Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants

Two chloroplastic proteins, named CDSP 32 and CDSP 34 for chloroplastic drought-induced stress protein of 32 and 34 kDa, were previously shown to be substantially synthesized in Solanum tuberosum plants subjected to water deficit. We investigated the localization of CDSPs in leaf chloroplasts from control and wilted potato plants using immunocytochemistry. Observation of electron micrographs did not reveal any important change in plastid structures of drought-stressed plants, except an increased number and a larger size of plastoglobuli. In well-watered plants, very little labeling corresponding to CDSP 32 was detected. Consecutively to water stress, a higher abundance of CDSP 32 was revealed, the protein being exclusively localized in the stroma. Immunocytochemical data indicated the presence of some CDSP 34 protein in well-watered plants and confirmed its accumulation upon water deficit. CDSP 34 was found to be preferentially associated with stromal lamellae thylakoids, but some protein was revealed in the stroma. No association of CDSP 34 with grana and plastoglobuli was noticed in chloroplasts from control and stressed plants.     

Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress

Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.     

Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants

In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.     

Identification and characterization of plastid trnF(GAA) pseudogenes in four species of Solanum (Solanaceae)

Non-functional trnF pseudogenes that rarely occur in embryophytes have been found in Solanaceae. We have sequenced the trnL-F intergenic spacer of four species of Solanum, and found duplicated regions of the original trnF gene. These repeats were 94–260 bp long causing large length variation in the trnL-F intergenic spacer resulting from differences in pseudogene copy number (2–4). The duplicated trnF regions are comprised of several highly structured motifs, which were partial residues, or entire parts of the Anticodon, T- and D-domains of the original gene, but all lacked the acceptor stems at the 5′- or 3′-end. Pseudogenes included several transitions and transversions in their sequences compared to the original trnF gene. Among pseudogene copies, T-domains were more frequent and fragmented than D-domain elements. Our results demonstrate that although chloroplast evolution is uniform such structural duplications in the sequences used for phylogenetic reconstructions should be treated with great caution.     

Response to biotic and oxidative stress in Arabidopsis thaliana: analysis of variably phosphorylated proteins

Protein phosphorylation plays a pivotal role in the regulation of many cellular events; increasing evidences indicate that this post-translational modification is involved in plant response to various abiotic and biotic stresses. Since phosphorylated proteins may be present at low abundance, enrichment methods are generally required for their analysis. We here describe the quantitative changes of phosphoproteins present in Arabidopsis thaliana leaves after challenging with elicitors or treatments mimicking biotic stresses, which stimulate basal resistance responses, or oxidative stress. Phosphoproteins from elicited and control plants were enriched by means of metal oxide affinity chromatography and resolved by 2D electrophoresis. A comparison of the resulting proteomic maps highlighted phosphoproteins showing quantitative variations induced by elicitor treatment; these components were identified by MALDI-TOF peptide mass fingerprinting and/or nanoLC-ESI-LIT-MS/MS experiments. In total, 97 differential spots, representing 75 unique candidate phosphoproteins, were characterized. They are representative of different protein functional groups, such as energy and carbon metabolism, response to oxidative and abiotic stresses, defense, protein synthesis, RNA processing and cell signaling. Ascertained protein phosphorylation found a positive confirmation in available Arabidopsis phosphoproteome database. The role of each identified phosphoprotein is here discussed in relation to plant defense mechanisms. Our results suggest a partial overlapping of the responses to different treatments, as well as a communication with key cellular functions by imposed stresses.     

Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment

Background

Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.

Results

We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.

Conclusions

We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.     

Evaluation of oxidative stress tolerance in maize (Zea mays L.) seedlings in response to drought

Seedlings of selected six genotypes of maize (Zea mays L.) differing in their drought sensitivity (LM5 and Parkash drought-tolerant and PMH2, JH3459, Paras and LM14 as drought-sensitive) were exposed to 72 h drought stress at two leaf stage. Alterations in their antioxidant pools combined with activities of enzymes involved in defense against oxidative stress were investigated in leaves. Activities of some reactive oxygen species (ROS)-scavenging enzymes, catalase (CAT) and ascorbate peroxidase (APX) were enhanced in tolerant genotypes in response to drought stress. Superoxide dismutase (SOD) activity was significantly decreased in sensitive genotypes, but remained unchanged in tolerant genotypes under stress. Peroxidase (POX) activity was significantly induced in tolerant, as well as sensitive genotypes. Imposition of stress led to increase in H2O2 and malondialdehyde (MDA, a marker for lipid peroxidation) content in sensitive genotypes, while in tolerant genotypes no change was observed. Significant increase in glutathione content was observed in sensitive genotypes. Ascorbic acid pool was induced in both tolerant and sensitive genotypes, but induction was more pronounced in tolerant genotypes. Significant activation of antioxidative defence mechanisms correlated with drought-induced oxidative stress tolerance was the characteristic of the drought tolerant genotypes. These studies provide a mechanism for drought tolerance in maize seedlings.     

Up-expression of NapA and other oxidative stress proteins is a compensatory response to loss of major Helicobacter pylori stress resistance factors

Twenty-six Helicobacter pylori targeted mutant strains with deficiencies in oxidative stress combating proteins, including 12 double mutant strains were analyzed via physiological and proteomic approaches to distinguish the major expression changes caused by the mutations. Mutations were introduced into both a Mtz^S and a Mtz^R strain background. Most of the mutations caused increased growth sensitivity of the strains to oxygen, and they all exhibited clear compensatory up-expression of oxidative stress resistance proteins enabling survival of the bacterium. The most frequent up-expressed oxidative stress resistance factor (observed in 16 of the mutants) was the iron-sequestering protein NapA, linking iron sequestration with oxidative stress resistance. The up-expression of individual proteins in mutants ranged from 2 to 10 fold that of the wild type strain, even when incubated in a low O₂ environment. For example, a considerably higher level of catalase expression (4 fold of that in the wild-type strain) was observed in ahpC napA and ahpC sodB double mutants. A Fur mutant up-expressed ferritin (Pfr) protein 20-fold. In some mutant strains the bacterial DNA is protected from oxidative stress damage apparently via overexpression of oxidative stress-combating proteins such as NapA, catalase or MdaB (an NADPH quinone reductase). Our results show that H. pylori has a variety of ways to compensate for loss of major oxidative stress combating factors.     

Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

Changes in gene expression, by application of H₂O₂, O₂°^–generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H₂O₂ and gamma irradiation, but not to O₂°^– generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I–IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H₂O₂ and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H₂O₂ action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H₂O₂ pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential.     

Deubiquitinating enzymes (DUBs): Regulation,homeostasis, and oxidative stress response

Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein–protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.     

Differences in the response of sunflower (Helianthus annuus) subjected to flooding and drought stress

Wample, R. L. and Thornton, R. K. 1984. Differences in the response of sunflower ( Helianthus annuus ) subjected to flooding and drought stress.
Comparison of drought- and flood-stressed sunflower plants ( Helianthus annuus L. hybrid 894) showed some similarities in response but differences in the mechanisms responsible for the responses to stress. Drought–stressed plants showed typical reductions in leaf water potential with increasing stress accompanied by increased leaf resistance. Photosynthesis declined while photorespiration increased after 48 and 96 h of drought stress. A primary reason for reduced photosynthesis in drought-stressed plants was increased stomatal resistance. No significant 0change in leaf water potential or in leaf resistance in flooded plants was found in this study. However, photosynthesis declined in a manner similar to that in drought-stressed plants and photorespiration showed only a transient increase at 48 h. Dark respiration was significantly higher at 48 and 96 h but the magnitude of the increase cannot account for the reduction in photosynthesis. Since the photosynthetic rate of flooded plants declined while stomata remained open, an effect at a more fundamental level is suggested and is thought to be related to disruption of carbohydrate transport.     

Dps proteins, an efficient detoxification and DNA protection machinery in the bacterial response to oxidative stress

The proteins belonging to the Dps (DNA-binding proteins from starved cells) family play an important role within the bacterial defence system against oxidative stress. They act on Fe(II) and hydrogen peroxide that are potentially toxic in the presence of air. Fe(II) forms spontaneously insoluble Fe(III) and reacts with molecular oxygen or its reduced forms to yield the highly damaging hydroxyl radicals. All Dps proteins have the distinctive capacity to annul the toxic combination of iron and hydrogen peroxide as they use the latter compound to oxidise Fe(II). In addition to this intrinsic DNA protection capacity, several members of the family, including the archetypical Escherichia coli Dps, protect DNA physically by shielding it in large Dps-DNA complexes. The structural and functional characteristics that endow Dps proteins with the chemical and physical protection mechanism are presented and discussed also in the framework of the varied situations that may be encountered in different bacterial species.      

Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT; EC 2. 1.1.77) catalyses the methyl esterification of the free alpha-carboxyl group of abnormal L-isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of L-isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t-butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane-cytoskeleton network stability. We conclude that the increased formation/exposure of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.     

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis

Recent work has highlighted the importance of protein post-translational modifications such as phosphorylation (enzymatic) and nitrosylation (nonenzymatic) in the early stages of apoptosis. In this study, we have investigated the levels of protein carbonylation, a nonenzymatic protein modification that occurs in conditions of cellular oxidative stress, during etopside-induced apoptosis of HL60 cells. Within 1 h of VP16 treatment, a number of proteins underwent carbonylation due to oxidative stress. This was inhibited by the antioxidant N-acetyl-L-cysteine. Among the proteins found to be carbonylated were glycolytic enzymes. Subsequently, we found that the rate of glycolysis was significantly reduced, probably due to a carbonylation mediated reduction in enzymatic activity of glycolytic enzymes. Our work demonstrates that protein carbonylation can be rapidly induced through cytotoxic drug treatment and may specifically inhibit the glycolytic pathway. Given the importance of glycolysis as a source of cellular ATP, this has severe implications for cell function.