Overexpression of <Emphasis Type="Italic">ThGSTZ1</Emphasis> from <Emphasis Type="Italic">Tamarix hispida</Emphasis> improves tolerance to exogenous ABA and methyl viologen

Key message

Molecular analysis of a zeta subfamily GST gene from T. hispida involved in ABA and methyl viologen tolerance in transgenic Arabidopsis and Tamarix.

Abstract

Glutathione S-transferase (GST) genes are important for the improvement of plant abiotic stress tolerance, and our previous study demonstrated that the ThGSTZ1 gene from Tamarix hispida improves plant salt and drought tolerance. To further understand the role of ThGSTZ1 in the response of plants to abscisic acid (ABA) and oxidative stress, three ThGSTZ1-overexpressing transgenic Arabidopsis thaliana lines were analyzed in the current study. The results showed that the transgenic lines exhibited higher biomass accumulation, higher activities of GST and other protective enzymes, and less reactive oxygen species (ROS) and cell damage than wild-type (WT) plants under ABA and methyl viologen (MV) stress. In addition, the analysis of a transgenic T. hispida line transiently expressing ThGSTZ1 confirmed these results. The activities of GST, glutathione peroxidase, and superoxide dismutase were markedly higher in the ThGSTZ1-overexpressing lines compared with the control lines under both ABA and MV treatments, and the transgenic lines also exhibited a lower degree of electrolyte leakage (EL) and a decreased H₂O₂ content. All these results suggested that ThGSTZ1 can also improve plant ABA and oxidation tolerance by regulating ROS metabolism and that ThGSTZ1 represents an excellent candidate gene for molecular breeding to increase plant stress tolerance.

     

Contents of Heavy Metals in Chinese Edible Herbs: Evidence from a Case Study of Epimedii Folium

Toxic heavy metal contamination in Chinese edible herbs has raised a worldwide concern. In this study, heavy metals in Epimedii Folium, an edible medicinal plant in China, were quantitatively analyzed. Variations of heavy metals in different species, in various organs (i.e., leaves, stems, and roots), in wild-growing and cultivated plants, and in 35 market samples of Epimedii Folium, were systematically investigated. In all of Epimedium samples, Hg (mercury) was not detectable (0.00 μg/g). Four species, Epimedium pubescens, Epimedium sagittatum, Epimedium brevicornu, and Epimedium wushanense, were found to contain Cu (copper) and Pb (lead). And contents of Cu and Pb in E. brevicornu were significantly higher than those in other species (P < 0.01). In wild-growing and cultivated Epimedium plants, Cd (cadmium) and As (arsenic) were not detectable, and concentrations of Cu and Pb in wild-growing plants were significantly higher than those in cultivated plants (P < 0.01). Cd was not detectable in leaves, roots, and stems, while organ specificity was apparent in the distribution of Cu, As, and Pb. And the highest levels of Cu and Pb were observed in roots and leaves, respectively. In Chinese markets, several samples of Epimedii Folium contained excessive Cu, Cd, As, and Pb beyond the national permissible limits. In summary, there was a large variation of heavy metals among Epimedii Folium samples, and Cu and Pb were the most important heavy metals contaminating the edible medicinal plant. Application of Epimedii Folium to drug and food industries will need to focus more on toxic heavy metal contamination.     

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis> exposed to cyanotoxins

Objectives

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications.

Results

Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l^?1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l^?1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected.

Conclusions

M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

     

Age-related changes in antioxidant and glutathione S-transferase enzyme activities in the Asian clam

Aging is accompanied by increased production of free oxygen radicals and impairment of normal cellular functions. The aim of this work was to provide preliminary data on age-related differences in the activities of antioxidant enzymes and phase II biotransformation enzyme glutathione S-transferase (GST) in a wild population of the Asian clam Corbicula fluminea. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), and GST were assessed in visceral mass of four age classes (0+-, 1+-, 2+-, and 3+-year-old) of C. fluminea clams. Age-related changes were seen in antioxidant enzyme status: levels of total SOD (totSOD) (P < 0.05), MnSOD, and CuZnSOD (P < 0.05) activities increased progressively during aging from younger to older clams. Changes in CAT and GR activities with advancing age were found, the levels being the highest in age class II, then being lower in age classes III and IV (P < 0.05). Activities of GPX and GST were lower in the senescent individuals (2+- and 3+-year-old clams) compared with young individuals (0+- and 1+-year-old clams). Overall, the decline of glutathione-dependent enzyme activities, coupled with higher and lower activities of totSOD and CAT, respectively, as the individual grows older, may render the older animals more susceptible to oxidative stress. Data reported here are not intended to be exhaustive since they concern only age/size structure of the population at one locality, so more detailed studies on both the developmental stages and levels of antioxidant enzymes of this new alien species in Serbian rivers are required.     

Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Gene Expression Analysis in Rice Plants Exposed to Metal Stresses

Environmental pollution by toxic heavy metals may lead to the possible contamination of the rice plant (Oryza sativa L.). Although gene expression analysis through real-time quantitative PCR (RT-qPCR) has increased our knowledge about biological responses to heavy metals, gene network that mediates rice plant responses to heavy metal stress remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving RT-qPCR. In this study, we analyzed the expression stability of eight commonly used housekeeping genes (GAPDH, Actin, eIF-4α, UBQ 5, UBQ 10, UBC, EF-1α and β-TUB) in rice leaves exposed to four kinds of heavy metals (Zn, Cu, Cd and Pb). The expression stability of these genes was determined using geNorm, NormFinder, BestKeeper and RefFinder algorithms. The results showed that UBQ 10 and UBC were the most stable reference genes across all the tested samples. We measured the expression profiles of the heavy metal-inducible gene O. sativa METALLOTHIONEIN2b (OsMT2b) using the two most stable and one least stable reference genes in all samples. The relative expression of OsMT2b varied greatly according to the different reference genes. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in rice plants.     

Activities of proteinases in invertebrate animals—Potential objects of fish nutrition. Effects of temperature,pH, and heavy metals

Differences in the degree of separate and combined effects of temperature, pH, and heavy metals (zinc, copper) on the trypsin-and chymotrypsin-like proteinase activities have been established in the whole body of some invertebrate animals—potential objects of fish nutrition: pond snail Lymnaeae stagnalis, orb snail Planorbarius purpura, zebra mussel Dreissena polymorpha, oligochaetes Tubifex sp. and Lumbriculus sp. in total, chironomid larvae Chironimus sp. and Ch. riparus, as well as crustacean zooplankton. It has been shown that enzymes of the potential prey at low temperature can compensate the low activity of intestinal proteinases of fish bentho- and planktophages.     

Toxic metals accumulation in <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> and <Emphasis Type="Italic">T. harzianum</Emphasis>

Heavy metal contamination represents an important environmental issue due to the toxic effects of metals on different organisms. Filamentous fungi play an important impact in the bioremediation of heavy metal-contaminated wastewater and soil. The purpose of this investigation was to observe fungal uptake behavior toward heavy metal. For this aim Trichoderma asperellum TS141 and T. harzianum TS103 at growth period were screened for their tolerance and uptake capability of cadmium (Cd), lead (Pb) and nickel (Ni) at different concentrations (0, 25, 50, 100, and 200 mg/L) in PDB media (potato dextrose broth as a complex medium). Results showed that both fungi were able to survive at the maximum concentration of 200 mg/L of the heavy metals, and remove them. T. asperellum had a better uptake capacity for Cd compared to Pb and Ni in the highest metal concentration in media. Maximum removal efficiency of Pb (68.4%) at 100 mg/L and Ni (78%) at 200 mg/L was performed by T. asperellum. For Cd, the highest removal efficiency (82.1%) was recorded by T. harzianum at 200 mg/L Cd in aqueous solution. The uptake of Cd was highly dependent on pH of solution than Pb and Ni so that the optimal pH of Cd uptake was 9 for T. asperellum and 4 for T. harzianum. Also, optimal temperature was 35°C for Cd and Pb uptake in both fungi, whereas for Ni uptake was 30 and 35°C in T. harzianum and T. asperellum, respectively. We propose that T. asperellum TS141 and T. harzianum TS103 can be used as a bioremediation agent for metal remediation from wastewater and heavy metal-contaminated soils.     

SlAGO4A,a core factor of RNA-directed DNA methylation (RdDM) pathway,plays an important role under salt and drought stress in tomato

In Arabidopsis, it has been clarified that AGO4 protein is implicated in a phenomenon termed RNA-directed DNA methylation (RdDM). Previously, four orthologs of AtAGO4 were cloned in tomato, designated as SlAGO4A–SlAGO4D. Here, we studied the role of the SlAGO4A gene in regulating salt and drought tolerance in tomato. SlAGO4A-down-regulating (AS) transgenic tomato plants showed enhanced tolerance to salt and drought stress compared to wild-type (WT) and SlAGO4A-overexpressing (OE) transgenic plants, as assessed by physiological parameters such as seed germination rate, primary root length, chlorophyll/proline/MDA/soluble sugar/RWC content, and survival rate. Moreover, several genes involved in ROS scavenging and plant defense, including CAT, SOD, GST, POD, APX, LOX, and PR1, were up- or down-regulated consistently under salt and drought stress. Notably, expression levels of some DNA methyltransferase genes and RNAi pathway genes were significantly lower in AS plants than in WT. Taken together, our results suggest that SlAGO4A gene plays a negative role under salt and drought stress in tomato probably through the modulation of DNA methylation as well as the classical RNAi pathway. Hence, it may serve as a useful biotechnological tool for the genetic improvement of stress tolerance in crops.     

In planta characterization of a tau class glutathione S-transferase gene from <Emphasis Type="Italic">Juglans regia</Emphasis> (<Emphasis Type="Italic">JrGSTTau1</Emphasis>) involved in chilling tolerance

Key message

JrGSTTau1 is an important candidate gene for plant chilling tolerance regulation.

Abstract

A tau subfamily glutathione S-transferase (GST) gene from Juglans regia (JrGSTTau1, GeneBank No.: KT351091) was cloned and functionally characterized. JrGSTTau1 was induced by 16, 12, 10, 8, and 6 °C stresses. The transiently transformed J. regia showed much greater GST, glutathione peroxidase (GPX), superoxide dismutase (SOD), and peroxidase (POD) activities and lower H₂O₂, malondialdehyde (MDA), reactive oxygen species (ROS), and electrolyte leakage (EL) rate than prokII (empty vector control) and RNAi::JrGSTTau1 under cold stress, indicating that JrGSTTau1 may be involved in chilling tolerance. To further confirm the role of JrGSTTau1, JrGSTTau1 was heterologously expressed in tobacco, transgenic Line5, Line9, and Line12 were chosen for analysis. The germinations of WT, Line5, Line9, and Line12 were similar, but the fresh weight, primary root length, and total chlorophyll content (tcc) of the transgenic lines were significantly higher than those of WT under cold stress. When cultivated in soil, the GST and SOD activities of transgenic tobacco were significantly higher than those of WT; however, the MDA and H₂O₂ contents of WT were on average 1.47- and 1.96-fold higher than those of Line5, Line9, and Line12 under 16 °C. The DAB, Evans blue, and PI staining further confirmed these results. Furthermore, the abundances of NtGST, MnSOD, NtMAPK9, and CDPK15 were elevated in 35S::JrGSTTau1 tobacco compared with WT. These results suggested that JrGSTTau1 improves the plant chilling tolerance involved in protecting enzymes, ROS scavenging, and stress-related genes, indicating that JrGSTTau1 is a candidate gene for the potential application in molecular breeding to enhance plant abiotic stress tolerance.

     

Metabolomics and Trace Element Analysis of Camel Tear by GC-MS and ICP-MS

Acne vulgaris is a very common skin disorder affecting human beings. There is a paucity of report on the role of heavy metals—lead (Pb) and cadmium (Cd)—globally, and trace metals—zinc (Zn) and copper (Cd)—particularly in Nigeria in the development/severity of acne vulgaris. This study is aimed to determine the blood levels of some heavy metals—cadmium and lead—and trace metals—zinc and copper—in acne vulgaris sufferers in a Nigerian population. Venous blood samples were collected from a total number of 90 non-obese female subjects consisting of 30 mild, 30 moderate and 30 severe acne vulgaris sufferers for blood Cd, Pb, Cu and Zn determination. They were age-matched with 60 females without acne vulgaris who served as the control subjects. Acne sufferers had significantly higher blood Cd and Pb (P = 0.0143 and P = 0.0001 respectively) and non-significantly different blood levels of Cu and Zn (P = 0.910 and P = 0.2140 respectively) compared to controls. There were significant progressive increases in blood levels of Cd and Pb (P = 0.0330 and P = 0.0001 respectively) and non-significant differences in the mean blood level of Cu and Zn (P = 0.1821 and P = 0.2728 respectively) from mild to moderate and severe acne vulgaris sufferers. Increases in blood Cd and Pb may play critical roles in the pathogenesis/severity of acne vulgaris, while Cu and Zn seem to play less significant roles in the development of this disorder in this environment.     

On background levels of heavy metals in agarophytes of the Black Sea

Ecological standards for commercial seaweeds are very important due to anthropogenic pollution of the marine sublittoral. The content of heavy metals in agarophyte red algae, seawater, and sediments are investigated by the method of atomic absorption spectroscopy. The level of Cd, Pb, and Ni in Gracilaria verrucosa was low, while the level of Zn, Pb, and Nu was comparatively higher in Gelidium latifolium and Phyllophora nervosa, especially in the Caucasus nearshore zone. There is no relationship between the concentration of metals in the environment and the concentration of metals in agarophytes. Background levels of dangerous metals are discussed in connection with commercial exploitation of red algae.     

Constitutive expression of <Emphasis Type="Italic">SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

Effects of temperature,pH, and heavy metals (copper,zinc) upon proteinase activities in digestive tract mucosa of typical and facultative piscivorous fish

Species-specific differences and effects of natural (temperature, pH) and anthropogenic (zinc, copper) factors upon activities of casein-and hemoglobinlytic proteinases have been studied in the gut mucosa in pike (Esox lucius), zander (Stizostedion lucioperca), burbot (Lota lota) and perch (Perca fluviatilis). It is revealed that the intestine mucosa proteinase activities differ considerably from those ones in the fish stomach mucosa and that studied factors affect these activities to different extents. The stomach mucosa proteinases are most tolerant to the effects of studied factors, which is especially true for pike. The least tolerant are the intestine caseinlytic proteinases (except for the enzymes in perch). The effects of temperature, pH, and heavy metals depend on fish species, factors’ combinations (mono-factor, bi-factor, or multi-factor action) and on factor variability.     

Alterations in photosynthetic pigments,protein, and carbohydrate metabolism in a wild plant <Emphasis Type="Italic">Coronopus didymus</Emphasis> L. (Brassicaceae) under lead stress

Coronopus didymus has been emerged as a promising wild, unpalatable plant species to alleviate lead (Pb) from the contaminated soils. This work investigated the hypothesis regarding various metabolic adaptations of C. didymus under lead (Pb) stress. In pot experiments, we assessed the effect of Pb at varied concentrations (500–2900 mg kg^?1) on growth, photosynthetic pigments, alteration of macromolecular (protein and carbohydrate) content, and activities of enzymes like protease, α-and β-amylase, peroxidase (POX), and polyphenol oxidase (PPO) in C. didymus for 6 weeks. Results revealed that Pb exposure enhanced the growth, protein, and carbohydrate level, but decreased the leaf pigment concentration and activities of hydrolytic enzymes. The activities of POX and PPO in roots increased progressively by ~337 and 675%, respectively, over the control, at 2900 mg kg^?1 Pb treatment. Likewise, contemporaneous findings were noticed in shoots of C. didymus, strongly indicating its inherent potential to cope Pb-induced stress. Furthermore, the altered plant biochemical status and upregulated metabolic activities of POX and PPO indulged in polyphenol peroxidation elucidate their role in allocating protection and conferring resistance against Pb instigated stress. The current work suggests that stress induced by Pb in C. didymus stimulated the POX and PPO activities which impart a decisive role in detoxification of peaked Pb levels, perhaps, by forming physical barrier or lignifications.     

Effects of exogenous methyl jasmonate-induced resistance in <Emphasis Type="Italic">Populus</Emphasis> × <Emphasis Type="Italic">euramericana</Emphasis> ‘Nanlin895’ on the performance and metabolic enzyme activities of <Emphasis Type="Italic">Clostera anachoreta</Emphasis>

Methyl jasmonate (MeJA) is a plant chemical elicitor that has been used to artificially induce chemical defense responses and trigger induced resistance against a broad range of arthropod herbivores. This study assessed the effects of exogenous MeJA on the growth performance, chemical detoxification, and antioxidant enzyme activities of Clostera anachoreta. After feeding C. anachoreta with 10^?5 mol/L MeJA solution-treated Populus × euramericana ‘Nanlin895’ leaves, we measured the larval and pupal development time, pupal weight, eclosion rate, fecundity, and nutritional physiology of the adults. We also measured superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, which are reactive oxygen species (ROS) scavengers, and glutathione S-transferase (GST) and carboxylesterase (CarE) activities, which are probably involved in the metabolism of induced plant allelochemicals. Methyl jasmonate (MeJA) treatment reduced larval performance in terms of prolonged developmental time of larvae and pupae and decreased growth rates, but had little effect on larval nutrition physiology. The activities of the SOD and POD antioxidant enzymes increased, but CAT activity declined at 36 and 48 h after C. anachoreta had fed on MeJA-treated leaves. The GST and CarE detoxification enzymes both were induced after the larvae had fed on MeJA-treated leaves. These results suggest that exogenous application of MeJA elicited induced resistance in Populus × euramericana ‘Nanlin895’ against C. anachoreta.     

A tomato plastidic ATP/ADP transporter gene <Emphasis Type="Italic">SlAATP</Emphasis> increases starch content in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

A plastidic ATP/ADP transporter (AATP) is responsible for importing ATP from the cytosol into plastids. Increasing the ATP supply is a potential way to facilitate anabolic synthesis in heterotrophic plastids of plants. In this work, a gene encoding the AATP protein, named SlAATP, was successfully isolated from tomato. Expression of SlAATP was induced by exogenous sucrose treatment in tomato. The coding region of SlAATP was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Constitutive expression of SlAATP significantly increased the starch accumulation in the transgenic plants. Real-time quantitative PCR (qRT-PCR) analysis showed that constitutive expression of StAATP up-regulated the expression of phosphoglucomutase (AtPGM), ADP-glucose pyrophosphorylase (AtAGPase), granule-bound starch synthase (AtGBSS I and AtGBSS II), soluble starch synthases (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) and starch branching enzyme (AtSBE I and AtSBE II) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses indicated that the major enzymes (AGPase, GBSS, SSS and SBE) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to the wild-type (WT). These findings suggest that SlAATP may improve starch content of Arabidopsis by up-regulating the expression of the related genes and increasing the activities of the major enzymes invovled in starch biosynthesis. The manipulation of SlAATP expression might be used for increasing starch accumulation of plants in the future.