence that a simple mutation is sufficient for establishment of C2 -specific gene expression in a C3 plant.

The Arabidopsis class II sirtuin is a lysine deacetylase and interacts with mitochondrial energy metabolism.

The posttranslational regulation of proteins by lysine (Lys) acetylation has recently emerged to occur not only on histones, but also on organellar proteins in plants and animals. In particular, the catalytic activities of metabolic enzymes have been shown to be regulated by Lys acetylation. The Arabidopsis (Arabidopsis thaliana) genome encodes two predicted sirtuin-type Lys deacetylases, of which only Silent Information Regulator2 homolog (SRT2) contains a predicted presequence for mitochondrial targeting. Here, we have investigated the function of SRT2 in Arabidopsis. We demonstrate that SRT2 functions as a Lys deacetylase in vitro and in vivo. We show that SRT2 resides predominantly at the inner mitochondrial membrane and interacts with a small number of protein complexes mainly involved in energy metabolism and metabolite transport. Several of these protein complexes, such as the ATP synthase and the ATP/ADP carriers, show an increase in Lys acetylation in srt2 loss-of-function mutants. The srt2 plants display no growth phenotype but rather a metabolic phenotype with altered levels in sugars, amino acids, and ADP contents. Furthermore, coupling of respiration to ATP synthesis is decreased in these lines, while the ADP uptake into mitochondria is significantly increased. Our results indicate that SRT2 is important in fine-tuning mitochondrial energy metabolism.

Metabolic recovery of Arabidopsis thaliana roots following cessation of oxidative stress.

To cope with the various environmental stresses resulting in reactive oxygen species (ROS) production plant metabolism is known to be altered specifically under different stresses. After overcoming the stress the metabolism should be reconfigured to recover basal operation however knowledge concerning how this is achieved is cursory. To investigate the metabolic recovery of roots following oxidative stress, changes in metabolite abundance and carbon flow were analysed. Arabidopsis roots were treated by menadione to elicit oxidative stress. Roots were fed with (13)C labelled glucose and the redistribution of isotope was determined in order to study carbon flow. The label redistribution through many pathways such as glycolysis, the tricarboxylic acid (TCA) cycle and amino acid metabolism were reduced under oxidative stress. After menadione removal many of the stress-related changes reverted back to basal levels. Decreases in amounts of hexose phosphates, malate, 2-oxoglutarate, glutamate and aspartate were fully recovered or even increased to above the control level. However, some metabolites such as pentose phosphates and citrate did not recover but maintained their levels or even increased further. The alteration in label redistribution largely correlated with that in metabolite abundance. Glycolytic carbon flow reverted to the control level only 18 h after menadione removal although the TCA cycle and some amino acids such as aspartate and glutamate took longer to recover. Taken together, plant root metabolism was demonstrated to be able to overcome menadione-induced oxidative stress with the differential time period required by independent pathways suggestive of the involvement of pathway specific regulatory processes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0296-1) contains supplementary material, which is available to authorized users.

Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis.

Acetylation of the ε-amino group of lysine (Lys) is a reversible posttranslational modification recently discovered to be widespread, occurring on proteins outside the nucleus, in most subcellular locations in mammalian cells. Almost nothing is known about this modification in plants beyond the well-studied acetylation of histone proteins in the nucleus. Here, we report that Lys acetylation in plants also occurs on organellar and cytosolic proteins. We identified 91 Lys-acetylated sites on 74 proteins of diverse functional classes. Furthermore, our study suggests that Lys acetylation may be an important posttranslational modification in the chloroplast, since four Calvin cycle enzymes were acetylated. The plastid-encoded large subunit of Rubisco stands out because of the large number of acetylated sites occurring at important Lys residues that are involved in Rubisco tertiary structure formation and catalytic function. Using the human recombinant deacetylase sirtuin 3, it was demonstrated that Lys deacetylation significantly affects Rubisco activity as well as the activities of other central metabolic enzymes, such as the Calvin cycle enzyme phosphoglycerate kinase, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase, and the tricarboxylic acid cycle enzyme malate dehydrogenase. Our results demonstrate that Lys acetylation also occurs on proteins outside the nucleus in Arabidopsis (Arabidopsis thaliana) and that Lys acetylation could be important in the regulation of key metabolic enzymes.

Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezers.

We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

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 metabolic response of Arabidopsis roots to oxidative stress is distinct from that of heterotrophic cells in culture and highlights a complex relationship between the levels of transcripts, metabolites, and flux.

Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study (Baxter et al., 2007), the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture: there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.

Role of Lon1 protease in post-germinative growth and maintenance of mitochondrial function in Arabidopsis thaliana.

Maintenance of protein quality control and turnover is essential for cellular homeostasis. In plant organelles this biological process is predominantly performed by ATP-dependent proteases. Here, a genetic screen was performed that led to the identification of Arabidopsis thaliana Lon1 protease mutants that exhibit a post-embryonic growth retardation phenotype. Translational fusion to yellow fluorescent protein revealed AtLon1 subcellular localization in plant mitochondria, and the AtLon1 gene could complement the respiratory-deficient phenotype of the yeast PIM1 gene homolog. AtLon1 is highly expressed in rapidly growing plant organs of embryonic origin, including cotyledons and primary roots, and in inflorescences, which have increased mitochondria numbers per cell to fulfill their high energy requirements. In lon1 mutants, the expression of both mitochondrial and nuclear genes encoding respiratory proteins was normal. However, mitochondria isolated from lon1 mutants had a lower capacity for respiration of succinate and cytochrome c via complexes II and IV, respectively. Furthermore, the activity of key enzymes of the tricarboxylic acid (TCA) cycle was significantly reduced. Additionally, mitochondria in lon1 mutants had an aberrant morphology. These results shed light on the developmental mechanisms of selective proteolysis in plant mitochondria and suggest a critical role for AtLon1 protease in organelle biogenesis and seedling establishment.

Functional characterisation of the peroxiredoxin gene family members of Synechococcus elongatus PCC 7942.

The genome of Synechococcus elongatus PCC 7942 encodes six peroxiredoxins (Prx). Single genes are present each for a 1-Cys Prx and a 2-Cys Prx, while four genes code for PrxQ-like proteins (prxQ-A1, -A2, -A3 and B). Their transcript accumulation varies with growth conditions in a gene-specific manner (Stork et al. in J Exp Bot 56:3193-3206, 2005). To address their functional properties, members of the prx gene family were produced as recombinant proteins and analysed for their peroxide detoxification capacity and quaternary structure by size exclusion chromatography. Independent of the reduction state, the 2-Cys Prx separated as oligomer, the 1-Cys Prx as dimer and the PrxQ-A1 as monomer. PrxQ-A2 was inactive in our assays, 1-Cys Prx activity was unaffected by addition of TrxA, while all others were stimulated to a variable extent by addition of E. coli thioredoxin. Sensitivity towards cumene hydroperoxide treatment of E. coli BL21 cells expressing the cyanobacterial PrxQ-A1 to A3 proteins was greatly reduced, while expression of the other Prx had no effect. The study shows differentiation of Prx functions in S. elongatus PCC 7942 which is discussed in relation to potential roles in site- and stress-specific defence.

Role of the cysteine residues in Arabidopsis thaliana cyclophilin CYP20-3 in peptidyl-prolyl cis-trans isomerase and redox-related functions.

Cyps (cyclophilins) are ubiquitous proteins of the immunophilin superfamily with proposed functions in protein folding, protein degradation, stress response and signal transduction. Conserved cysteine residues further suggest a role in redox regulation. In order to get insight into the conformational change mechanism and functional properties of the chloroplast-located CYP20-3, site-directed mutagenized cysteine-->serine variants were generated and analysed for enzymatic and conformational properties under reducing and oxidizing conditions. Compared with the wild-type form, elimination of three out of the four cysteine residues decreased the catalytic efficiency of PPI (peptidyl-prolyl cis-trans isomerase) activity of the reduced CYP20-3, indicating a regulatory role of dithiol-disulfide transitions in protein function. Oxidation was accompanied by conformational changes with a predominant role in the structural rearrangement of the disulfide bridge formed between Cys(54) and Cys(171). The rather negative E(m) (midpoint redox potential) of -319 mV places CYP20-3 into the redox hierarchy of the chloroplast, suggesting the activation of CYP20-3 in the light under conditions of limited acceptor availability for photosynthesis as realized under environmental stress. Chloroplast Prx (peroxiredoxins) were identified as interacting partners of CYP20-3 in a DNA-protection assay. A catalytic role in the reduction of 2-Cys PrxA and 2-Cys PrxB was assigned to Cys(129) and Cys(171). In addition, it was shown that the isomerization and disulfide-reduction activities are two independent functions of CYP20-3 that both are regulated by the redox state of its active centre.

S-nitrosylation of peroxiredoxin II E promotes peroxynitrite-mediated tyrosine nitration.

Nitric oxide (NO) is a free radical product of cell metabolism that plays diverse and important roles in the regulation of cellular function. S-Nitrosylation is emerging as a specific and fundamental posttranslational protein modification for the transduction of NO bioactivity, but very little is known about its physiological functions in plants. We investigated the molecular mechanism for S-nitrosylation of peroxiredoxin II E (PrxII E) from Arabidopsis thaliana and found that this posttranslational modification inhibits the hydroperoxide-reducing peroxidase activity of PrxII E, thus revealing a novel regulatory mechanism for peroxiredoxins. Furthermore, we obtained biochemical and genetic evidence that PrxII E functions in detoxifying peroxynitrite (ONOO-), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and O2- that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO- detoxification activity of PrxII E, causing a dramatic increase of ONOO--dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO-, indicating that the PrxII E modulation of ONOO- bioactivity is biologically relevant. We conclude that NO regulates the effects of its own radicals through the S-nitrosylation of crucial components of the antioxidant defense system that function as common triggers for reactive oxygen species- and NO-mediated signaling events.

The function of peroxiredoxins in plant organelle redox metabolism.

In 1996, cDNA sequences referred to as plant peroxiredoxins (Prx), i.e. a 1-Cys Prx and a 2-Cys Prx, were reported from barley. Ten years of research have advanced our understanding of plant Prx as thiol-based peroxide reductases with a broad substrate specificity, ranging from hydrogen peroxide to alkyl hydroperoxides and peroxinitrite. Prx have several features in common. (i) They are abundant proteins that are routinely detected in proteomics approaches. (ii) They interact with proteins such as glutaredoxins, thioredoxins, and cyclophilins as reductants, but also non-dithiol-disulphide exchange proteins. By work with transgenic plants, their activity was shown to (iii) affect metabolic integrity, (iv) protect DNA from damage in vitro and as shown here in vivo, and (v) modulate intracellular signalling related to reactive oxygen species and reactive nitrogen species. (vi) In all organisms Prx are encoded by small gene families that are of particular complexity in higher plants. A comparison of the Prx gene families in rice and Arabidopsis thaliana supports previous suggestions on Prx function in specific subcellular and metabolic context. (vii) Prx gene expression and activity are subjected to complex regulation realized by an integration of various signalling pathways. 2-Cys Prx expression depends on redox signals, abscisic acid, and protein kinase cascades. Besides these general properties, the chloroplast Prx have acquired specific roles in the context of photosynthesis. The thioredoxin-dependent peroxidase activity can be measured in crude plant extracts and contributes significantly to the overall H(2)O(2) detoxification capacity. Thus organellar Prx proteins enable an alternative water-water cycle for detoxification of photochemically produced H(2)O(2), which acts independently from the ascorbate-dependent Asada-Halliwell-Foyer cycle. 2-Cys Prx and Prx Q associate with thylakoid membrane components. The mitochondrial PrxII F is essential for root growth under stress. Following a more general introduction, the paper summarizes present knowledge on plant organellar Prx, addressing Prx in signalling, and also suggests some lines for future research.

Peroxiredoxin Q of Arabidopsis thaliana is attached to the thylakoids and functions in context of photosynthesis.

Peroxiredoxin Q (Prx Q) is one out of 10 peroxiredoxins encoded in the genome of Arabidopsis thaliana, and one out of four that are targeted to plastids. Peroxiredoxin Q functions as a monomeric protein and represents about 0.3% of chloroplast proteins. It attaches to the thylakoid membrane and is detected in preparations enriched in photosystem II complexes. Peroxiredoxin Q decomposes peroxides using thioredoxin as an electron donor with a substrate preference of H(2)O(2) > cumene hydroperoxide >> butyl hydroperoxide >> linoleoyl hydroperoxide and insignificant affinity towards complex phospholipid hydroperoxide. Plants with decreased levels of Prx Q did not have an apparently different phenotype from wildtype at the plant level. However, similar to antisense 2-cysteine (2-Cys) Prx plants [Baier, M. et al. (2000)Plant Physiol., 124, 823-832], Prx Q-deficient plants had a decreased sensitivity to oxidants in a leaf slice test as indicated by chlorophyll a fluorescence measurements. Increased fluorescence ratios of photosystem II to I at 77 K and modified transcript levels of plastid- and nuclear-encoded proteins show that regulatory mechanisms are at work to compensate for the lack of Prx Q. Apparently Prx Q attaches to photosystem II and has a specific function distinct from 2-Cys peroxiredoxin in protecting photosynthesis. Its absence causes metabolic changes that are sensed and trigger appropriate compensatory responses.