Lipid peroxidation and antioxidative response in Arabidopsis thaliana exposed to cadmium and copper

Formation of lipid hydroperoxides, malondialdehyde (MDA) and hydroxyalkenals (HAEs), membrane damages and antioxidative response of plants expressed as changes in glutathione S-transferase activity (GST) and anthocyanin accumulation were studied in Arabidopsis thaliana (L.) Heynh cv. Columbia plants treated for 7 days with various concentrations: 5, 25, 50, 100 μM Cd and Cu. Increased lipid hydroperoxide content was metal concentration-dependent. The level of MDA + HAE was elevated in Cd- and Cu- treated plants, but it was metal concentration-dependent under Cu stress. Electrolyte leakage measurements showed a larger membrane damage under Cu- than Cd-treatment. In Cu-stressed plants, GST activity was always enhanced in comparison with control, while in plants exposed to Cd it dropped slightly at lower metal concentrations; but at 100 μM Cd it was even higher than in plants treated with the same Cu concentration. Anthocyanin accumulation was considerably higher under Cu than Cd stress. Both lipid peroxidation and antioxidative response was stronger in Cu- than Cd-treated Arabidopsis thaliana plants. Various mechanisms of defense against the lipid peroxidation products, depending on the metal type, are discussed.     

Redox potentials of trinuclear μ-oxo ruthenium acetate clusters with N-heterocyclic ligands

The trinuclear clusters of general composition [Ru₃O(OOCCH₃)₆(N-Het)₃], where N-Het=pyridine and pyrazine derivatives, exhibit a series of reversible waves in the range of −1.8 to 2.4 V versus SHE, in acetonitrile, ascribed to the successive [cluster]^{−2/−1/0/+1/+2/+3} redox couples. The redox potentials decrease with the pK_a of the N-heterocyclic ligands according to the equations E°(+3/+2)= 2.24−0.023 pK_a; E°(+2/+1)=1.34−0.029 pK_a; E°(+1/0)=0.36−0.039 pK_a and E°(0/−1)=−0.68− 0.074 pK_a. The dependence is greater at lower oxidation states, reflecting the role of π-backbonding in the complexes.     

Fe modulates Cd-induced oxidative stress and the expression of stress responsive proteins in the nodules of Vigna radiata

The aim of this study was to investigate the protective role of Fe in providing tolerance against Cd-stress in root nodules of Vigna radiata, because Cd may be more deleterious in the absence of Fe. Biochemical, histological and proteomic responses to Cd-exposure (50?μM CdCl₂) were examined under Fe-sufficient (+Fe/+Cd) or Fe-deficient (?Fe/+Cd) soils by comparing non ?Cd exposed control (+Fe/?Cd) plants with additional control of Fe-deficient and non-exposed Cd plants (?Fe/?Cd). Cd-exposure negatively affected on growth and some physiological parameters of host plant and nodules, and also induced oxidative stress with the decline of antioxidative enzyme activities. The negative effects of Cd-exposure in +Fe/+Cd plants were much less than those in ?Fe/+Cd and ?Fe/?Cd ones. When compared with ?Fe/Cd and ?Fe/?Cd plants, a marked improvement of bacteriod development and cell division was observed and deformation of cell wall remarkably alleviated in the nodules of (+Fe/Cd) plants. Proteomic study revealed that 20 proteins were differentially expressed by Fe/Cd combined treatment. Eleven proteins of interest were identified and classified as precursor for RNA metabolism, storage of seeds, hypothetical proteins, and unknown proteins. These results indicate that Fe plays a pivotal role in alleviating Cd-stress, as evidence by reduction in oxidative damage and protection of cell wall and bacteriods in nodules.     

Responses of two kidney bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) cultivars to the combined stress of sulfur deficiency and cadmium toxicity

Plants suffer from combined stress of sulfur deficiency and cadmium toxicity in some agricultural lands. However, little is known about the reaction in plants, such as responses in antioxidant enzymes and non-protein thiol compounds, to such combined stress. Therefore, in this study, four treatments, S-sufficiency (T_S?Cd), S-deficiency (T_?S?Cd), Cd stress (T_S+Cd) and combined stress of S-deficiency and Cd stress (T_?S+Cd), were set up to investigate (1) the effects of sulfur deficiency or sulfur sufficiency on Cd toxicity to kidney bean cultivar seedlings and the related mechanisms, and (2) the responses of two kidney bean cultivars to combined stress of S-deficiency and Cd-tolerance. The results showed significant increases in hydrogen peroxide (H₂O₂) and malondialdehyde contents and significant increases in antioxidant enzyme (superoxide dismutase, catalase, peroxidase, and glutathione S-transferase) activities and non-protein thiol compounds (non-protein thiols, reduced glutathione, phytochelatins) synthesis in the plants in T_S+Cd and T_?S+Cd. On the tissue level, higher proportion of Cd was found to be immobilized/deposited in roots, while on the sub-cell level, higher proportion of Cd was located in cell walls and vacuole fractions with lower in cell organelles. Taken together, the results indicated that Cd detoxification was achieved by the two kidney bean cultivars through antioxidant enzyme activation, non-protein thiol compound synthesis and sub-cellular compartmentalization. In addition, the results indicated that sufficient S supply helped to relieve Cd toxicity, which is of special significance for remediation or utilization of Cd-contaminated soils as S is a plant essential nutrient.     

Changes in expression of proteins involved in alleviation of Fe-deficiency by sulfur nutrition in Brassica napus L.

In order to characterize the significance of sulfur (S) nutrition in protein expression under iron (Fe)-deficient conditions, gel-based proteomic analysis was performed with the leaves of Brassica napus exposed to S and Fe combined treatments: sufficient in S and Fe (+S/+Fe, control), sufficient S but Fe deprived (+S/?Fe), deprived S but sufficient Fe (?S/+Fe), and deprived S and Fe (?S/?Fe). The resulting data showed that 15 proteins were down-regulated due to production of oxidative damage as indicated by H₂O₂ and O ₂ ^?1 localizations and due to leaf chlorosis in leaves in S-deprived leaves either in presence (?S/+Fe) or absence of Fe (?S/?Fe), whereas these down-regulated proteins were well expressed in the presence of S (+S/?Fe) compared to control (+S/+Fe). In addition, two proteins were up-regulated under S-deprived condition in presence (?S/+Fe) and absence of (?S/?Fe) Fe. The functional classification of these identified proteins was estimated that 40 % of the proteins belong to chloroplast precursor, and rest of the proteins belongs to hypothetical proteins, RNA binding, secondary metabolism and unknown proteins. On the other hand, five protein spots from S deprived (?S/+Fe) and ten spots from Fe deprived (?S/?Fe) conditions were absent, whereas they were well expressed in presence of S (+S/?Fe) compared to control plants (+S/+Fe). These results suggest that sulfur nutrition plays an important role in alleviating protein damage in Fe-deficient plants and adaptation to Fe-deficiency in oilseed rape.     

Ascorbate plays a key role in alleviating low temperature-induced oxidative stress in Arabidopsis

Low temperature has a negative impact on plant cells and results in the generation of reactive oxygen species (ROS). In order to study the role of ascorbate under chilling stress, the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc2-1 to low temperature (2°C) was investigated. After chilling stress, vtc2-1 mutants exhibited oxidative damage. An increase in the H₂O₂ generation and the production of thiobarbituric acid reactive substances (TBARS), and a decrease in chlorophyll content, the maximal photochemical efficiency of PSII (F_v/F_m) and oxidizable P₇₀₀ were also noted. The ratio of ascorbate/dehydroascorbate and reduced glutathione/oxidzed glutathione in the vtc2-1 mutants were reduced, compared with the wild type (WT) plants. The activities of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), and soluble antioxidants were lower in the vtc2-1 mutants than those in WT plants. These results suggested that the ascorbate-deficient mutant vtc2-1 was more sensitive to chilling treatment than WT plants. The low temperature-induced oxidative stress was the major cause of the decrease of PSII and PSI function in the vtc2-1 mutants. Ascorbate plays a critical role of defense without which the rest of the ROS defense network is unable to react effectively.     

Tissue analyses guidelines for diagnosing sulfur deficiency in white wheat

Summary Visual identification of S deficiency in white wheat is difficult since deficiency symptoms are nearly identical with those of N deficiency. In this study, S deficiency was best identified by determining the total N/S ratio rather than S concentration in vegetative tissue. Vegetative growth generally decreased from tillering to boot when the whole plant N/S ratio exceeded 17. The N/S ratio in S-sufficient plants declined gradually with age, implying that the critical N/S ratio may decline with advancing growth. Changes in stem: leaf ratio could have been responsible for the decline since the N/S ratio in stem tissue at heading was less than that of green leaf tissue.Sulphur concentration less reliably indicated S-deficiency, because differences in S levels between S-deficient and S-sufficient wheat, were often less than year-to-year variation of S concentration of plants sampled at the same growth stage. In addition, S concentration in whole plants declined sharply between tillering and heading. These factors make it difficult to designate a critical S level. Sulfur distribution among various plant organs suggests that critical S levels might best be obtained by utilizing green leaf tissue.Nitrogen concentration in S-sufficient wheat plants also decreased quite rapidly with growth, which indicates a similar difficulty for determining critical N percentages. Consequently, the most reliable distinction between N and S deficiency in wheat was accomplished by evaluation of the total N/S ratio in whole plant tissue.Contribution from the Agricultural Research Service, USDA, in cooperation with the Agricultural Experiment Station, Oregon State University. Technical Paper No.3953 of the latter.     

Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana

Aims

Metallothioneins are cysteine-rich, metal-binding proteins, but their exact functions are not fully understood. In this study, we isolated two metallothionein genes, BcMT1 and BcMT2 from Brassica campestris to increase our understanding of metal tolerance mechanisms in Brassica plants.

Methods

Semi-quantitative RT-PCR was used to analyze expression of the two BcMTs genes. BcMT1 and BcMT2 were ectopically expressed in Arabidopsis thaliana. Quantitative real-time RT-PCR and GUS-staining method were used to select transgenic Arabidopsis plants. Cd and Cu concentrations were analyzed by flame atomic absorption spectrometry. Histochemical detection of H₂O₂ and O₂ ^?? were conducted by 3,3-diaminobenzidine and nitroblue tetrazoliu-staining methods.

Results

BcMT1 is expressed predominantly in roots, whereas BcMT2 is expressed mainly in leaves of B. campestris. Expression of BcMT1 was induced by both Cd and Cu, but expression of BcMT2 was enhanced only by Cd. Ectopic expression of BcMT1 and BcMT2 in Arabidopsis thaliana enhanced the tolerance to Cd and Cu and increased the Cu concentration in the shoots of the transgenic plants. Transgenic Arabidopsis accumulated less reactive oxygen species (ROS) than wild-type plants.

Conclusions

BcMT1 and BcMT2 increased Cd and Cu tolerance in transgenic Arabidopsis, and decreased production of Cd- and Cu-induced ROS, thereby protecting plants from oxidative damage.     

The Arabidopsis thaliana Immunophilin ROF1 Directly Interacts with PI(3)P and PI(3,5)P2 and Affects Germination under Osmotic Stress

A direct interaction of the Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the rof1⁻ knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WSrof1⁻/2⁻). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.     

Arabidopsis thaliana Glyoxalase 2-1 Is Required during Abiotic Stress but Is Not Essential under Normal Plant Growth

The glyoxalase pathway, which consists of the two enzymes, GLYOXALASE 1 (GLX 1) (E.C.: 4.4.1.5) and 2 (E.C.3.1.2.6), has a vital role in chemical detoxification. In Arabidopsis thaliana there are at least four different isoforms of glyoxalase 2, two of which, GLX2-1 and GLX2-4 have not been characterized in detail. Here, the functional role of Arabidopsis thaliana GLX2-1 is investigated. Glx2-1 loss-of-function mutants and plants that constitutively over-express GLX2-1 resemble wild-type plants under normal growth conditions. Insilico analysis of publicly available microarray datasets with ATTEDII, Mapman and Genevestigator indicate potential role(s) in stress response and acclimation. Results presented here demonstrate that GLX2-1 gene expression is up-regulated in wild type Arabidopsis thaliana by salt and anoxia stress, and by excess L-Threonine. Additionally, a mutation in GLX2-1 inhibits growth and survival during abiotic stresses. Metabolic profiling studies show alterations in the levels of sugars and amino acids during threonine stress in the plants. Elevated levels of polyamines, which are known stress markers, are also observed. Overall our results suggest that Arabidopsis thaliana GLX2-1 is not essential during normal plant life, but is required during specific stress conditions.     

Arabidopsis desaturase 2 gene is involved in the regulation of cadmium and lead resistance

Aims

It was shown previously that Arabidopsis (Arabidopsis thaliana) desaturase 2 (ADS2) cDNA was isolated and it was shown that the expression of ADS2 was organ-dependent and up-regulated by low temperature. However, little is known about the role of ADS2 gene in heavy metal resistance in plants. In this study, we showed that ADS2 gene is involved in the regulation of cadmium (Cd) and lead (Pb) resistance.

Methods

For heavy metal resistance tests, seeds were germinated and grown on 1/2 MS media supplemented with the indicated concentrations of metal ions. To quantify root length, plants were grown vertically in plates. For heavy metal treatments, two-week old wild-type seedlings grown on MS media were treated with cadmium (Cd) or lead (Pb) for 24 h, and then sampled for metal content measurement and qPCR analysis.

Results

ADS2 was strongly repressed by Cd(II), and ads2-1 mutant plants showed increased Cd(II) resistance. A lower Cd content was detected in ads2-1 plants than in wild-type plants subjected to Cd(II) treatment, which was associated with activation in expression of AtPDR8 gene, a pump excluding Cd(II) and/or Cd(II)-containing toxic compounds from the cytoplasm, suggesting that ADS2-mediated Cd(II) resistance is AtPDR8 dependent. We also found that ads2-1 plants showed increased Pb(II) sensitivity, and ADS2 was strongly repressed by hydrogen peroxide (H₂O₂) but not by Pb(II). The ads2-1 mutant showed increased sensitivity to oxidative stresses mediated by H₂O₂ and paraquat, and higher levels of H₂O₂ accumulation were observed in leaves of ads2-1 plants than those of wild-type plants when subjected to Pb(II) and H₂O₂, indicating that ADS2 mediates Pb(II) resistance indirectly by impaired ROS scavenging.

Conclusions

ADS2 gene mediates Cd(II) and Pb(II) resistance, at least in part, through two distinct mechanisms, an AtPDR8-dependent mechanism and a ROS detoxification system-mediated mechanism, respectively.     

Balancing defense and growth—Analyses of the beneficial symbiosis between Piriformospora indica and Arabidopsis thaliana

The mutualistic interaction between the endophytic and root-colonizing fungus Piriformospora indica and Arabidopsis thaliana is a nice model system to study beneficial and non-benefical traits in a symbiosis. Colonized Arabidopsis plants are taller, produce more seeds and are more resistant against biotic and abiotic stress. Based on genetic, molecular and cellular analyses, Arabidopsis mutants were identified which are impaired in their beneficial response to the fungus. Several mutants are smaller rather than bigger in the presence of the fungus and are defective in defense responses. This includes mutants with defects in defense-signaling components, defense proteins and enzymes, and defense metabolites. The mutants cannot control root colonization and are often over-colonized by P. indica. As a consequence, the benefits for the plants are lost and they try to restrict root colonization by activating unspecific defense responses against P. indica. These observations raise the question as to how the plants balance defense gene activation or development and what signaling molecules are involved. P. indica promotes the synthesis of phosphatidic acid (PA), which binds to the 3-PHOSPHOINOSITIDE-DEPENDENT-KINASE1 (PDK1). This activates a kinase pathway which might be crucial for balancing defense and growth responses. The review describes plant defense compounds which are necessary for the mutualistic interaction between the two symbionts. Furthermore, it is proposed that the PA/PDK1 pathway may be crucial for balancing defense responses and growth stimulation during the interaction with P. indica.     

Mitogen-Activated Protein Kinases 3 and 6 Are Required for Full Priming of Stress Responses in Arabidopsis thaliana

In plants and animals, induced resistance (IR) to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. It has been hypothesized that cell priming involves accumulation of latent signaling components that are not used until challenge exposure to stress. However, the identity of such signaling components has remained elusive. Here, we show that during development of chemically induced resistance in Arabidopsis thaliana, priming is associated with accumulation of mRNA and inactive proteins of mitogen-activated protein kinases (MPKs), MPK3 and MPK6. Upon challenge exposure to biotic or abiotic stress, these two enzymes were more strongly activated in primed plants than in nonprimed plants. This elevated activation was linked to enhanced defense gene expression and development of IR. Strong elicitation of stress-induced MPK3 and MPK6 activity is also seen in the constitutive priming mutant edr1, while activity was attenuated in the priming-deficient npr1 mutant. Moreover, priming of defense gene expression and IR were lost or reduced in mpk3 or mpk6 mutants. Our findings argue that prestress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for full induction of defense responses during IR.     

Green peach aphid infestation induces Arabidopsis PHYTOALEXIN-DEFICIENT4 expression at site of insect feeding

The Arabidopsis thaliana PHYTOALEXIN-DEFICIENT4 (PAD4) protein, which has homology to lipases, is required for phloem-based resistance against the green peach aphid (GPA; Myzus persicae Sülzer). PAD4 modulates antibiotic and antixenotic defenses against GPA. PAD4 in conjunction with its interacting partner ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) also functions in basal resistance to bacterial and oomycete pathogens by promoting salicylic acid (SA)-dependent and SA-independent defenses. By contrast, neither EDS1 nor SA is required for PAD4-controlled defense against GPA. Distinct molecular activities of PAD4 are involved in different aspects of Arabidopsis defense against GPA and pathogens. Histochemical analysis of plants containing a PAD4_p:GUS chimera, which expresses the GUS reporter from the PAD4 promoter, indicated strong PAD4 promoter activity at the site of penetration of the vasculature by the insect stylet. GUS activity was also observed in non-vascular tissues of GPA-infested leaves, thus raising the possibility that a combination of distinct PAD4 activities in vascular and non-vascular tissues contribute to Arabidopsis defense against GPA.