In vivo and in vitro studies on fluorophore-specificity

In vivo and in situ microscopy is a selective and easy method for detecting reactive oxygen (ROS)- and nitrogen species (RNS). Of the several fluorescent indicators developed in the last 30 years, the specificity and sensitivity of 4-amino-5-methylamino-2’-7’-difluorofluorescein diacetate (DAF-FM DA) as a nitric oxide (NO) indicator was tested by spectrofluorimetry and fluorescence microscopy. The peroxynitrite (ONOO-)-dependence of aminophenyl fluorescein (APF) and the hydrogen peroxide (H2O2)-sensitivity of 2’-7’-dichlorodihydrofluorescein diacetate (H2DCF DA) and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was also determined. The results show that DAF-FM is a suitable fluorophore for detecting NO in plant tissues and aminophenyl fluorescein can be used as a ONOO- -responsive dye. It was also found that DCF does not detect NO in solutions, but its fluorescence emission is strongly sensitive to H2O2. Moreover, the DCF fluorescence was found to be ONOO—sensitive, as well. In vivo studies revealed that Amplex Red can be applied as a H2O2-sensitive and -selective fluorophore in plant tissues

Nitric oxide (NO) generation during vegetative/generative transition of the apical meristem in wheat

The phase transition from vegetative to reproductive development of the shoot apical meristem (SAM) is a critical event during the life cycle of seed-propagated plants. Nitric oxide (NO) as a general plant signal plays a role during growth and development and under biotic and abiotic stress conditions. In this study, apical meristems of spring wheat (Triticum aestivum L. cv. Thaifun) were isolated during their development and NO was detected by DAF-FM using a fluorescent microscope. To test the NO specificity of DAF-FM, in vivo and in vitro experiments were also carried out. In vegetative apices NO level was very low and it significantly enhanced during transition (in the „double ridge” phase). After transition, NO level decreased and proved to be low in the generative phase (11th-13th week). Our results show that DAF-FM is a suitable and specific indicator of NO. The significant increase in NO level during the vegetative/generative transition of wheat SAM suggests its regulatory function

Nitric oxide as a potent signalling molecule in plants

The role of NO in stress responses in plants came in the focus of plant science in the last decade. Better understanding of plant stress responses is very important in the light of increasing intensities of stressors like drought, salinity and others, due to global climatic and environmental changes. Our knowledge, concerning signal transduction pathways is very scarce, especially in terms of NO-related alterations in proteins and gene expression as well as regulation. In this review we consider different NO-reactions, signalling pathways, NO – plant hormone interactions and NO-induced and -mediated signalization under osmotic stress in relation with the development of root architecture

Involvement of nitric oxide (NO) in plant responses to metalloids

Plants respond to the limited or excess supply of metalloids, boron (B), silicon (Si), selenium (Se), arsenic (As), and antimony (Sb) via complex signaling pathways that are mainly regulated by nitric oxide (NO). The absorption of metalloids from the soil is facilitated by pathways that involve aquaporins, aquaglyceroporins, phosphate, and sulfate transporters; however, their regulation by NO is poorly understood. Using in silico software, we predicted the S-nitrosation of known metalloid transporters, proposing NO-dependent regulation of metalloid transport systems at the posttranslational level. NO intensifies the stress-mitigating effect of Si, whereas in the case of Se, As, and Sb, the accumulation of NO or reactive nitrogen species contributes to toxicity. NO promotes the beneficial effect of low Se concentrations and mitigates the damage caused by B deficiency. In addition, the exogenous application of NO donor, sodium nitroprusside, reduces B, Se, and As toxicity. The primary role of NO in metalloid stress response is to mitigate oxidative stress by activating antioxidant defense at the level of protein activity and gene expression. This review discusses the role of NO in plant responses to metalloids and suggests future research directions. © 2021 The Author

Nitric oxide production induced by heavy metals in Brassica juncea L. Czern. and Pisum sativum L.

In plants, nitric oxide (NO) has multiple roles in defence reactions under abiotic stresses, including heavy metal load. Literature data suggest that there is a causal relationship between NO and iron metabolism but the effects of essential micronutrients/toxic heavy metals on NO production have not been investigated. In this study our aim is to demonstrate the possible role of NO in the plant response to heavy metals in the metal accumulator Brassica juncea and the crop plant Pisum sativum grown in the presence of either 100 μM cadmium, copper or zinc. NO production was measured in the root tips with fluorescent method, using 4,5-diaminofluorescein diacetate (DAF-2 DA), a specific dye to nitric oxide. We obtained different NO levels with the different heavy metal load: the most effective metal were copper and cadmium, in this case the NO production became double after one week treatment. In case of copper load, two-phase kinetics was found: a fast NO burst in the first six hours was followed by a slower, gradual increase. The fast appearance of NO in the presence of cupric ions suggest that it can be a novel reaction hitherto not studied in plants under heavy metal stress. After long-term treatment, NO levels were inversely related to the nitrite concentrations originated from nitrate reductase activity suggesting the conversion of nitrite to nitric oxide by the known enzymatic ways

Generation of nitric oxide in roots of Pisum sativum, Triticum aestivum and Petroselinum crispum plants under osmotic and drought stress

The concentration-, time- and tissue-dependent generation of nitric oxide (NO) was investigated in roots of Pisum sativum L. and Triticum aestivum L.. under osmotic stress, as well as in Petroselinum crispum L. under drought stress. Osmotic stress for pea and wheat was induced by polyethylene glycol (PEG) treatments in nutrient solution, while drought stress was caused by withdrawal of watering of soil-grown parsley. NO was detected by the NO-specific fluorescent dye, 4,5-diaminofluorescein-diacetate (DAF-2DA), using Zeiss Axiowert 200 M type fluorescent microscope. Changes in nitrate reductase activity was determined in the same series of treatments. Our results show that NO generation was proportional to the osmotic concentration of PEG both in pea and wheat roots and to the severity of drought in parsley root. The sites of NO production were in the regions of meristemic and elongation zones, and in case of wheat, root cap was also involved. In parsley root, the exodermis and the central cylinder showed the most intensive NO accumulation. In wheat and pea, time course revealed a fast transient (several hours) and a slow permanent increase in NO production. It is suggested that the fast kinetics may be due to non-enzymatic, while the constant increase was caused by enzymatic reactions. In parsley, long term experiments were carried out including the regeneration process after rewatering. It is concluded that NO plays a role as signaling molecule under osmotic and drought stress conditions

Early nitric oxide (NO) responses to osmotic stress in pea, Arabidopsis and wheat

Nitric oxide (NO) is a novel diffusible, lipophylic gas, which acts in diverse physiological processes in plants. The 4,5-diaminofluorescein diacetate (DAF-2DA)- based in vivo and in situ fluorescence method of NO detection gives an excellent opportunity to investigate the transient NO generations in plant tissues. During our work with the help of this method time-dependent kinetics of NO formation were investigated in osmotic stress treated-Pisum sativum L., Triticum aestivum L. and Arabidopsis thaliana L. roots. Osmotic stress was provoked by addition polyethylene glycol (PEG 6000) to the nutrient solution. In the case of pea plants an early phase of NO generation was distinguishable, which reached a maximum point at 24th hours after PEG treatment. This transient NO accumulation was followed by a slower but more significant NO formation. In Arabidopsis roots NO was formed already in the first 12 hours, the highest NO level was detected only 36 hours after treatment. Interestingly, in PEG-treated wheat roots the first NO peak appeared already after 1 hour treatment, which slightly moderated in the following 12 hours. In the cases of all the investigated plant species two phased NO generation was observed. The significant transient NO bursts were followed by slower NO accumulations. These early NO formations could have an important role in acclimation of plants to osmotic stress