Production Of Non-protein Thiols Research Articles

Nitric Oxide- and Sulfur-Mediated Reversal of Cadmium-Inhibited Photosynthetic Performance Involves Hydrogen Sulfide and Regulation of Nitrogen, Sulfur, and Antioxidant Metabolism in Mustard

This study aimed to test the role of hydrogen sulfide (H2S) in the responses regarding the nitric oxide- (NO) and sulfur (S)-mediated improvement in photosynthesis and growth under cadmium (Cd) stress in mustard (Brassica juncea L. cv. Giriraj), and integrate the mechanisms of S, nitrogen (N), and antioxidant metabolism. The plants grown with Cd (200 mg Cd kg−1 soil) exhibited reduced assimilation of S and N and diminished photosynthetic performance, which was associated with higher Cd accumulation-induced excess reactive oxygen species (ROS) production. The application of 100 μM of sodium nitroprusside (SNP, a NO donor) together with a more prominent concentration of S resulted in increased photosynthetic S- and N-use efficiency, production of non-protein thiols and phytochelatins, efficiency of enzymatic (superoxide dismutase, ascorbate peroxidase, and glutathione reductase), non-enzymatic antioxidants (ascorbate and glutathione) limiting Cd accumulation and, thus, reduced oxidative stress (superoxide radical, hydrogen peroxide, and thiobarbituric acid reactive species content). The benefit of NO together with S was manifested through a modulation in H2S production. The use of 100 μM of hypotaurine (HT; H2S scavenger) or 100 μM of cPTIO (2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) in plants treated with NO plus S reversed the action of NO plus S, with a higher reduction in photosynthesis and growth with the use of HT, suggesting that H2S plays a significant role in the NO- and S-mediated alleviation of Cd stress. The interplay of NO and ES with H2S may be used in augmenting the photosynthesis and growth of Cd-grown mustard plants.

Functional and mechanistic insights into the differential effect of the toxicant ‘Se(IV)’ in the cyanobacterium Anabaena PCC 7120

Selenium, an essential trace element for animals, poses a threat to all forms of life above a threshold concentration. The ubiquitously present cyanobacteria, a major photosynthetic biotic component of aquatic and other ecosystems, are excellent systems to study the effects of environmental toxicants. The molecular changes that led to beneficial or detrimental effects in response to different doses of selenium oxyanion Se(IV) were analyzed in the filamentous cyanobacterium Anabaena PCC 7120. This organism showed no inhibition in growth up to 15mg/L sodium selenite, but above this dose i.e. 20-100mg/L of Se(IV), both growth and photosynthesis were substantially inhibited. Along with the increased accumulation of non-protein thiols, a consistent reduction in levels of ROS was observed at 10mg/mL dose of Se(IV). High dose of Se(IV) (above 20mg/L) enhanced endogenous reactive oxygen species (ROS)/lipid peroxidation, and decreased photosynthetic capability. Treatment with 100mg/L Se(IV) downregulated transcription of several photosynthesis pathways-related genes such as those encoding photosystem I and II proteins, phycobilisome rod-core linker protein, phycocyanobilin, phycoerythrocyanin-associated proteins etc. Interestingly, at a dose range of 10-15mg/L Se(IV), Anabaena showed an increase in PSII photosynthetic yield and electron transport rate (at PSII), suggesting improved photosynthesis. Se was incorporated into the Anabaena cells, and Se-enriched thylakoid membranes showed higher redox conductivity than the thylakoid membranes from untreated cells. Overall, the data supports that modulation of photosynthetic machinery is one of the crucial mechanisms responsible for the dose-dependent contrasting effect of Se(IV) observed in Anabaena.

Integrative response of arsenic uptake, speciation and detoxification by Salix atrocinerea

Despite arsenic (As) being very toxic with deleterious effects on metabolism, it can be tolerated and accumulated by some plants. General genetic mechanisms responsible for As tolerance in plants, including Salix species, have been described in transcriptomic analysis, but further experimental verification of the significance of particular transcripts is needed. In this study, a Salix atrocinerea clone, able to thrive in an As-contaminated brownfield, was grown hydroponically in controlled conditions under an As concentration similar to the bioavailable fraction of the contaminated area (18 mg kg−1) for 30 days. At different time points, i.e. short-term and long-term exposure, biometric data, As accumulation, phytochelatin synthesis, non-protein thiol production and expression of target genes related to these processes were studied. Results showed that S. atrocinerea presents a great tolerance to As and accumulates up to 2400 mg As kg−1 dry weight in roots and 25 mg As kg−1 dry weight in leaves. Roots reduce As V to As III rapidly, with As III being the predominant form of As accumulated in root tissues, whereas in the leaves it is As V. After 1 d of As exposure, roots and leaves show de novo synthesis and an increase in non-protein thiols as compared to the control. Integrating these data on As accumulation in the plant and its speciation, non-protein thiol production and the kinetic gene expression of related target genes, a fundamental role is highlighted for these processes in As accumulation and tolerance in S. atrocinerea. As such, this study offers new insights in the plant tolerance mechanisms to As, which provides important knowledge for future application of high-biomass willow plants in phytoremediation of As-polluted soils.

Salicylic acid alleviates glyphosate-induced oxidative stress in Hordeum vulgare L

Glyphosate (GLY) is considered the most used herbicide in the world and has been associated with several environmental contamination risks. Despite being partially degraded by soil microorganisms, its residues can negatively affect the growth of valuable non-target plants. Thus, there is a need to find new strategies that minimize its impacts and enhance crop tolerance to GLY, allowing a more advantageous and safer, use of this herbicide. Salicylic acid (SA) is a hormone-like substance, able to enhance the efficiency of the antioxidant (AOX) system in plants and their tolerance to oxidative stress. This study aimed to unveil the effects of SA (100 μM) on the oxidative status of Hordeum vulgare L. in response to GLY (30 mg kg−1). After 14 days of growth, the presence of GLY led to a significant inhibition of growth, an accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2-), an increase in lipid peroxidation (LP), proline and non-protein thiols, a decrease of the content of reduced ascorbate (AsA) and an upregulation of AOX enzymes. The exogenous application of SA mitigated the effects of GLY on growth, amount of H2O2 and degree of PL. It has also contributed to the reduction of AsA content, production of non-protein thiols and increased AOX enzymatic activity, particularly superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and gluthatione S-transferase (GST). These results show a positive role of SA against GLY induced oxidative stress, by modulating the AOX capacity of barley plants. However, the observed phytotoxicity of GLY was so pronounced, that the ameliorating effect of SA on AOX defenses was not enough to significantly overcome the herbicide-induced oxidative damage.

Sulfur Protects Pakchoi (Brassica chinensis L.) Seedlings against Cadmium Stress by Regulating Ascorbate-Glutathione Metabolism.

Cadmium (Cd) pollution in food chains pose a potential health risk for humans. Sulfur (S) is a significant macronutrient that plays a significant role in the regulation of plant responses to diverse biotic and abiotic stresses. However, no information is currently available about the impact of S application on ascorbate-glutathione metabolism (ASA-GSH cycle) of Pakchoi plants under Cd stress. The two previously identified genotypes, namely, Aikangqing (a Cd-tolerant cultivar) and Qibaoqing (a Cd-sensitive cultivar), were utilized to investigate the role of S to mitigate Cd toxicity in Pakchoi plants under different Cd regimes. Results showed that Cd stress inhibited plant growth and induced oxidative stress. Exogenous application of S significantly increased the tolerance of Pakchoi seedlings suffering from Cd stress. This effect was demonstrated by increased growth parameters; stimulated activities of the antioxidant enzymes and upregulated genes involved in the ASA-GSH cycle and S assimilation; and by the enhanced ASA, GSH, phytochelatins, and nonprotein thiol production. This study shows that applying S nutrition can mitigate Cd toxicity in Pakchoi plants which has the potential in assisting the development of breeding strategies aimed at limiting Cd phytoaccumulation and decreasing Cd hazards in the food chain.

Phytotoxicity and detoxification mechanism differ among inorganic and methylated arsenic species in Arabidopsis thaliana

Plants are able to take up inorganic arsenic (As) and methylated As, but whether the mode of phytotoxicity and the detoxification mechanism differ between different As species remains unclear. This study aimed to investigate the differences in phytotoxicity and detoxification mechanism between arsenate [As(V)], monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)]. Arabidopsis thaliana was grown in agar-solidified medium, hydroponic or soil pot experiments. Root and shoot growth, seed production, As accumulation and oxidative stress indicators of wild-type plants exposed to As(V), MMA(V) and DMA(V) were compared. The role of thiols in As detoxification was investigated using a specific inhibitor of glutathione (GSH) biosynthesis and the mutants defective in GSH synthesis (cad2-1), phytochelatin (PC) synthesis (cad1-3) or tonoplast transporters for As(III)-PCs (abcc1-2). Methylated As species, especially DMA(V), were more toxic than As(V) for growth and seed production in A. thaliana. Methylated As species were more efficiently translocated from roots to shoots and from shoots to seeds than As(V). DMA(V) exposure resulted in a greater oxidative stress than other As species. As(V) and MMA(V) induced the production of non-protein thiols (NPT), but DMA(V) did not. The GSH inhibitor BSO greatly enhanced the sensitivity to As(V) and MMA(V), but decreased the sensitivity to DMA(V). The mutants cad1-3, cad2-1 and abcc1-2 were similarly hypersensitive to As(V) and MMA(V), but not to DMA(V). As(V) and MMA(V) enhanced the expression of the sulphur assimilation genes encoding ATP Sulphurylase (ATPS) and adenosine-5′-phosphosulphate reductase (APR1) more than DMA(V). DMA(V) is more toxic to A. thaliana than As(V) or MMA(V). The detoxification mechanism for MMA(V) is similar to that for As(V), involving thiol production, complexation with PCs and vacuolar sequestration. This mechanism is ineffective for the detoxification of DMA(V).

Biosynthesis of silver nanoparticles by marine bacterium, Idiomarina sp. PR58-8

Metal-tolerant microorganisms have been exploited in recent years to synthesize nanoparticles due to their potential to offer better size control through peptide binding and compartmentalization. In this paper, we report the intracellular synthesis of silver nanoparticles (SNPs) by the highly silver-tolerant marine bacterium, Idiomarina sp. PR58-8 on exposure to 5 mM silver nitrate. SNPs were characterized by UV-visible spectrophotometry, X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). UV-visible absorption scan of a 48 h culture exposed to 5 mM silver nitrate revealed a broad peak at 450 nm indicative of the surface plasmon resonance of SNPs. XRD analysis confirmed the presence of elemental silver and the crystallite size was calculated to be 25 nm using Scherrer formula. The average particle size as per TEM analysis was found to be 26 nm. Metal stress is known to induce the production of non-protein thiols (NP–SHs) which sequester metal ions. In this study, the production of NP–SHs was followed from 6–48 h, wherein it was observed that the NP–SH levels in the silver-exposed culture were consistently higher (261% on an average) than in the unexposed culture.

Influence of salicylic acid pre-treatment on cadmium tolerance and its relationship with non-protein thiol production in flax root

Dose-dependent changes in cadmium (Cd) tolerance, non-protein thiol (NP-SH) production and their relationship were investigated in sixteen-day-old flax ( Linum usitatissimum L.) seedlings derived from seeds pre-soaked with various salicylic acid (SA) doses and grown hydroponically under increased Cd concentrations (0, 50 and 100 μM CdCl 2 ). The results show that single Cd subjection decreased root elongation as expressed by tolerance index (TI). Moreover, an overproduction of NP-SH was detected in both roots and shoots. These Cd toxicity effects were directly related to the high levels of Cd amounts in flax tissues as expressed by root and shoot Cd bioaccumulation factors (BAF). In addition, Cd-tolerance of roots TI was negatively correlated with changes in root BAF but positively correlated with shoot BAF. However, positive correlation was illustrated between root TI and NP-SH contents. SA considerably reversed the Cd-induced decrease in root growth parameters and TI. Moreover, in Cd-treated plants, SA pre-soaking prevented Cd accumulation in the shoot as consequence of significant decreases in BAF of roots, Cd transport estimated by the translocation factor (TF) and shoot BAF, respectively. Interestingly, SA pre-treatment reduced BAF of roots and shoots, enhanced NP-SH production in roots and decreased it in leaves. These results suggest that SA might play a preventive role in Cd uptake, sequestration and translocation processes based primarily in roots where SA-enhanced NP-SH contribute to the improvement of flax tolerance to Cd stress. Key words: Cadmium, salicylic acid, bioaccumulation, growth, non-protein thiols, Linum usitatissimum .

Cadmium Accumulation in Relation to Organic Acids and Nonprotein Thiols in Leaves of the Recently Found Cd Hyperaccumulator Rorippa globosa and the Cd-accumulating Plant Rorippa islandica

It has been proposed that organic acid and nonprotein thiol are involved in the hyperaccumulation of metals. In this study, Cd accumulation, organic acid, and nonprotein thiol production and their relationships in the leaves of Cd-hyperaccumulator Rorippa globosa were examined and compared with a closely related species, Rorippa islandica. The results showed that there was no reduction in biomass of R. globosa when treated with 25 μg Cd g−1 (T2), despite Cd accumulation in the leaves was up to 158.2 μg g−1 DW. On the other hand, the growth of Cd-treated R. islandica was obviously inhibited as it accumulated more than 100 μg g−1 DW in the leaves. Therefore, R. islandica behaved as a Cd-accumulating plant. The Cd treatments could significantly induce the synthesis of acetic acid in both species, suggesting that acetic acid, as the most abundant organic acid, might be related to the Cd accumulation. Significant positive correlations between Cd concentrations and both tartaric and malic acid concentrations in the leaves of R. globosa were observed. There was a significant positive correlation between Cd concentrations and acetic acid concentrations in the leaves of R. islandica. This trend of tartaric and malic acids in the leaves of R. globosa and acetic acid in the leaves of R. islandica might be related to Cd accumulation. In addition, a quadratic relationship was obviously observed for NP-SH contents and total Cd concentrations in the leaves of R. globosa, indicating that NP-SH was significantly related to Cd accumulation and tolerance.

Response of two terrestrial green microalgae (Chlorophyta, Trebouxiophyceae) isolated from Cu-rich and unpolluted soils to copper stress

Some algae inhabit Cu-polluted soils. Intracellular Cu-accumulation and production of non-protein thiols in response to copper stress were compared in Stichococcus minor and Geminella terricola isolated from Cu-polluted and unpolluted soils, respectively. Cu-exposed (0.5 μM) S. minor accumulated lower amounts of copper (0.38 mM) than G. terricola (4.20 mM) and maintained 8.5-fold higher level of glutathione (GSH) than G. terricola. The ratio GSH/0.5 GSSG in the Cu-treated S. minor (7.21) was 7-times higher than in G. terricola. Reduced and oxidized forms of phytochelatins were found in both algae. Under copper stress (5 μM) the ratio –SH total/Cu intracellular in S. minor ranged from 2.3 to 6.2, while it was lower than 1.0 in G. terricola. Low intracellular Cu-accumulation and maintenance of high GSH level concomitant with PCs production seem to be responsible for a higher Cu-resistance of S. minor than G. terricola.

Overexpression ofArabidopsis phytochelatin synthase (atpcsi) does not change the maximum capacity for non-protein thiol production induced by Cadmium

Phytochelatins (PCs) play an important role in heavy-metal homeostasis and detoxification. However, we previously reported that the overexpression of PC synthase inArabidopsis does not lead to increased tolerance of cadmium but, rather, plants show higher Cd sensitivity. Here, we compared the maximum capacity for non-protein thiol (NPT) production at various concentrations of Cd in order to estimate PC synthesis indirectly for both transgenic (pcs9) and wild-type plants. The pcs9 line produced the highest level of NPT when treated with 200 p.M Cd for 3 d. In comparison, the maximum productivity by the wild type was in response to 500 µM Cd. Nevertheless, the absolute amounts of NPT produced did not differ significantly between those two genotypes. Furthermore, exogenous application of 1 mM GSH did not dramatically change the capacity for either pcs9 or wild-type plants. These results suggest that Cd hypersensitivity in the transgenic pcs9 may not be caused by supraoptimal intracellular concentrations of PC, but may, instead, be due to overexpressed PC synthase itself because that enzyme can bind metals. This action, therefore, may lead to some unknown disruption in cellular metal homeostasis under Cd stress.

Response to copper stress in aposymbiotically grown lichen mycobiont Cladonia cristatella: uptake, viability, ergosterol and production of non-protein thiols

The mycobiont of lichens usually determines the morphology of the symbiotic organism and is also dominates in terms of biomass. However, its role for sensitivity or tolerance of lichens to heavy metals is almost unknown. In the present study, the influence of copper (Cu) on the aposymbiotically-grown mycobiont of Cladonia cristatella was assessed. Intracellular Cu uptake was correlated with increasing Cu concentrations over a 24-h exposure time. Viability, measured as the degree of reduction of triphenyltetrazolium chloride to triphenyl formazan, as well as to ergosterol levels, decreased with growing Cu concentrations tested. Reduced glutathione (GSH) was found to be the most abundant low-molecular-weight thiol in the hyphae of C. cristatella and its intracellular content increased at concentrations of 10 μ m Cu. Higher Cu concentrations caused a significant decrease in GSH, possibly due to heavy metal-induced oxidation of GSH to glutathione disulphide (GSSG). Free cysteine levels were relatively constant. As expected, we did not observe the production of phytochelatins in the mycobiont, contrary to what is found in intact lichens and axenic cultures of their photobionts.

Transient Versus Sustained Phosphorylation and Nuclear Accumulation of ERKs Underlie Anti-Versus Pro-apoptotic Effects of Estrogens

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17beta-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17beta-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17beta-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17beta-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.

Expression of Brassica napus L. gamma-Glutamylcysteine Synthetase and Low-and High-Affinity Sulfate Transporters in Response to Excess Cadmium

: In both the roots and leaves of Brassica napus L. cv. Youyan No. 8 under treatment with 30 μmol/L Cd, massive production of non-protein thiols (NPT; mainly containing glutathione (GSH) and phytochelatins (PCs)) was induced, together with an increase in γ-glutamylcysteine synthetase (γ-ECS) mRNA transcripts. Because γ-ECS is the key enzyme catalyzing the first step in GSH biosynthesis, which, in turn, is converted to PCs, the Cd-induced increase in γ-ECS expression may be responsible for the observed increase in the production of NPT. Using a quantitative reverse transcription polymerase chain reaction (RT-PCR) approach, the expression of genes encoding a putative low-affinity sulfate transporter (LAST) and a putative high-affinity sulfate transporter (HAST) was determined at the transcriptional level. The RT-PCR analysis of relative transcript amounts indicates that the LAST gene in B. napus leaves showed a constitutive expression, which was hardly affected by Cd treatment. However, treatment with 30 μmol/L Cd for 2 or 3 d induced a marked increase in the expression of LAST in roots. Transcriptional expression of the HAST gene occurred in roots, but not in leaves. The expression of HAST only in the roots suggests that it has a specific function in sulfate uptake from soil and that the putative LAST may be responsible for the transport of sulfate from the roots to the shoots, as well as for the uptake of sulfate from soil. These results indicate that changes in transcriptional expression for sulfate transporters were required for the increased demand for sulfate during Cd stress. (Managing editor: Li-Hui ZHAO)