Salicylic acid (SA) plays a key role in plant disease resistance and hypersensitive cell death but is also implicated in hardening responses to abiotic stressors. Cadmium (Cd) exposure increased the free SA contents of barley (Hordeum vulgare) roots by a factor of about 2. Cultivation of dry barley caryopses presoaked in SA-containing solution for only 6 h or single transient addition of SA at a 0.5 mm concentration to the hydroponics solution partially protected the seedlings from Cd toxicity during the following growth period. Both SA treatments had little effect on growth in the absence of Cd, but increased root and shoot length and fresh and dry weight and inhibited lipid peroxidation in roots, as indicated by malondialdehyde contents, in the presence of Cd. To test whether this protection was due to up-regulation of antioxidant enzymes, activities and transcript levels of the H 2 O 2 -metabolizing enzymes such as catalase and ascorbate peroxidase were measured in control and SA-treated seedlings in the presence or absence of 25 m Cd. Cd stress increased the activity of these enzymes by variable extent. SA treatments strongly or completely suppressed the Cd-induced up-regulation of the antioxidant enzyme activities. Slices from leaves treated with SA for 24 h also showed an increased level of tolerance toward high Cd concentrations as indicated by chlorophyll a fluorescence parameters. The results support the conclusion that SA alleviates Cd toxicity not at the level of antioxidant defense but by affecting other mechanisms of Cd detoxification.Cd is a highly toxic and persistent environmental poison for plants and animals (di Toppi and Gabbrielli, 1999). Cd interferes with many cellular functions mainly by complex formation with side groups of organic compounds such as proteins resulting in inhibition of essential activities. Although the mechanisms of cytoplasmic toxicity are identical in all organisms, different plant species and varieties show a wide range of plasticity in Cd tolerance, reaching from the high degree of sensitivity of most plants on the one hand to the hyperaccumulating phenotype of some tolerant higher plants on the other hand (McGrath et al., 2001). On an expanded concentration scale, even sensitive species vary considerably in their response to Cd. For example pea (Pisum sativum) is considerably more sensitive to Cd than barley (Hordeum vulgare cv Gerbel), which still grows well at concentrations above 10 m under nutrient rich conditions. Cd induces genetic and biochemical changes in plant metabolism that are related to general and Cd-specific stress responses (Blinda et al., 1997). Cd tolerance is correlated with intracellular compartmentalization and hence specific transport processes that allow the toxic effects of low Cd levels to decrease at least (Brune et al., 1995; Gonzalez et al., 1999). The activation of the cellular antioxidant metabolism belongs to the general stress responses induced by heavy metals (Dietz et al., 1999). Although an active antioxidative metabolism does not...

Acetylation of the «-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.

Peroxiredoxins (prxs) are peroxidases with broad substrate specificity. The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application. The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions. The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate. prx Q also responded to peroxides and diamide treatment. prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate. The strongest responses were observed for prx II C, which was induced with all treatments. The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli. In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction. The midpoint redox potentials (E m Ј) of Prx II B, Prx II C, and Prx II E were around Ϫ290 mV and, thus, less negative than E m Ј of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (Ϫ307 to Ϫ322 mV). The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge. Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light. The results are discussed in context with the subcellular localization of the Prx gene products.Peroxiredoxins (Prxs) are enzymes that reduce hydrogen peroxide (H 2 O 2 ) and alkyl hydroperoxides. They are grouped in four classes: (a) 2-Cys Prx; (b) Prx Q; (c) Prx II, which all contain two catalytic Cys residues in distinct sequence environment; and (d) 1-Cys Prx with one conserved Cys residue only (Dietz, 2003). A phylogenetic distance analysis suggests that 2-Cys Prx, Prx Q, and 1-Cys Prx are related proteins, whereas the group of Prx II is likely to have evolved independently (Verdoucq et al., 1999;Horling et al., 2002). The catalytic Cys residues undergo oxidation during the peroxide reduction reaction and need to be reduced by electron donors such as glutaredoxins, thioredoxins, or cyclophilins before the next catalytic cycle (Lee et al., 2001;Rouhier et al., 2001;Kö nig et al., 2002). For the bacterial and animal homologs, a broad substrate specificity has been described (Nogoceke et al., 1997; Bryk et al., 2000; Hillas et al., 2000). In in vitro tests, these Prx proteins reduced H 2 O 2 , lipid peroxides, such as butyl hydroperoxide, phospholipid peroxides and cumene hydroperoxide, and peroxynitrite. For plant Prxs, the catalytic properties have only poorly been investigated.The Arabidopsis genome encodes 10 open reading frames (ORFs) for peroxiredoxins. Based on sequence similarities, they can be assigned to the four subgroups of peroxiredoxins: two ORFs code for 2...

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 À ), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and O 2 À that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO À detoxification activity of PrxII E, causing a dramatic increase of ONOO À -dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO À , indicating that the PrxII E modulation of ONOO À 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.

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 “helper” NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

Peroxiredoxins (Prx) have recently moved into the focus of plant and animal research in the context of development, adaptation, and disease, as they function both in antioxidant defense by reducing a broad range of toxic peroxides and in redox signaling relating to the adjustment of cell redox and antioxidant metabolism. At-PrxII F is one of six type II Prx identified in the genome of Arabidopsis thaliana and the only Prx that is targeted to the plant mitochondrion. Therefore, it might be assumed to have functions similar to the human 2-Cys Prx (PRDX3) and type II Prx (PRDX5) and yeast 1-Cys Prx that likewise have mitochondrial localizations. This paper presents a characterization of PrxII F at the level of subcellular distribution, activity, and reductive regeneration by mitochondrial thioredoxin and glutaredoxin. By employing tDNA insertion mutants of A. thaliana lacking expression of AtprxII F (KO-AtPrxII F), it is shown that under optimal environmental conditions the absence of PrxII F is almost fully compensated for, possibly by increases in activity of mitochondrial ascorbate peroxidase and glutathione-dependent peroxidase. However, a stronger inhibition of root growth in KO-AtPrxII F seedlings as compared with wild type is observed under stress conditions induced by CdCl 2 as well as after administration of salicylhydroxamic acid, an inhibitor of cyanide-insensitive respiration. Simultaneously, major changes in the abundance of both nuclear and mitochondria-encoded transcripts were observed. These results assign a principal role to PrxII F in antioxidant defense and possibly redox signaling in plants cells.

The time of seed germination is a major decision point in the life of plants determining future growth and development. This timing is controlled by seed dormancy, which prevents germination under favourable conditions. The plant hormone abscisic acid (ABA) and the protein DELAY OF GERMINATION 1 (DOG1) are essential regulators of dormancy. The function of ABA in dormancy is rather well understood, but the role of DOG1 is still unknown. Here, we describe four phosphatases that interact with DOG1 in seeds. Two of them belong to clade A of type 2C protein phosphatases: ABA-HYPERSENSITIVE GERMINATION 1 (AHG1) and AHG3. These phosphatases have redundant but essential roles in the release of seed dormancy epistatic to DOG1. We propose that the ABA and DOG1 dormancy pathways converge at clade A of type 2C protein phosphatases.

Summary Mitochondria host vital cellular functions, including oxidative phosphorylation and co‐factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity‐based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage‐Dependent Anion Channel 1 to sub‐stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.