Ascorbate peroxidase-mediated in situ labelling of proteins in secreted exosomes.

The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Here we report an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome-generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule-derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63-fused APEX variant.

Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane.

In the study of intracellular bacteria that reside within a membrane-bound vacuole, there are many questions related to how prokaryotic or eukaryotic transmembrane or membrane-associated proteins are organized and function within the membranes of these pathogen-containing vacuoles. Yet this host-pathogen interaction interface has proven difficult to experimentally resolve. For example, one method to begin to understand protein function is to determine the protein-binding partners; however, examining protein-protein interactions of hydrophobic transmembrane proteins is not widely successful using standard immunoprecipitation or coimmunoprecipitation techniques. In these scenarios, the lysis conditions that maintain protein-protein interactions are not compatible with solubilizing hydrophobic membrane proteins. In this chapter, we outline two proximity labeling systems to circumvent these issues to study (1) eukaryotic proteins that localize to the membrane-bound inclusion formed by Chlamydia trachomatis using BioID, and (2) chlamydial proteins that are inserted into the inclusion membrane using APEX2. BioID is a promiscuous biotin ligase to tag proximal proteins with biotin. APEX2 is an ascorbate peroxidase that creates biotin-phenoxyl radicals to label proximal proteins with biotin or 3,3'-diaminobenzidine intermediates for examination of APEX2 labeling of subcellular structures using transmission electron microscopy. We present how these methods were originally conceptualized and developed, so that the user can understand the strengths and limitations of each proximity labeling system. We discuss important considerations regarding experimental design, which include careful consideration of background conditions and statistical analysis of mass spectrometry results. When applied in the appropriate context with adequate controls, these methods can be powerful tools toward understanding membrane interfaces between intracellular pathogens and their hosts.

Pre-harvest methyl jasmonate treatments increase antioxidant systems in lemon fruit without affecting yield or other fruit quality parameters.

BACKGROUND: Jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA) are hormones involved in the regulation of many processes in plants and act (when applied as a post- or pre-harvest treatment) to increase fruit bioactive compounds with antioxidant potential. However, there is no literature available regarding the effect of pre-harvest MeJA treatment on lemon fruit antioxidant systems, which was the aim of the present study. RESULTS: MeJA treatment (0.1, 0.5 and 1.0 mmol L-1 ) increased antioxidant compounds, such as phenolics, in the juice and flavedo of 'Fino' and 'Verna' lemons at harvest, with the most effective concentration being 0.1 mmol L-1 in both cultivars. In addition, catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) activities were also increased by MeJA treatment, with the highest increases being also found with 0.1 mmol L-1 . The increases in APX and CAT were maintained from one treatment to another during fruit development on the tree, whereas the increase on POD disappeared after 8-10 days of each treatment. For both antioxidant systems, the highest increases were found in lemon harvested at the commercial ripening stage. By contrast, crop yield, fruit ripening process and quality parameters were generally not affected by MeJA treatment. CONCLUSION: Preharvest MeJA treatment could be a useful tool for increasing antioxidant potential and the health beneficial effects of lemon fruit consumption, given the relationship between these properties and phenolic content. Moreover, the increased concentration of phenolics and the activity of antioxidant enzymes in the flavedo of MeJA treated fruit could increase lemon tolerance to chilling injury and decay during postharvest storage. © 2019 Society of Chemical Industry.

Effect of foliar application with sodium selenate on selenium biofortification and fruit quality maintenance of 'Starking Delicious' apple during storage.

BACKGROUND: Selenium (Se) is an essential micronutrient due to its anticarsinogenic properties and positive influence on human immune system. Fortification of some fruits based on their rates of consumption and availability all year round appears to be an effective way to supplement Se in the human diet. In this study the possibility of augmenting Se content in 'Starking Delicious' apple fruit during two growing seasons was investigated. In 2016, the impact of 0, 0.5, 1 and 1.5 mg Se L-1 by foliar application on Se accumulation and fruit ripening as well as quality attributes was investigated. In 2017, the effects of 1.5 mg Se L-1 foliar application on fruit Se content and changes in the antioxidant system and storability were studied with a 30-day interval during 6 months storage at 0 ± 1 °C. RESULTS: Foliar application of Se significantly increased both leaf and fruit Se concentration. The increase in Se content enhanced the flesh firmness, titrable acidity, and soluble solid content of the fruit. The activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were markedly amplified by Se treatments as compared to the control, resulting in lower superoxide anion radical (O2 -• ) and hydrogen peroxide (H2 O2 ) contents, correspondingly higher membrane integrity as revealed by lower ion leakage and malondialdehyde accumulation and the fruit with lower water core. CONCLUSION: Application of Se was efficient in increasing fruit Se content and nutraceutical properties, retarding the flesh firmness reduction, and postponing fruit ripening resulting from lower ethylene biosynthesis rate, thereby positively affecting apple fruit quality and storability. © 2019 Society of Chemical Industry.

Impact of lead tolerant plant growth promoting rhizobacteria on growth, physiology, antioxidant activities, yield and lead content in sunflower in lead contaminated soil.

Present study was conducted to evaluate the effect of lead tolerant plant growth promoting rhizobacteria (LTPGPR) on growth, physiology, yield, antioxidant activities and lead uptake in sunflower in soil contaminated with lead under pot conditions. Three pre-characterized LTPGP strains (S2 (Pseudomonas gessardii strain BLP141), S5 (Pseudomonas fluorescens A506) and S10 (Pseudomonas fluorescens strain LMG 2189)) were used to inoculate sunflower growing in soil contaminated with different levels (300, 600 and 900 mg kg-1) of lead by using lead nitrate salt as source of lead. Treatments were arranged according to completely randomized design with factorial arrangements. At harvesting, data regarding growth attributes (root shoot length, root shoot fresh and dry weights), yield per plant, physiological attributes (Chlorophyll 'a', 'b' and carotenoids content), antioxidant activities (Ascorbate peroxidase, catalase, superoxide dismutase and glutathione reductase), proline and malanodialdehyde content, and lead content in root, shoot and achenes of sunflower were recorded. Data were analysed by standard statistical procedures. Results showed that lead contamination reduced the plants growth, physiology and yield at all levels of lead stress. But application of LTPGPR in soil contaminated with lead improved plant growth, physiology, yield, and antioxidant activities, proline, and reduced the malanodialdehyde content (that is reduced by the application of different strains in lead contamination) of sunflower as compared to plants grown in soil without inoculation. Inoculation also promoted the uptake of lead in root, shoots and reduced the uptake of lead in achenes of plants as compared to plants in lead contamination without inoculation.

Impairment of ntcA gene revealed its role in regulating iron homeostasis, ROS production and cellular phenotype under iron deficiency in cyanobacterium Anabaena sp. PCC 7120.

Iron deficiency ends up into several unavoidable consequences including damaging oxidative stress in cyanobacteria. NtcA is a global nitrogen regulator controls wide range of metabolisms in addition to regulation of nitrogen metabolism. In present communication, NtcA based regulation of iron homeostasis, ROS production and cellular phenotype under iron deficiency in Anabaena 7120 has been investigated. NtcA regulates the concentration dependent iron uptake by controlling the expression of furA gene. NtcA also regulated pigment synthesis and phenotypic alterations in Anabaena 7120. A significant increase in ROS production and corresponding reduction in the activities of antioxidative enzymes (SOD, CAT, APX and GR) in CSE2 mutant strain in contrast to wild type Anabaena 7120 also suggested the possible involvement of NtcA in protection against oxidative stress in iron deficiency. NtcA has no impact on the expression of furB and furC in spite of presence of consensus NtcA binding site (NBS) and -10 boxes in their promoter. NtcA also regulates the thylakoid arrangement as well as related photosynthetic and respiration rates under iron deficiency in Anabaena 7120. Overall results suggested that NtcA regulates iron acquisition and in turn protect Anabaena cells from the damaging effects of oxidative stress induced under iron deficiency.

Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq).

UNLABELLED: Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels. BIOLOGICAL SIGNIFICANCE: In this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a key enzyme in glycolysis. Both l-ascorbate peroxidase and superoxide dismutase play a role in anti-oxidative scavenging defense systems. These proteins have potential for use as biomarkers to screen for high and low embryogenic oil palm samples.

The role of ascorbate peroxidase, guaiacol peroxidase, and polysaccharides in cassava (Manihot esculenta Crantz) roots under postharvest physiological deterioration.

This study aimed to investigate the role of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), polysaccharides, and protein contents associated with the early events of postharvest physiological deterioration (PPD) in cassava roots. Increases in APX and GPX activity, as well as total protein contents occurred from 3 to 5 days of storage and were correlated with the delay of PPD. Cassava samples stained with Periodic Acid-Schiff (PAS) highlighted the presence of starch and cellulose. Degradation of starch granules during PPD was also detected. Slight metachromatic reaction with toluidine blue is indicative of increasing of acidic polysaccharides and may play an important role in PPD delay. Principal component analysis (PCA) classified samples according to their levels of enzymatic activity based on the decision tree model which showed GPX and total protein amounts to be correlated with PPD. The Oriental (ORI) cultivar was more susceptible to PPD.

Postharvest Exogenous Application of Abscisic Acid Reduces Internal Browning in Pineapple.

Internal browning (IB) is a postharvest physiological disorder causing economic losses in pineapple, but there is no effective control measure. In this study, postharvest application of 380 µM abscisic acid (ABA) reduced IB incidence by 23.4-86.3% and maintained quality in pineapple fruit. ABA reduced phenolic contents and polyphenol oxidase and phenylalanine ammonia lyase activities; increased catalase and peroxidase activities; and decreased O2(·-), H2O2, and malondialdehyde levels. This suggests ABA could control IB through inhibiting phenolics biosynthesis and oxidation and enhancing antioxidant capability. Furthermore, the efficacy of IB control by ABA was not obviously affected by tungstate, ABA biosynthesis inhibitor, nor by diphenylene iodonium, NADPH oxidase inhibitor, nor by lanthanum chloride, calcium channel blocker, suggesting that ABA is sufficient for controlling IB. This process might not involve H2O2 generation, but could involve the Ca(2+) channels activation. These results provide potential for developing effective measures for controlling IB in pineapple.

Cadmium toxicity affects chlorophyll a and b content, antioxidant enzyme activities and mineral nutrient accumulation in strawberry.

BACKGROUND: Cadmium (Cd) is well known as one of the most toxic metals affecting the environment and can severely restrict plant growth and development. In this study, Cd toxicities were studied in strawberry cv. Camarosa using pot experiment. Chlorophyll and malondialdehyde (MDA) contents, catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) activities and mineral nutrient concentrations were investigated in both roots and leaves of strawberry plant after exposure Cd. RESULTS: Cd content in both roots and leaves was increased with the application of increasing concentrations of Cd. We found higher Cd concentration in roots rather than in leaves. Chlorophyll a and b was decreased in leaves but MDA significantly increased under increased Cd concentration treatments in both roots and leaves. SOD and CAT activities was also increased with the increase Cd concentrations. K, Mn and Mg concentrations were found higher in leaves than roots under Cd stress. In general, increased Cd treatments increased K, Mg, Fe, Ca, Cu and Zn concentration in both roots and leaves. Excessive Cd treatments reduced chlorophyll contents, increased antioxidant enzyme activities and changes in plant nutrition concentrations in both roots and leaves. CONCLUSION: The results presented in this work suggested that Cd treatments have negative effect on chlorophyll content and nearly decreased 30% of plant growth in strawberry. Strawberry roots accumulated higher Cd than leaves. We found that MDA and antioxidant enzyme (CAT, SOD and APX) contents may have considered a good indicator in determining Cd tolerance in strawberry plant.

Cadmium toxicity affects chlorophyll a and b content, antioxidant enzyme activities and mineral nutrient accumulation in strawberry

BACKGROUND: Cadmium (Cd) is well known as one of the most toxic metals affecting the environment and can severely restrict plant growth and development. In this study, Cd toxicities were studied in strawberry cv. Camarosa using pot experiment. Chlorophyll and malondialdehyde (MDA) contents, catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) activities and mineral nutrient concentrations were investigated in both roots and leaves of strawberry plant after exposure Cd. RESULTS: Cd content in both roots and leaves was increased with the application of increasing concentrations of Cd. We found higher Cd concentration in roots rather than in leaves. Chlorophyll a and b was decreased in leaves but MDA significantly increased under increased Cd concentration treatments in both roots and leaves. SOD and CAT activities was also increased with the increase Cd concentrations. K, Mn and Mg concentrations were found higher in leaves than roots under Cd stress. In general, increased Cd treatments increased K, Mg, Fe, Ca, Cu and Zn concentration in both roots and leaves. Excessive Cd treatments reduced chlorophyll contents, increased antioxidant enzyme activities and changes in plant nutrition concentrations in both roots and leaves. CONCLUSION: The results presented in this work suggested that Cd treatments have negative effect on chlorophyll content and nearly decreased 30% of plant growth in strawberry. Strawberry roots accumulated higher Cd than leaves. We found that MDA and antioxidant enzyme (CAT, SOD and APX) contents may have considered a good indicator in determining Cd tolerance in strawberry plant.

Maturity-dependent chilling tolerance regulated by the antioxidative capacity in postharvest cucumber (Cucumis sativus L.) fruits.

BACKGROUND: The aim of the present study was to reveal the effect of fruit maturity on the chilling tolerance of cucumber (Cucumis sativus L.) fruit and the oxidative and antioxidative mechanisms involved. Chinese mini-cucumber (cv. Hangcui-1) fruits were harvested at four developmental stages: Immature (3-8 days after anthesis (DAA)), Mature (9-16 DAA), Breaker (17-22 DAA) and Yellow (35-40 DAA). All fruits were stored at 2 °C for 9 days and rewarmed at 20 °C for 2 days. RESULTS: The chilling injury index declined with advancing fruit maturity. High superoxide anion radical production rate and hydrogen peroxide content were observed in Immature fruits after cold storage and rewarming. Under chilling stress, superoxide dismutase showed an early response. Fruits at earlier maturity stages exhibited higher catalase, ascorbate peroxidase and monodehydroascorbate reductase activities and glutathione content as well as its redox state, and lower peroxidase, dehydroascorbate reductase and glutathione reductase activities and ascorbate content as well as its redox state. CONCLUSION: Fruits at the earlier developmental stage are more susceptible to chilling injury, which is related to increased oxidative stress. High peroxidase activity and ascorbate content and maintenance of the latter's redox state appear critical to the chilling tolerance of cucumber fruits at later developmental stages.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide.

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

The level of ascorbate peroxidase is enhanced in benznidazole-resistant populations of Trypanosoma cruzi and its expression is modulated by stress generated by hydrogen peroxide

Ascorbate peroxidases (APX) are class I heme-containing enzymes that convert hydrogen peroxide into water molecules. The gene encoding APX has been characterized in 11 strains of Trypanosoma cruzi that are sensitive or resistant to benznidazole (BZ). Bioinformatic analysis revealed the presence of two complete copies of the T. cruzi APX (TcAPX) gene in the genome of the parasite, while karyotype analysis showed that the gene was present in the 2.000-kb chromosome of all of the strains analyzed. The sequence of TcAPX exhibited greater levels of similarity to those of orthologous enzymes from Leishmania spp than to APXs from the higher plant Arabidopsis thaliana. Northern blot and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed no significant differences in TcAPX mRNA levels between the T. cruzi strains analyzed. On the other hand, Western blots showed that the expression levels of TcAPX protein were, respectively, two and three-fold higher in T. cruzi populations with in vitro induced (17 LER) and in vivo selected (BZR) resistance to BZ, in comparison with their corresponding susceptible counterparts. Moreover, the two BZ-resistant populations exhibited higher tolerances to exogenous hydrogen peroxide than their susceptible counterparts and showed TcAPX levels that increased in a dose-dependent manner following exposure to 100 and 200 µM hydrogen peroxide.

Biochemical effect of carbaryl on oxidative stress, antioxidant enzymes and osmolytes of cyanobacterium Calothrix brevissima.

Carbaryl is used in Indian agriculture for control of rice field pests and it is next to Benzene hexachloride in pesticide consumption. In present study, carbaryl (0, 10, 20, 30 and 40 mg/L) induced toxic effects were observed after 21 days exposure on a non target rice field biofertilizer Calothrix brevissima with special reference to oxidative stress, antioxidant enzymes and osmolytes. At 40 mg/L carbaryl the decrease in carotenoid, chlorophyll, phycobilin and protein were 63%, 43%, 40% and 40% respectively in comparison to control. Total carbohydrate, malondialdehyde, superoxide dismutase, ascorbate peroxidase, catalase and osmolytes showed enhancement at all the treated concentration. Increased amount of MDA (46% at 40 mg/L) indicated free radical mediated deleterious effect of carbaryl. Enhancement of SOD, APX, CAT and osmolytes in presence of carbaryl indicated their involvement in free radical scavenging. SOD, CAT and APX showed maximum activities (79%, 64% and 39% respectively) at 40 mg/L carbaryl. The order of enhancement in osmolytes was glycine-betaine (66%) > proline (54%) > sucrose (50%) at 40 mg/L which might be another adaptive defense strategy of the cyanobacterium against the pesticide.