The ABA/LANCL1-2 Hormone/Receptors System Controls ROS Production in Cardiomyocytes through ERRα.

Rat H9c2 cardiomyocytes overexpressing the abscisic acid (ABA) hormone receptors LANCL1 and LANCL2 have an increased mitochondrial proton gradient, respiration, and vitality after hypoxia/reoxygenation. Our aim was to investigate the role of the ABA/LANCL1-2 system in ROS turnover in H9c2 cells. H9c2 cells were retrovirally infected to induce the overexpression or silencing of LANCL1 and LANCL2, without or with the concomitant silencing of the transcription factor ERRα. Enzymes involved in radical production or scavenging were studied by qRT-PCR and Western blot. The mitochondrial proton gradient and ROS were measured with specific fluorescent probes. ROS-generating enzymes decreased, ROS-scavenging enzymes increased, and mitochondrial ROS were reduced in LANCL1/2-overexpressing vs. control cells infected with the empty vector, while the opposite occurred in LANCL1/2-silenced cells. The knockdown of ERRα abrogated all beneficial effects on ROS turnover in LANCL1/2 overexpressing cells. Taken together, these results indicate that the ABA/LANCL1-2 system controls ROS turnover in H9c2 via ERRα. The ABA/LANCL system emerges as a promising target to improve cardiomyocyte mitochondrial function and resilience to oxidative stress.

DNP and ATP regulate the pulp breakdown development in Phomopsis longanae Chi-infected longan fruit through modulating the ROS metabolism.

Compared with the P. longanae-infected longan, the DNP-treated P. longanae-infected fruit represented a higher pulp breakdown index, a higher O2 -. production rate, and a higher MDA content, but the lower activities of APX, SOD and CAT, the lower transcript levels of DlAPX6, DlSOD1, DlSOD2, DlSOD3 and DlCAT1, the lower values of AsA, GSH, flavonoid and total phenolics, a lower scavenging ability of DPPH radical, and a lower value of reducing power. Whereas, the ATP-treated P. longanae-infected samples showed the contrary results. The above findings indicated that the DNP-promoted the pulp breakdown in P. longanae-infected longan was because DNP weakened the capacity of scavenging ROS, raised the O2 -. level, and accelerated the membrane lipids peroxidation. However, the ATP-suppressed the pulp breakdown in P. longanae-infected longan was because ATP improved the capacity of scavenging ROS, reduced the O2 -. level, and reduced the membrane lipids peroxidation.

Exploring plant growth-promoting rhizobacteria as stress alleviators: a methodological insight.

Rhizospheric and root-endophyte bacteria can stimulate plant growth through diverse biochemical mechanisms and pathways, particularly under biotic and abiotic stresses. For this reason, biotechnological trends on plant growth-promoting rhizobacteria (PGPR) application as biofertilizers, bioremediators, and stress alleviators are gaining increasing interest as ecofriendly strategies for sustainable agriculture management and soil restoration. The first steps needed to implement these technologies are isolation, screening, and characterization of PGPR that can be potentially applied as bioinoculants to alleviate biotic and/or abiotic stresses. Therefore, a complete and accurate methodological study and laboratory techniques are required to warrant the correct achievement of these steps. This review compiles and details the fundamentals, methods, and procedures of key protocols used in isolation and characterization of PGPR for plant stress alleviation.

Theanine Improves Salt Stress Tolerance via Modulating Redox Homeostasis in Tea Plants (Camellia sinensis L.).

Theanine, a unique non-proteinogenic amino acid, is one of the most abundant secondary metabolites in tea. Its content largely determines green tea quality and price. However, its physiological roles in tea plants remain largely unknown. Here, we showed that salt stress significantly increased the accumulation of glutamate, glutamine, alanine, proline, and γ-aminobutyric acid, as well as theanine, in the new shoots of tea plants. We further found that salt stress induced the expression of theanine biosynthetic genes, including CsGOGATs, CsAlaDC, and CsTSI, suggested that salt stress induced theanine biosynthesis. Importantly, applying theanine to the new shoots significantly enhanced the salt stress tolerance. Similar effects were also found in a model plant Arabidopsis. Notably, exogenous theanine application increased the antioxidant activity of the shoots under salt stress, suggested by reduced the reactive oxygen species accumulation and lipid peroxidation, as well as by the increased SOD, CAT, and APX activities and expression of the corresponding genes. Finally, genetic evidence supported that catalase-mediated antioxidant scavenging pathway is required for theanine-induced salt stress tolerance. Taken together, this study suggested that salt stress induces theanine biosynthesize in tea plants to enhance the salt stress tolerance through a CAT-dependent redox homeostasis pathway.

Postharvest melatonin treatment inhibited longan (Dimocarpus longan Lour.) pericarp browning by increasing ROS scavenging ability and protecting cytomembrane integrity.

Postharvest melatonin treatments have been reported to improve the quality and storability, especially to inhibit browning in many fruits, but the effect had not been systematically investigated on longan fruit. In this study, the effect of 0.4 mM melatonin (MLT) dipping on the quality and pericarp browning of longan fruits stored at low temperature was investigated. The MLT treatment did not influence the TSS content of longan fruits but lead to increased lightness and h° value while decreased a* value of pericarp. More importantly, the treatment significantly delayed the increase in electrolyte leakage and malonaldehyde accumulation, inhibited the activities of polyphenol oxidase and peroxidase, and thus retarded pericarp browning. In addition, the treatment significantly inhibited the production of O2 •- and H2O2 while promoted the accumulation of glutathione, flavonoids, and phenolics at earlier storage stages in longan pericarp. Interestingly, the activities of ascorbate peroxidase (APX) and superoxide dismutase (SOD) were significantly upregulated but activities of catalase were downregulated in the MLT-treated longan pericarp. MLT treatment effectively enhanced APX and SOD activities, increased flavonoid, phenolics, and glutathione content, protected cytomembrane integrity, inhibited the production of O2 •- and H2O2 and browning-related enzymes, and thus delayed the longan pericarp browning.

Micro RNAs in Regulation of Cellular Redox Homeostasis.

In living cells Reactive Oxygen Species (ROS) participate in intra- and inter-cellular signaling and all cells contain specific systems that guard redox homeostasis. These systems contain both enzymes which may produce ROS such as NADPH-dependent and other oxidases or nitric oxide synthases, and ROS-neutralizing enzymes such as catalase, peroxiredoxins, thioredoxins, thioredoxin reductases, glutathione reductases, and many others. Most of the genes coding for these enzymes contain sequences targeted by micro RNAs (miRNAs), which are components of RNA-induced silencing complexes and play important roles in inhibiting translation of their targeted messenger RNAs (mRNAs). In this review we describe miRNAs that directly target and can influence enzymes responsible for scavenging of ROS and their possible role in cellular redox homeostasis. Regulation of antioxidant enzymes aims to adjust cells to survive in unstable oxidative environments; however, sometimes seemingly paradoxical phenomena appear where oxidative stress induces an increase in the levels of miRNAs which target genes which are supposed to neutralize ROS and therefore would be expected to decrease antioxidant levels. Here we show examples of such cellular behaviors and discuss the possible roles of miRNAs in redox regulatory circuits and further cell responses to stress.

Inactivation of the auto-inhibitory domain in Arabidopsis AtCPK1 leads to increased salt, cold and heat tolerance in the AtCPK1-transformed Rubia cordifolia L cell cultures.

Calcium-dependent protein kinases (CDPKs) are essential regulators of plant growth and development, biotic and abiotic stress responses. Inactivation of the auto-inhibitory domain (AID) of CDPKs provides the constitutive activity. This study investigated the effect of overexpressed native and constitutive active (AtCPK1-Ca) forms of the AtCPK1 gene on abiotic stress tolerance and the ROS/redox system in Rubia cordifolia transgenic callus lines. Overexpression of the native AtCPK1 increased tolerance to salinity and cold almost in two times, when AtCPK1-Ca - in three times compare to control culture. A more interesting effect of overexpression of the AtCPK1 and AtCPK1-Ca was observed for heat resistance. The native form of AtCPK1 increased resistance to heating by 45%, while the AtCPK1-Ca increased by 80%. At the same time, another type of mutation of the AID (AtCPK1-Na, not active) did not affect the tolerance of the cell culture to stresses. We suppose, in this process, the ROS/redox system might be involved. Levels of intracellular ROS, ROS-generating enzymes expression and activities (Rbohs, Prx) and ROS-detoxifying enzymes (SOD, Cat, Apx and Prx) changed in a coordinated manner and in strict interconnection, depending of the callus growth phase and correlated with improved stress tolerance caused by AtCPK1. Because overexpression of both the AtCPK1 and AtCPK1-Ca did not significantly change callus growth, we propose that inactivation of AID of the AtCPK1 or its ortholog, might be an interesting instrument for improvement of plant cells resistance to abiotic stress.

Chitosan postharvest treatment suppresses the pulp breakdown development of longan fruit through regulating ROS metabolism.

The influences of Kadozan, a novel chitosan formulation, on the pulp breakdown and ROS metabolism in postharvest 'Fuyan' longans were studied. Compared with control longans, the longans treated with 1:500 Kadozan dilution (VKadozan: VKadozan + Water) exhibited the suppressed development of pulp breakdown, higher AsA and GSH amounts, higher activities of ROS-scavenging enzymes like SOD, CAT, APX and POD, higher reducing power, and higher scavenging ability for DPPH radical, but a lower MDA amount, lower levels of ROS including O2- and H2O2. These findings indicated that the application of 1:500 Kadozan dilution (VKadozan: VKadozan + Water) for harvested longans could enhance the ROS-scavenging capacity to decrease the generation and accumulation of ROS, and a lower level of ROS could slow down the peroxidation progress of membrane lipids, alleviate the damage of longan pulp cellular membrane structure, and ultimately suppress pulp breakdown occurrence of harvested longans.

Transcriptome Analyses and Antioxidant Activity Profiling Reveal the Role of a Lignin-Derived Biostimulant Seed Treatment in Enhancing Heat Stress Tolerance in Soybean.

Soybean (Glycine max Merr.) is a worldwide important legume crop, whose growth and yield are negatively affected by heat stress at germination time. Here, we tested the role of a biostimulant based on lignin derivatives, plant-derived amino acids, and molybdenum in enhancing soybean heat stress tolerance when applied on seeds. After treatment with the biostimulant at 35 °C, the seed biometric parameters were positively influenced after 24 h, meanwhile, germination percentage was increased after 72 h (+10%). RNA-Seq analyses revealed a modulation of 879 genes (51 upregulated and 828 downregulated) in biostimulant-treated seeds as compared with the control, at 24 h after incubation at 35 °C. Surprisingly, more than 33% of upregulated genes encoded for ribosomal RNA (rRNA) methyltransferases and proteins involved in the ribosome assembly, acting in a specific protein network. Conversely, the downregulated genes were involved in stress response, hormone signaling, and primary metabolism. Finally, from a biochemical point of view, the dramatic H2O2 reduction 40%) correlated to a strong increase in non-protein thiols (+150%), suggested a lower oxidative stress level in biostimulant-treated seeds, at 24 h after incubation at 35 °C. Our results provide insights on the biostimulant mechanism of action and on its application for seed treatments to improve heat stress tolerance during germination.

Effects of Arbuscular Mycorrhizal Fungi on Watermelon Growth, Elemental Uptake, Antioxidant, and Photosystem II Activities and Stress-Response Gene Expressions Under Salinity-Alkalinity Stresses.

Salinity-alkalinity stress has caused severe environment problems that negatively impact the growth and development of watermelon (Citrullus lanatus L.). In this study, watermelon seedlings were inoculated with the arbuscular mycorrhizal fungi (AMF) Funneliformis mosseae to investigate its effect on watermelon growth and development. The main measurements included morphological traits, elemental and water uptake, the level of reactive oxygen species, antioxidant enzyme and photosynthesis activities, and relative expression levels of stress response genes. Under salinity-alkalinity stresses, watermelon morphological traits, elemental and water uptake were all significantly alleviated after incubation with AMF. Antioxidant abilities of watermelon were significantly improved after incubation with AMF in salinity-alkalinity stresses. Under normal conditions, all photosynthesis related parameters were significantly increased after incubation of AMF. In contrast, they were all significantly reduced under salinity-alkalinity stresses and were all significantly alleviated after incubation of AMF. Salinity-alkalinity stresses impacted the chloroplast structure and AMF significantly alleviated these damages. Under salinity-alkalinity stresses, the relative expression level of RBCL was significantly reduced and was significantly alleviated after AMF treatment. The relative expression level of PPH was significantly increased and was further significantly reduced after AMF treatment. For the relative expression levels of antioxidant response related genes Cu-Zn SOD, CAT, APX, GR, their relative expression levels were significantly increased and were further significantly increased after AMF treatment. Our study demonstrated the beneficial effects of AMF under salinity-alkalinity stresses, which could be implicated in the management of watermelon cultivation under salinity-alkalinity regions.

Safe Cultivation of Medicago sativa in Metal-Polluted Soils from Semi-Arid Regions Assisted by Heat- and Metallo-Resistant PGPR.

Soil contamination with heavy metals is a constraint for plant establishment and development for which phytoremediation may be a solution, since rhizobacteria may alleviate plant stress under these conditions. A greenhouse experiment was conducted to elucidate the effect of toxic metals on growth, the activities of ROS (reactive oxygen species)-scavenging enzymes, and gene expression of Medicago sativa grown under different metal and/or inoculation treatments. The results showed that, besides reducing biomass, heavy metals negatively affected physiological parameters such as chlorophyll fluorescence and gas exchange, while increasing ROS-scavenging enzyme activities. Inoculation of M. sativa with a bacterial consortium of heat- and metallo-resistant bacteria alleviated metal stress, as deduced from the improvement of growth, lower levels of antioxidant enzymes, and increased physiological parameters. The bacteria were able to effectively colonize and form biofilms onto the roots of plants cultivated in the presence of metals, as observed by scanning electron microscopy. Results also evidenced the important role of glutathione reductase (GR), phytochelatin synthase (PCS), and metal transporter NRAMP1 genes as pathways for metal stress management, whereas the gene coding for cytochrome P450 (CP450) seemed to be regulated by the presence of the bacteria. These outcomes showed that the interaction of metal-resistant rhizobacteria/legumes can be used as an instrument to remediate metal-contaminated soils, while cultivation of inoculated legumes on these soils is still safe for animal grazing, since inoculation with bacteria diminished the concentrations of heavy metals accumulated in the aboveground parts of the plants to below toxic levels.

Salicylic acid-induced differential resistance to the Tomato yellow leaf curl virus among resistant and susceptible tomato cultivars.

BACKGROUND: In higher plants, salicylic acid (SA) plays important roles in inducing resistance to biotic and abiotic stresses. Tomato yellow leaf curl virus (TYLCV) causes a highly devastating viral disease in plants, particularly in tomato. However, the roles of SA in inducing tomato plant resistance to TYLCV remain unclear. RESULTS: In this study, we investigated whether the exogenous application of SA can improve the resistance of tomato plants to TYLCV in two tomato cultivars, resistant 'Zhefen-702' and susceptible 'Jinpeng-1'. The impacts of SA on the accumulation of ascorbic acid (AsA) and biosynthetic gene expression, the activity of some important reactive oxygen species (ROS)-scavenging enzymes, and the expression patterns of stress-related genes were also determined. Results indicated that SA can effectively regulate the accumulation of AsA, especially in 'Jinpeng-1'. Similarly, the expression patterns of most of the AsA biosynthetic genes showed a negative relationship with AsA accumulation in the resistant and susceptible tomato cultivars. In the two tomato cultivars, the activities of ascorbate peroxidase (APX) and peroxidase (POD) in the SA + TYLCV treated plants were increased during the experiment period except at 14 days (APX in 'Jinpeng-1' was also at 4 days) post infected (dpi) with TYLCV. Simultaneously, the activity of SOD was reduced in 'Jinpeng-1' and increased in 'Zhefen-702' after treatment with SA + TYLCV. SA can substantially induce the expression of ROS-scavenging genes at different extents. From 2 to 10 dpi, the virus content in the SA + TYLCV treated plants was remarkably lower than those in the TYLCV treated plants in 'Jinpeng-1'and Zhefen-702'. CONCLUSIONS: The above results suggest that SA can enhance tomato plant resistance by modulating the expression of genes encoding for ROS-scavenging players, altering the activity of resistance-related enzymes, and inducing the expression of pathogenesis-related genes to produce systemic acquired resistance. Simultaneously, these results confirm that SA is a resistance-inducing factor against TYLCV infection that can be effectively applied in tomato plants.

iTRAQ-based analysis of leaf proteome identifies important proteins in secondary metabolite biosynthesis and defence pathways crucial to cross-protection against TMV.

Cross-protection is a phenomenon in which infection with a mild virus strain protects host plants against subsequent infection with a closely related severe virus strain. This study showed that a mild strain mutant virus, Tobacco mosaic virus (TMV)-43A could cross protect Nicotiana benthamiana plants against wild-type TMV. Furthermore, we investigated the host responses at the proteome level to identify important host proteins involved in cross-protection. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyze the proteome profiles of TMV, TMV-43A and cross-protected plants at different time-points. Our results showed that TMV-43A can cross-protect N. benthamiana plants from TMV. In cross-protected plants, photosynthetic activities were augmented, as supported by the increased accumulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) enzymes, which are crucial for chlorophyll biosynthesis. The increased abundance of ROS scavenging enzymes like thioredoxins and L-ascorbate peroxidase would prevent oxidative damage in cross-protected plants. Interestingly, the abundance of defence-related proteins (14-3-3 and NbSGT1) decreased, along with a reduction in virus accumulation during cross-protection. In conclusion, we have identified several important host proteins that are crucial in cross-protection to counter TMV infection in N. benthamiana plants. BIOLOGICAL SIGNIFICANCE: TMV is the most studied model for host-virus interaction in plants. It can infect wide varieties of plant species, causing significant economic losses. Cross protection is one of the methods to combat virus infection. A few cross-protection mechanisms have been proposed, including replicase/coat protein-mediated resistance, RNA silencing, and exclusion/spatial separation between virus strains. However, knowledge on host responses at the proteome level during cross protection is limited. To address this knowledge gap, we have leveraged on a global proteomics analysis approach to study cross protection. We discovered that TMV-43A (protector) protects N. benthamiana plants from TMV (challenger) infection through multiple host pathways: secondary metabolite biosynthesis, photosynthesis, defence, carbon metabolism, protein translation and processing and amino acid biosynthesis. In the secondary metabolite biosynthesis pathway, enzymes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) play crucial roles in chlorophyll biosynthesis during cross protection. In addition, accumulation of ROS scavenging enzymes was also found in cross-protected plants, providing rescues from excessive oxidative damage. Reduced abundance of plant defence proteins is correlated to reduced virus accumulation in host plants. These findings have increased our knowledge in host responses during cross-protection.

Enhanced storability of blueberries by acidic electrolyzed oxidizing water application may be mediated by regulating ROS metabolism.

This study aimed to investigate the effects of acidic electrolyzed oxidizing water (AEW) treatment on storability and metabolism of reactive oxygen species (ROS) in blueberries cv. 'Brightwell' during storage at 4 °C. Results showed that, compared with the control blueberries, AEW treated-blueberries exhibited lower incidence of fruit decay, higher rate of commercially acceptable fruit, higher fruit firmness and skin hardness, and higher anthocyanin and total phenolics contents, along with higher activities of SOD, CAT and APX, higher antioxidant activity, but lower generation rate of superoxide anion and cell membrane permeability. These results demonstrated that AEW treatment for enhancing storability of harvested blueberries during storage may be mediated by regulating ROS metabolism, manifested as AEW increasing ROS scavenging capacity and reducing ROS accumulation, and thereby maintained the structural integrity of cellular membrane, which indicated that AEW treatment was a facile postharvest method for extending the shelf life of harvested blueberries.

Investigating the mechanisms underlying phytoprotection by plant growth-promoting rhizobacteria in Spartina densiflora under metal stress.

Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS-scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated. Sediments of the metal-polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP-OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm-forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively. Metal excess induced SOD (five-fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal-resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non-exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation. The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75- and five-fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.

Phenotypical and biochemical characterisation of resistance for parasitic weed (Orobanche foetida Poir.) in radiation-mutagenised mutants of chickpea.

BACKGROUND: Some radiation-mutagenised chickpea mutants potentially resistant to the broomrape, Orobanche foetida Poir., were selected through field trials. The objectives of this work were to confirm resistance under artificial infestation, in pots and mini-rhizotron systems, and to determine the developmental stages of broomrape affected by resistance and the relevant resistance mechanisms induced by radiation mutagenesis. RESULTS: Among 30 mutants tested for resistance to O. foetida, five shared strong resistance in both pot experiments and mini-rhizotron systems. Resistance was not complete, but the few individuals that escaped resistance displayed high disorders of shoot development. Results demonstrated a 2-3-fold decrease in stimulatory activity of root exudates towards broomrape seed germination in resistant mutants in comparison with non-irradiated control plants and susceptible mutants. Resistance was associated with an induction of broomrape necrosis early during infection. When infested, most of the resistant mutants shared enhanced levels of soluble phenolic contents, phenylalanine ammonia lyase activity, guaiacol peroxidase activity and polyphenol oxidase activity, in addition to glutathione and notably ascorbate peroxidase gene expression in roots. CONCLUSION: Results confirmed enhanced resistance in chickpea radiation-mutagenised mutants, and demonstrated that resistance is based on alteration of root exudation, presumed cell-wall reinforcement and change in root oxidative status in response to infection. © 2016 Society of Chemical Industry.

Oxidative stress and antioxidant systems in Guava (Psidium guajava L.) fruits during ripening.

Two varieties of guava viz., L-49 and Hisar Safeda differing in their shelf lives were analyzed for various components of oxidative stress and of enzymatic and non-enzymatic antioxidative system at different stages of fruit ripening. Indices of oxidative stress viz., lipoxygenase activity, malondialdehyde value and H2O2 content increased throughout during ripening in both the varieties. The extent of oxidative stress was more pronounced in Hisar Safeda (shelf life 3-4 days) than in L-49 (shelf life 7-8 days). Except for superoxide dismutase, activities of all other antioxidative enzymes viz., catalase, peroxidase, ascorbate peroxidase and glutathione reductase increased up to color turning stage and decreased thereafter. Superoxide dismutase activity, however, increased upto ripe stage followed by a decline. Contents of ascorbic acid and glutathione (total, oxidized and reduced) were found to be the maximum at turning and mature stage, respectively. It is inferred that ripening of guava fruit is accompanied by a progressive increase in oxidative/peroxidative stress which induces antioxidant system but not until later stages of ripening. Over-accumulation of ROS due to dysfunctioning of ROS scavenging system at later stages of fruit ripening appears to be responsible for loss of tissue structure as observed in ripened and over-ripened fruits.