RESUMEN
MAIN CONCLUSION: Nitric oxide sustains root tip surface redox activity and restricts lipid peroxidation-triggered cell death in the root tips. In order to gain more insight into the involvement of nitric oxide (NO) in plant response to sudden flooding-induced hypoxic stress, we studied the effect of two NO donors, sodium nitroprusside and S-nitroso-L-glutathione, on short-term partial submergence-induced root growth inhibition, alteration in root surface redox activity, lipid peroxidation and cell death in two barley cultivars (cv.) at their early seedling stage. The short-term hypoxic stress induces root growth arrest in cv. Karmel, accompanied by increased lipid peroxidation and cell death. By contrast, in cv. Slaven, short-term hypoxic conditions cause only reduced root growth rate, associated with elevated extracellular NO level in the root tips. The root tip surface redox activity decreases with the increasing timespan of hypoxic conditions in both cultivars; however, this decrease in redox activity started earlier and was greater in the cv. Karmel in comparison with cv. Slaven. Application of NO donors during hypoxic stress sustains the root redox activity and eliminates the hypoxia-induced lipid peroxidation, accompanied by a partial restoration of root growth after short-term hypoxic stress. These results suggest that extracellular NO plays a key role in maintaining the root tip surface redox activity and in the restriction of lipid peroxidation and cell death under short-term hypoxic stress in the root tips of barley seedlings.
Asunto(s)
Hordeum , Meristema , Meristema/metabolismo , Óxido Nítrico/metabolismo , Hordeum/metabolismo , Plantones/metabolismo , Hipoxia/metabolismo , Oxidación-Reducción , Raíces de Plantas/metabolismoRESUMEN
MAIN CONCLUSION: NBT and HE may be efficiently used for the detection of superoxide, while DCDHF-DA and DHR123 for the detection of peroxynitrite in intact barley root tips, only if PRXs and oxidoreductases are inhibited to avoid false-positive reactions. Strong peroxidase (PRX) and oxidoreductase activities were observed in the barley root tips that were markedly inhibited by NaN3. Rapid and strong nitro-blue tetrazolium chloride (NBT) reduction is associated mainly with the vital functions of root cells but not with superoxide formation. In turn, the inhibition of root surface redox activity by NaN3 strongly reduced the formation of formazan, but its slight accumulation, observed in the root elongation zone, was a result of NADPH oxidase-mediated apoplastic superoxide formation. A longer staining time period with NBT was required for the detection of antimycin A-mediated superoxide formation inside the cells. This antimycin A-induced superoxide was clearly detectable by hydroethidine (HE) after the inhibition of PRXs by NaN3, and it was restricted into the root transition zone. TEMPOL, a superoxide scavenger, strongly inhibited both NBT reduction and HE oxidation in the presence of NaN3. Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN3 and was detectable mainly in the root transition zone. This fluorescence signal was not influenced by the application of pyruvate but was strongly reduced by urea, a peroxynitrite scavenger. The presented results suggest that if the root PRXs and oxidoreductases are inhibited, both NBT and HE detect mainly superoxide, whereas both DCDHF-DA and DHR123 may be efficiently used for the detection of peroxynitrite in intact barley root tips. The inhibition of PRXs and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in the intact barley root tip.
Asunto(s)
Hordeum , Meristema , Oxidorreductasas , Peroxidasas , Especies Reactivas de Oxígeno , SuperóxidosRESUMEN
KEY MESSAGE: Already a short-term Cd treatment induces changes in gene expression in barley root tips via IAA and ROS signaling during mild and severe Cd stress, respectively. Even a short, 30 min, Cd treatment of roots induced a considerable alteration in gene expression in the barley root tips within an hour after the treatments. The very early activation of MYB1 transcription factor expression is partially regulated by auxin signaling in mildly stressed seedlings. An increase in allene oxide cyclase and NADPH oxidase expression was a distinguishing feature of root tips response to mild Cd stress and their expression is activated via IAA signaling. Meanwhile, early changes in the level of dehydrin transcripts were detected in moderately and severely stressed root tips, and their induction is related to altered ROS homeostasis in cells. The early activation of glutathione peroxidase expression by mild Cd stress indicates the involvement of IAA in the signaling process. In contrast, early ascorbate peroxidase expression was induced only with Cd treatment causing severe stress and ROS play central roles in its induction. The expression of cysteine protease was activated similarly in both mildly and severely Cd-stressed roots; consequently, both increased IAA and ROS levels take part in the regulation of cysteine protease expression. The Cd-evoked accumulation of BAX Inhibitor-1 mRNA was characteristic for moderately and severely stressed roots. Whereas decreased IAA level did not affect its expression, rotenone-mediated ROS depletion markedly reduced the Cd-induced expression of BAX Inhibitor-1. An early increase of alternative oxidase levels in the root tip cells indicated that the reduction of mitochondrial superoxide generation is an important component of barley root response to severe Cd stress.
Asunto(s)
Cadmio/toxicidad , Hordeum/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Western Blotting , Expresión Génica/efectos de los fármacos , Hordeum/anatomía & histología , Hordeum/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/anatomía & histología , Raíces de Plantas/metabolismo , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismoRESUMEN
MAIN CONCLUSION: Activation of GPX and enhanced NO level play a key role in IBA-mediated enhanced Cd tolerance in young barley roots. Application of exogenous indole-3-acetic acid (IAA) or an IAA precursor improves the tolerance of plants to heavy metals. However, the physiology of these tolerance mechanisms remains largely unknown. Therefore, we studied the priming effect of indole-3-butyric acid (IBA), an IAA precursor, on mild and severe cadmium (Cd) stress-induced responses in roots of young barley seedlings. IBA, similarly to mild Cd stress, significantly increased the glutathione peroxidase (GPX) activity in the apexes of barley roots, which remained elevated after the IBA pretreatment as well. IBA pretreatment-evoked high nitric oxide generation in roots effectively reduced the high superoxide level under the severe Cd stress, leading to less toxic peroxynitrite accumulation accompanied by markedly reduced Cd-induced cell death. On the other hand, the IBA-evoked changes in IAA homeostasis resulted in root growth reorientation from longitudinal elongation to radial swelling. However, the application of an IAA signaling inhibitor, following the activation of defense responses by IBA, was able to promote root growth even at high concentrations of Cd. Based on the results, it can be concluded that the application of IBA, as an effective activator of Cd tolerance mechanisms in young barley roots, and the subsequent use of an IAA signaling inhibitor for the inhibition of root morphogenic responses induced by altered auxin metabolism, results in a high degree of root Cd tolerance, helping it to withstand even the transient exposure to lethal Cd concentration without the absolute inhibition of root growth.
Asunto(s)
Cadmio/toxicidad , Hordeum/efectos de los fármacos , Indoles/farmacología , Óxido Nítrico/metabolismo , Raíces de Plantas/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Hordeum/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Plantones/efectos de los fármacos , Plantones/metabolismoRESUMEN
The presented review is a summary on the current knowledge about metal induced stress response in plants, focusing on the roles of reactive oxygen species, auxin and nitric oxide in roots. The article focuses mainly on the difference between defence and toxicity symptoms of roots during metal-induced stress. Nowadays, pollution of soils by heavy metals is a rapidly growing issue, which affects agriculture and human health. In order to deal with these problems, we must first understand the basic mechanisms and responses to environmental conditions in plants growing under such conditions. Studies so far show somewhat conflicting data, interpreting the same stress responses as both symptoms of defence and toxicity. Therefore, the aim of this review is to give a report about current knowledge of heavy metal-induced stress research, and also to differentiate between toxicity and defence, and outline the challenges of research, focusing on reactive oxygen and nitrogen species, auxin, and the interplay among them. There are still remaining questions on how reactive oxygen and nitrogen species, as well as auxin, can activate either symptoms of toxicity or defence, and adaptation responses.
Asunto(s)
Ácidos Indolacéticos/metabolismo , Metales Pesados/toxicidad , Óxido Nítrico/metabolismo , Raíces de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Raíces de Plantas/efectos de los fármacosRESUMEN
The aim of this study was to observe the possible function of increased superoxide and NO production in the response of barley root tip to the harmful level of Cd. While superoxide generation was detected only in the transition zone, the formation of NO was observed in the apical elongation zones of the control root tips. However, the root region with the superoxide generation was also associated with peroxynitrite specific fluorescence signal. Superoxide, H2O2 and peroxynitrite generation increased with Cd treatment in a dose-dependent manner. In turn, NO level increased at low 10-20 µM but decreased at high 50-60 µM Cd concentrations in comparison with the control. While co-treatment of roots with rotenone markedly attenuated the Cd-induced superoxide generation and lipid peroxidation, it increased the level of NO in the root tips. Although rotenone did not influence the Cd-induced increase of GPX activity at 10-30 µM Cd concentrations, it markedly reversed the high 40-60 µM Cd concentrations-induced decline of GPX activity. Cd-induced cell death was associated with robust superoxide generation, but not with a high level of peroxynitrite. The Cd-evoked inhibition of root growth was significantly reversed by a strong antioxidant N-acetyl cysteine but not by a peroxynitrite scavenger uric acid, suggesting that similarly to Cd-induced cell death, an imbalance in the ROS homeostasis and not an enhanced level of peroxynitrite is responsible for the Cd-induced root growth inhibition. Based on these findings, it can be assumed that NO acts mainly in the regulation of superoxide level in the tips of root. Under Cd stress, the enhanced NO level is involved in the scavenging of highly toxic superoxide through the formation of peroxynitrite, thus reducing the superoxide-mediated cell death in barley root.
Asunto(s)
Cadmio/toxicidad , Hordeum/efectos de los fármacos , Meristema/efectos de los fármacos , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxidos/metabolismo , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hordeum/metabolismo , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Meristema/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In order to gain more insight into the involvement of mitochondrial complex III in the Cd-induced stress, we studied the effect of complex III inhibitors, antimycin A (AA), and myxothiazol (MYXO), on the Cd-induced ROS and NO generation in the barley root tip. Short-term exposure of barley roots to either MYXO or AA provoked a dose-dependent increase in both H2O2 and NO formation. In contrast to H2O2 generation, an enhanced superoxide formation in the transition zone of the root was a characteristic feature of AA-treated roots. MYXO and AA co-treatment had an additive effect on the amount of both H2O2 and NO formed in roots. On the other hand, AA-induced superoxide formation was markedly reversed in roots co-treated with MYXO. Both AA and MYXO exacerbated the Cd-mediated H2O2 or NO generation in the root tip. On the contrary, while AA also exacerbated the Cd-induced superoxide generation, MYXO dose-dependently attenuated it. These data provide strong evidence that ROS generation, a very early symptom of Cd toxicity in roots, is originated in mitochondria. Cd, similarly to AA, generates superoxide by blocking the mitochondrial electron transport chain (ETC) at complex III. In turn, the site of Cd-induced NO generation is not associated with complex III, but ROS formed in mitochondria at this third complex of ETC are probably responsible for enhanced NO generation in barley root under Cd stress.
Asunto(s)
Antimicina A/metabolismo , Cadmio/metabolismo , Hordeum/química , Peróxido de Hidrógeno/metabolismo , Óxido Nítrico/metabolismo , Raíces de Plantas/química , Superóxidos/metabolismo , Metacrilatos/metabolismo , Tiazoles/metabolismoRESUMEN
Exposure of barley roots to mM concentrations of L-NAME for 30â¯min caused a considerable root growth inhibition in a dose-dependent manner. The inhibition of root growth was higher in seedlings co-treated with Cd and L-NAME, compared with roots treated with Cd alone, despite the fact that L-NAME markedly reduced the uptake of Cd by roots. Treatment of roots with L-NAME evoked a decrease in NO level in both control and Cd-treated root tips only after a relatively long lag period, which overlaps with an increase in superoxide and H2O2 levels and peroxynitrite generation. L-NAME-induced root growth inhibition is alleviated not only by the application of the NO donor SNP but also by the ROS and peroxynitrite scavengers. Our results indicate that L-NAME, a NOS inhibitor in the animal kingdom, indeed evokes NO depletion also in the plant tissues; however, it does not occur due to the action of L-NAME as an inhibitor of NOS or NOS-like activity, but as a consequence of L-NAME-induced enhanced superoxide generation, leading to increased peroxynitrite level in the root tips due to the reaction between superoxide and NO.
Asunto(s)
Cadmio/toxicidad , Hordeum/efectos de los fármacos , Meristema/efectos de los fármacos , NG-Nitroarginina Metil Éster/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Arginina/administración & dosificación , Arginina/metabolismo , Hordeum/crecimiento & desarrollo , Hordeum/metabolismo , Meristema/crecimiento & desarrollo , Meristema/metabolismo , NG-Nitroarginina Metil Éster/administración & dosificación , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
As future scientists, university students need to learn how to avoid making errors in their own manuscripts, as well as how to identify flaws in papers published by their peers. Here we describe a novel approach on how to promote students' ability to critically evaluate scientific articles. The exercise is based on instructing teams of students to write intentionally flawed manuscripts describing the results of simple experiments. The teams are supervised by instructors advising the students during manuscript writing, choosing the 'appropriate' errors, monitoring the identification of errors made by the other team and evaluating the strength of their arguments in support of the identified errors. We have compared the effectiveness of the method with a journal club-type seminar. Based on the results of our assessment we propose that the described seminar may effectively complement the existing approaches to teach critical scientific thinking. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):22-30, 2018.