Antioxidant enzyme activities and hormonal status in response to Cd stress in the wetland halophyte Kosteletzkya virginica under saline conditions

Salt marshes constitute major sinks for heavy metal accumulation but the precise impact of salinity on heavy metal toxicity for halophyte plant species remains largely unknown. Young seedlings of Kosteletzkya virginica were exposed during 3 weeks in nutrient solution to Cd 5 µM in the presence or absence of 50 mM NaCl. Cadmium (Cd) reduced growth and shoot water content and had major detrimental effect on maximum quantum efficiency (F_v/F_m), effective quantum yield of photosystem II (Y(II)) and electron transport rates (ETRs). Cd induced an oxidative stress in relation to an increase in O₂^?? and H₂O₂ concentration and lead to a decrease in endogenous glutathione (GSH) and α‐tocopherol in the leaves. Cd not only increased leaf zeatin and zeatin riboside concentration but also increased the senescing compounds 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and abscisic acid (ABA). Salinity reduced Cd accumulation already after 1 week of stress but was unable to restore shoot growth and thus did not induce any dilution effect. Salinity delayed the Cd‐induced leaf senescence: NaCl reduced the deleterious impact of Cd on photosynthesis apparatus through an improvement of F_v/F_m, Y(II) and ETR. Salt reduced oxidative stress in Cd‐treated plants through an increase in GSH, α‐tocopherol and ascorbic acid synthesis and an increase in glutathione reductase (EC 1.6.4.2) activity. Additional salt reduced ACC and ABA accumulation in Cd+NaCl‐treated leaves comparing to Cd alone. It is concluded that salinity affords efficient protection against Cd to the halophyte species K. virginica, in relation to an improved management of oxidative stress and hormonal status.     

Heat tolerance in rice mutants is associated with reduced accumulation of reactive oxygen species

Four mutants induced by ethylmethane sulphonate (N22-H-dgl56, N22-H-dgl101, N22-H-dgl162 and N22-H-dgl219) with conspicuous dark green leaves were identified in the drought and heat-tolerant rice cultivar Nagina22 (N22), when screened under prolonged drought and heat conditions in field. During dark-induced senescence, these mutants maintained higher chlorophyll and carotenoid contents, and photochemical efficiency of photosystem 2 in comparison with N22. Following heat treatment, these mutants accumulated less reactive oxygen species (assayed by histochemical staining for H₂O₂ and superoxide radicals) and maintained higher chlorophyll content than N22.     

Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury

Purified anthocyanin extracts show strong antioxidant properties in vitro, but it is not known whether they can scavenge reactive oxygen in living cells. The oxidative responses in red and green portions of Pseudowintera colorata leaf laminae were compared by the real‐time imaging of H₂O₂ in cells after mechanical injury. An oxidative burst was elicited almost immediately from chloroplasts in the palisade mesophyll, as evidenced using the fluorochromes dichlorofluorescein and scopoletin. H₂O₂ accumulated in green lamina regions for 10 min, and then decreased slowly. By contrast, red regions recovered rapidly, and maintained consistently low levels of H₂O₂. Infusion of cells with N‐acetyl‐l ‐cysteine accelerated the depletion of H₂O₂ from green regions. Wounded leaves ultimately developed a localized necrotic lesion and an intense anthocyanic band. The red regions were enriched in anthocyanins, flavonols, dihydroflavonols, and hydroxycinnamic acids. Only the anthocyanins were suitably located to account for the enhanced rates of H₂O₂ scavenging. The data support the hypothesis that red cells have elevated antioxidant capabilities in vivo.     

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H2O2 in Arabidopsis plants

Background

Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.

Methods

We studied the effect of wounding and MeJA treatment on the levels of H₂O₂ and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).

Results

The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H₂O₂ accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H₂O₂ were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

General significance

This is the first report demonstrating that H₂O₂-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.     

The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H₂O₂) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H₂O₂ and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H₂O₂ in leaves. In this study, the M strain of Cucumber mosaic virus (M‐CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock‐inoculated leaves, H₂O₂ accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H₂O₂ accumulation was always higher than in healthy leaves. Subcellular H₂O₂ localization observed using CLSM showed that H₂O₂ in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H₂O₂ was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H₂O₂ and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.     

Seed biopriming with drought tolerant isolates of Trichoderma harzianum promote growth and drought tolerance in Triticum aestivum

Green house study was aimed to investigate the effect of seed biopriming with drought tolerant isolates of Trichoderma harzianum, viz. Th 56, 69, 75, 82 and 89 on growth of wheat under drought stress and to explore the mechanism underlying plant water stress resilience in response to Trichoderma inoculation. Measurements of relative water content, osmotic potential, osmotic adjustment, leaf gas exchange, chlorophyll fluorescence and membrane stability index were performed. In addition, analysis of the phenolics, proline, lipid peroxidation and measurements of phenylalanine ammonia‐lyase activity were carried out. Seed biopriming enhanced drought tolerance of wheat as drought induced changes like stomatal conductance, net photosynthesis and chlorophyll fluorescence were delayed. Drought stress from 4 to 13 days of withholding water induced an increase in the concentration of stress induced metabolites in leaves, while Trichoderma colonisation caused decrease in proline, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and an increase in total phenolics. A common factor that negatively affects plants under drought stress conditions is accumulation of toxic reactive oxygen species (ROS), and we tested the hypothesis that seed biopriming reduced damages resulting from accumulation of ROS in stressed plants. The enhanced redox state of colonised plants could be explained by higher l ‐phenylalanine ammonia‐lyase (PAL) activity in leaves after 13 days of drought stress in Trichoderma treated plants. Similar activity was induced in untreated plants in response to drought stress but to a lower extent in comparison to treated plants. Our results support the hypothesis that seed biopriming in wheat with drought tolerant T. harzianum strains increased root vigour besides performing the process of osmoregulation. It ameliorates drought stress by inducing physiological protection in plants against oxidative damage, due to enhanced capacity to scavenge ROS and increased level of PAL, a mechanism that is expected to augment tolerance to abiotic stresses.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.     

Antioxidative protection in the leaves of dark-senescing intact barley seedlings

In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H₂O₂ and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H₂O₂ was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.     

Possible Participation of Active Oxygen Species in the Induction of Cyanide-Resistant Pathway at the Initial Senescence of Tobacco Callus

Changes in active oxygen species (AOS) and respiration, with special reference to the alternative pathway of respiration, were followed in callus of tobacco (Nicotiana rusticaL.) during its growth and senescence. Two peaks of the total respiration rate were observed: the first one appeared on day 11, and the second one on day 19 (in senescing callus). H₂O₂and O^– ₂contents increased gradually and reached the greatest values when callus senescence initiated (by day 11 and day 15, respectively), and then declined. The peaks of H₂O₂and O^– ₂before the onset of senescence coincided with the peaks of capacity V _altand activity V _altof alternative pathway, respectively. After the onset of senescence, ·OH accumulated abundantly and maintained at a relatively high level from then on, accompanied by the decrease in both V _altand V _alt. The conclusion is that these three active oxygen species operated coordinately to regulate the alternative pathway during growth and senescence of tobacco callus, as confirmed by the callus treatments with AOS scavengers, exogenous ·OH, and inhibitors of antioxidant enzymes. The possibility of AOS-induced alternative pathway respiration at the initial senescence of tobacco callus is discussed.     

Cultivation of Arabidopsis cell cultures in a stirred bioreactor at variable oxygen levels: Influence on tocopherol production

Abstract

In plants, an increased production of toxic oxygen species is commonly observed under low oxygen stress, but cellular responses still have to be fully investigated. Plant cell cultures can be a valuable tool to study plant metabolic responses to various environmental stresses including low oxygen condition. Arabidopsis suspension cultures growing in shake flasks were subjected to hypoxia by stopping shaking for different intervals, showing an increase of the antioxidant metabolite α‐tocopherol. In order to obtain a more controlled condition, cultivation of Arabidopsis suspension cultures was established in a 5‐l stirred bioreactor. A constant aeration of 20% dissolved oxygen was found to be the most suitable for cell growth. A 4‐h anoxic shock was induced by suspending the aeration and flushing into the vessel with nitrogen. During the anoxic stress, tocopherol levels resulted increased at the end of the treatment, indicating that the complete oxygen deprivation, indeed, induced a defence response involving antioxidant metabolism. The presence of an oxidative stress as a consequence of anoxic condition was also confirmed by the increased levels of H₂O₂. Overall, these results indicate that Arabidopsis suspension cultures grown in a stirred bioreactor can be a useful in vitro system for investigating low oxygen stress.     

Physiological significance of isoprenoids and phenylpropanoids in drought response of Arundinoideae species with contrasting habitats and metabolism

Physiological, biochemical and morpho‐anatomical traits that determine the phenotypic plasticity of plants under drought were tested in two Arundinoideae with contrasting habitats, growth traits and metabolism: the fast‐growing Arundo donax, which also is a strong isoprene emitter, and the slow‐growing Hakonechloa macra that does not invest on isoprene biosynthesis. In control conditions, A. donax displayed not only higher photosynthesis but also higher concentration of carotenoids and lower phenylpropanoid content than H. macra. In drought‐stressed plants, photosynthesis was similarly inhibited in both species, but substantially recovered only in A. donax after rewatering. Decline of photochemical and biochemical parameters, increased concentration of CO₂ inside leaves, and impairment of chloroplast ultrastructure were only observed in H. macra indicating damage of photosynthetic machinery under drought. It is suggested that volatile and non‐volatile isoprenoids produced by A. donax efficiently preserve the chloroplasts from transient drought damage, while H. macra invests on phenylpropanoids that are less efficient in preserving photosynthesis but likely offer better antioxidant protection under prolonged stress.     

Polyphenols suppress hydrogen peroxide‐induced oxidative stress in human bone‐marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are considered a highly promising candidate cell type for cell‐based tissue engineering and regeneration because of their self‐renewal and multi‐lineage differentiation characteristics. Increased levels of reactive oxygen/nitrogen species (ROS/RNS) are associated with tissue injury and inflammation, impact a number of cellular processes, including cell adhesion, migration, and proliferation, and have been linked to cellular senescence in MSCs, potentially compromising their activities. Naturally occurring polyphenolic compounds (polyphenols), epigallocatechin‐3‐gallate (EGCG), and curcumin, block ROS/RNS and are potent inflammation‐modulating agents. However, their potential protective effects against oxidative stress in hMSCs have not been examined. In this study, we carried out a systematic analysis of the effects of polyphenols on hMSCs in their response to oxidative stress in the form of treatment with H₂O₂ and S‐nitroso‐N‐acetylpenicillamine (SNAP), respectively. Parameters measured included colony forming activity, apoptosis, and the levels of antioxidant enzymes and free reactive species. We found that polyphenols reversed H₂O₂‐induced loss of colony forming activity in hMSCs. In a dose‐dependent manner, polyphenols inhibited increased levels of ROS and NO, produced by H₂O₂ or SNAP, respectively, in MSCs. Notably, polyphenols rapidly and almost completely blocked H₂O₂‐induced ROS in the absence of significant direct effect on H₂O₂ itself. Polyphenols also protected the antioxidant enzymes and reduced apoptotic cell death caused by H₂O₂ exposure. Taken together, these findings demonstrate that EGCG and curcumin are capable of suppressing inducible oxidative stress in hMSCs, and suggest a possible new approach to maintain MSC viability and potency for clinical application. J. Cell. Biochem. 114: 1163–1173, 2013. © 2012 Wiley Periodicals, Inc.     

Heat shock protein 70 regulates the abscisic acid-induced antioxidant response of maize to combined drought and heat stress

We investigated the interaction between heat shock protein 70 (HSP70) and abscisic acid (ABA)-induced antioxidant response of maize to the combination of drought and heat stress. First, the increased activities of enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), induced by drought were less than those by heat or combined drought and heat stress, except some individual cases (e.g. CAT in leaves, GR in roots). Second, both HSP70 synthesis and H₂O₂ production increased prominently under drought, heat or their combination stress; the increase in leaves induced by drought and heat combination was the highest, followed by heat and by drought, while the increase in roots had not visible difference. Third, either in leaves or roots, pretreatment with ABA inhibitor, HSP70 inhibitor and H₂O₂ scavenger, significantly arrested the stress-induced increase of antioxidant enzyme activities, and ABA inhibitor and H₂O₂ scavenger obviously suppressed HSP70 synthesis, while HSP70 inhibitor slightly heightened H₂O₂ accumulation. Finally, 100 μM ABA significantly enhanced the activities of antioxidant enzymes, HSP70 expression and H₂O₂ production under stresses in comparison with ABA-deficient mutant vp5 maize plants without pretreatment. Thus, ABA-induced H₂O₂ production enhances the HSP70 synthesis and up-regulates the activities of antioxidant enzymes, resulting in the suppression of cellular reactive oxygen species (ROS) levels. Our results suggest that HSP70 may play a crucial role in ABA-induced antioxidant defense of maize to drought and heat combination.     

Nitric oxide interferes with plant photo-oxidative stress by detoxifying reactive oxygen species

Oxidative stress within chloroplasts is originated due to light‐dependent O₂ reduction. This may be exacerbated by bipyridinium herbicides, which act at photosystem I as artificial electron acceptors. Their oxidation produces a superoxide anion that further dismutates to H₂O₂ and then, by the Fenton reaction, H₂O₂ may be reduced to the hydroxyl radical (OH^?). Reactive oxygen species (ROS), when produced in high amounts, provoke severe damage to the plant cell. Herein it is reported that two nitric oxide (NO) donors, sodium nitroprusside (100 µm ) and S‐nitroso‐N‐acetylpenicillamine (200 µm ), greatly reduced lipid peroxidation and the protein loss caused by the application of a high dose of the bipyridinium herbicide diquat to potato leaf pieces or isolated chloroplasts. Nitric oxide donors also protected the RNA against oxidative damage. Photo‐oxidative toxicity was correlated with an increase in photosynthetic electron transport and ROS production, but the rate of electron transport was restored and the ROS free amount was markedly reduced in the presence of NO. The specific activity of superoxide dismutase was not affected by diquat or NO donors, whereas just a small increase in catalase activity was observed after 24 h of treatment. These results provide strong evidence that NO is a potent antioxidant in plants and that its action may, at least in part, be explained by its ability to directly scavenge ROS.     

CUL4B impedes stress-induced cellular senescence by dampening a p53-reactive oxygen species positive feedback loop

Tumor suppressor p53 is known to regulate the level of intracellular reactive oxygen species (ROS). It can either alleviate oxidative stress under physiological and mildly stressed conditions or exacerbate oxidative stress under highly stressed conditions. We here report that a p53–ROS positive feedback loop drives a senescence program in normal human fibroblasts (NHFs) and this senescence-driving loop is negatively regulated by CUL4B. CUL4B, which can assemble various ubiquitin E3 ligases, was found to be downregulated in stress-induced senescent cells, but not in replicative senescent cells. We observed that p53-dependent ROS production was significantly augmented and stress-induced senescence was greatly enhanced when CUL4B was absent or depleted. Ectopic expression of CUL4B, on the other hand, blunted p53 activation, reduced ROS production, and attenuated cellular senescence in cells treated with H₂O₂. CUL4B was shown to promote p53 ubiquitination and proteosomal degradation in NHFs exposed to oxidative stress, thus dampening the p53-dependent cellular senescence. Together, our results established a critical role of CUL4B in negatively regulating the p53–ROS positive feedback loop that drives cellular senescence.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.